Peripheral blood mononuclear cells increase the permeability of dengue virus-infected endothelial...

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Peripheral blood mononuclear cells increase thepermeability of dengue virus-infected endothelialcells in association with downregulation of vascularendothelial cadherin

Beti Ernawati Dewi,1,2,3 Tomohiko Takasaki1 and Ichiro Kurane1,3

Correspondence

Ichiro Kurane

[email protected]

1Laboratory of Vector-Borne Viruses, Department of Virology 1, National Institute of InfectiousDiseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

2Department of Microbiology, Medical Faculty, University of Indonesia, Jalan Pegangsaan Timur no.16, Jakarta 10320, Indonesia

3Department of Infection Biology, Institute of Basic Medical Sciences, University of Tsukuba,Tsukuba 305-8575, Japan

Received 6 August 2007

Accepted 30 October 2007

Plasma leakage is one of the characteristic features of dengue haemorrhagic fever. The interaction

among peripheral blood mononuclear cells (PBMCs), dengue virus and endothelial cells was

analysed in vitro. Human umbilical vein endothelial cells (HUVECs) were infected with dengue-2

virus (DV-2) at an m.o.i. of 0.5 p.f.u. per cell. PBMCs were added to DV-2-infected HUVECs, and

transendothelial electrical resistance (TEER) and transalbumin permeability were assessed.

Dengue virus infection at an m.o.i. of 0.5 p.f.u. per cell alone did not decrease the TEER, but

addition of PBMCs decreased the TEER, increased the albumin permeability and induced

morphological changes of HUVECs. The extent of the decrease was more profound with adherent

PBMCs than with non-adherent PBMCs. The expression of vascular endothelial cadherin

(VE-cadherin) was examined using real-time RT-PCR and immunofluorescence. Addition of

PBMCs to DV-2-infected HUVECs decreased the levels of mRNA transcripts and cell-surface

expression of VE-cadherin. The results indicate that PBMCs increased the permeability of

DV-2-infected HUVECs and that the increased permeability was concomitant with morphological

change and the decrease in VE-cadherin expression. The results suggest that functional

impairment of the DV-2-infected HUVEC monolayer was caused by interaction with PBMCs.

INTRODUCTION

Dengue viruses are a serious cause of morbidity andmortality in tropical and subtropical areas of the world(Halstead, 1988). Dengue viruses usually cause a mild tosevere febrile illness, dengue fever (DF), but may causedengue haemorrhagic fever (DHF) and dengue shocksyndrome (DSS) in some cases (Gubler, 1998; Halstead,1979). It is estimated that up to 100 million individuals areinfected with dengue viruses, with 500 000 cases of DHFand 25 000 deaths annually (Gubler, 1998). It has beensuggested that antibody-dependent enhancement of den-gue virus infection contributes to the pathogenesis of DHF(Halstead, 1979, 1988). It has also been proposed that theseverity of the disease is related to the virulence of thedengue virus strain (Leitmeyer et al., 1999; Rico-Hesseet al., 1997).

Although the mechanism of pathogenesis of DHF has notbeen completely elucidated, it is apparent that profoundplasma leakage is one of the characteristic physiological

features. Due to the lack of a good animal model,endothelial cells have been used as a surrogate to elucidatethe pathogenesis of dengue virus interactions. Endothelialcells can be infected with dengue virus in vitro (Bunyaratvejet al., 1997; Huang et al., 2003; Killen & O’Sullivan, 1993).Dengue virus infection induces cytokine production(Avirutnan et al., 1998; Bosch et al., 2002) and alters theexpression of adhesion molecules on endothelial cells(Krishnamurti et al., 2002). Although endothelial cells arenot massively infected, there is evidence of dengue virusinfection of endothelial cells in vivo (Gubler & Zaki, 1998;Hall et al., 1991; Jessie et al., 2004). It is possible that lowlevels of dengue virus infection of endothelial cells mayinduce profound effects locally, along with other factorssuch as cytokines and peripheral blood mononuclear cells(PBMCs).

Endothelial cells form a continuous cell layer along the wallof blood vessels and control the movement of solutes andfluid from the vascular space to the tissue (Tretiach et al.,2003). Two main types of junctional structures, adherence

Journal of General Virology (2008), 89, 642–652 DOI 10.1099/vir.0.83356-0

642 0008-3356 G 2008 SGM Printed in Great Britain


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and tight junctions, are involved in cell–cell contact. Theadherence junction is a multimolecular complex composedpredominantly of transmembrane proteins termed cadher-ins (Navarro et al., 1995; Steinberg & McNutt, 1999) and isa major contributor to the maintenance of monolayerintegrity (Allport et al., 2000; Dejana et al., 1999; DelMaschio et al., 1996). Vascular endothelial cells express aunique member of the cadherin family, vascular endothe-lial cadherin (VE-cadherin). VE-cadherin is a novelmember of the cadherin family and is selectively expressedat intercellular boundaries of all types of endothelial cells,but is not expressed by any other cell types (Dejana et al.,2001; Lampugnani et al., 1992). VE-cadherin plays animportant role in the maintenance of endothelial mono-layer integrity. Blocking of VE-cadherin functions withspecific antibodies results in increased vascular permeab-ility (Corada et al., 2001; Hordijk et al., 1999).

Activation of endothelial cells facilitates the adherence ofleukocytes to endothelial cells (McIntyre et al., 1985, 1997).Moreover, activated endothelial cells, in turn, activateadherent cells (McIntyre et al., 1985, 1997). Therefore, wehypothesized that adherence of leukocytes to endothelialcells infected with dengue virus might change the functionsof endothelial cells and increase permeability. In the presentstudy, we attempted to determine the effect of PBMCs onthe permeability of dengue virus-infected human umbilicalvein endothelial cells (HUVECs). A transendothelial elec-trical resistance (TEER) assay and albumin permeabilityassay were used to assess the permeability of HUVECs invitro (Dewi et al., 2004). In addition, we examined therelationship between the permeability of endothelial cellsand VE-cadherin expression.

METHODS

Human PBMCs. PBMCs were obtained from two healthy adult

donors. Donor A was dengue antibody-positive and had been

immunized with Japanese encephalitis vaccine 2 years previously.

Donor B was Japanese encephalitis antibody-positive but dengue

antibody-negative. PBMCs were separated by Ficoll-Paque

(Pharmacia Biotech AB) gradient centrifugation in LEUCOSEP tubes

(Greiner Labortechnik) according to the manufacturer’s instructions.

A ring of PBMCs in the tube were collected and washed twice in

30 ml RPMI 1640 medium. PBMCs were resuspended at a

concentration of 16106 cells ml21 in RPMI 1640 medium (Sigma)

supplemented with 10 % heat-inactivated fetal bovine serum (FBS)

(Sigma), L-glutamine and NaHCO3 (Gibco Invitrogen), 100 mg

streptomycin ml21 and 100 U penicillin ml21 (Gibco Invitrogen)

(Scott et al., 1980).

Separation of adherent and non-adherent PBMCs. Adherent

PBMCs were isolated as described by Malkovsky et al. (1987). One

million PBMCs in 1 ml RPMI supplemented with 10 % FBS were

added to 12-well tissue-culture plates (Nunc). After 2 h of incubation

at 37 uC in 5 % CO2, non-adherent cells were removed by pipetting and

plates were washed twice with RPMI medium without FBS. Adherent

PBMCs in the wells were removed by gentle scraping with a rubber cell

scraper. Adherent and non-adherent PBMCs were washed and

resuspended at a concentration of 16106 cells ml21 in RPMI

supplemented with 10 % FBS. Fifty microlitres non-adherent or

adherent PBMCs at a concentration of 16106 cells ml21 was addedto HUVECs on transwell membranes in the experiments. Thepercentage of monocytes in fractionated PBMCs was determined byimmunofluorescence with FITC-conjugated, anti-CD14 antibody(Biomedia). Anti-CD14 antibody reacts with the 55 kDa single-chainmembrane glycoprotein that is expressed primarily on monocytes andmacrophages.

Infection of HUVECs with dengue virus. HUVECs were purchasedfrom Clonetics (catalogue no. CC-2517) and propagated insupplemented endothelial cell basal medium-2 (Dewi et al., 2004).HUVECs were infected with dengue-2 virus (DV-2) New Guinea Cstrain on transwell membranes at an m.o.i. of 0.5 p.f.u. per cell.Briefly, medium in the upper transwell polycarbonate membraneculture dish was removed and the cells were infected by adding 50 mlDV-2 per well. Cells were incubated for 2 h at 37 uC in 5 % CO2, andwashed with PBS without CaCl2 and MgCl2 [PBS (2)]. Culturesupernatant fluid from uninfected C6/36 cells and heat-inactivatedDV-2 (Putnak et al., 1991) were used as controls. HUVECs were alsoinfected with DV-2 on slide chambers for determining the percentageof DV-2 antigen-positive cells and for immunofluorescence stainingof VE-cadherin.

Effect of PBMCs and culture supernatant fluids from DV-2-

infected HUVECs and PBMCs on the permeability of HUVECs.HUVECs were infected with DV-2 for 2 h, as described above, and50 ml of 106 to 102 PBMCs ml21 was added to the infected HUVECs.In other experiments, HUVECs on transwell membranes were washedand 100 ml culture supernatant fluid from DV-2-infected HUVECsand PBMCs were added. After various incubation times, the TEERwas assessed. Culture supernatant fluids from uninfected HUVECstreated with PBMCs were used as controls. Human TNF-a (1 mgml21) with a titre of 26107 U mg21 was used as a positive control(Dewi et al., 2004).

Transendothelial electrical resistance (TEER). The TEER ofHUVECs was measured using the electrical cell sensor systemEndohm chamber (Millicell-ERS; Millipore) (World PrecisionInstruments), as reported previously (Dewi et al., 2004). The TEERwas first measured at 24 h after seeding, and this time point wasdefined as 0 h. The TEER was measured at various incubation timepoints after addition of PBMCs or culture supernatant fluids fromDV-2-infected HUVECs and PBMCs. The TEER included theresistance of the interelectrode solution and blank membrane;therefore, a transwell polycarbonate membrane culture dish withoutcells was always included as the blank. Every test was done induplicate and repeated at least three times. The mean and SD werecalculated from two independent experiments with two transwellculture dishes per group.

Transendothelial albumin permeability. Transendothelial albuminpermeability on transwell polycarbonate membrane culture disheswas also examined using trypan blue-bound BSA as described byBonner & O’Sullivan (1998) with a minor modification (Dewi et al.,2004). Two transwell culture dishes per group were washed with PBSand 100 ml trypan blue-bound BSA was added to the upper chamberof each transwell polycarbonate membrane culture dish containingHUVEC monolayers (Dewi et al., 2004). The amount of trypan blue-bound BSA detected in the lower chamber was considered torepresent the level of HUVEC permeability.

Counting the PBMCs attached to HUVECs. In order to determinewhether dengue virus infection increases the number of PBMCsadherent to HUVECs, HUVEC-attached PBMCs were counted.HUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cellfor 2 h. PBMCs were added to HUVECs and incubated for 24 h. Cellswere washed twice with PBS (2), fixed with methanol at 37 uC for

Permeability of dengue virus-infected endothelial cells

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30 min and washed twice with PBS (2). Cells were then stained with

Giemsa for 30 min at room temperature and washed once with water

and once with 30 % methanol in PBS (2). After air drying, the

PBMCs on HUVECs were counted in five fields for each treatment

with a magnification of 6200 under a light microscope.

Morphology of HUVECs. HUVECs were seeded into 96-well flat-

bottomed microplates (Nunc) and infected with DV-2 at an m.o.i. of

0.5 p.f.u. per cell. PBMCs were added and incubated for 24 h.

HUVECs were then fixed and stained as reported previously (Dewi

et al., 2004). After air drying, the morphology of HUVECs was

observed under a light microscope with magnification of 6200.

Indirect immunofluorescence staining. PBMCs and HUVECs

were stained for DV-2 antigen by indirect immunofluorescence

staining as described previously (Dewi et al., 2004). Cells were

incubated with 1 : 250-diluted hyperimmune mouse ascites fluid to

DV-2 (Brandt et al., 1967) at 37 uC for 60 min. After washing, the

cells were reacted with 1 : 500-diluted FITC-conjugated sheep anti-

mouse IgG (Cappel) in PBS containing 5 % Block Ace (AbD Serotec)

at 37 uC for 60 min. The slide was washed and covered with Syva

microtech mounting fluid which contained PBS with glycerol

(Boehring Diagnostic Inc.). Cells were observed under a fluorescence

microscope.

Immunofluorescence staining of VE-cadherin. HUVECs on slide

chambers were washed with PBS and air-dried for 20 min. Cells were

fixed in acetone at –20 uC for 20 min and rinsed in PBS. Cells

were treated with Block Ace at 37 uC for 60 min. After washing

three times for 3 min with PBS, cells were incubated with 1 : 500-

diluted, FITC-labelled antibody to VE-cadherin (Bender

MedSystems) in PBS containing 5 % Block Ace at 37 uC for

60 min. Slides were washed and covered with Syva microtech

mounting fluid containing phosphate buffer with glycerol (Boehring

Diagnostic Inc.). Cells were observed under a fluorescence micro-

scope at 6400 magnification.

RNA isolation from HUVECs. HUVECs on 12-well plates (Nunc)

were lysed with 1 ml Isogen (Wako Nippon Gene) and 200 ml

chloroform was then added. RNA isolation was done according to the

manufacturer’s instructions (Wako Nippon Gene). The RNA was

dissolved in 20 ml ultrapure DNase-free, RNase-free distilled water

(Invitrogen Life Technologies) and then treated with DNase I

(Invitrogen Life Technologies) at room temperature for 15 min to

remove contaminating DNA. The RNA concentration was measured

using a spectrophotometer and adjusted to 100 mg ml21 and used for

real-time RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) was used as a housekeeping gene to confirm the use of

equal amounts of HUVEC mRNA.

Real-time RT-PCR. TaqMan primers and probes for detection of the

VE-cadherin gene were prepared according to the sequence of

GenBank accession no. NM_001795 and those for the GAPDH gene

used GenBank accession no. BC004109. Primers used for amplifica-

tion of VE-cadherin were forward primer 59-TCACCTGGTCGCCA-

ATCC-39 and reverse primers 59-AGGCCACATCTTGGGTTCCT-39.

The probe specific for VE-cadherin was 59-TCTCTTTCTTTTCTC-

TGTCTACTCCTTATCCCTTGGTT-39. Primers used for amplifica-

tion of GAPDH were forward primer 59-GAAATCCCATCACC-

ATCTTCCA-39 and reverse primer 59-CCAGCATCGCCCCACTT-39.

The probe specific for GAPDH was 59-AGCGAGATCCCTCC-39.

The ABI PRISM 7000 sequence detection system (Applied

Biosystems) was used for one-step RT-PCR and hybridization.

Aliquots (5–10 ng) of purified RNA were used as the template in

25 ml total reaction volumes. The reagents were added to the TaqMan

one-step RT-PCR master mix reagents (PE Applied Biosystems). The

real-time RT-PCR program was: initial incubation for 30 min at48 uC for reverse transcription and denaturation at 95 uC for 10 minfollowed by 45 cycles of 95 uC for 15 s and 57 uC for 1 min. All testswere performed in duplicate.

Statistical analysis. The significance of differences was determinedby Student’s t-tests compared with control HUVECs withouttreatment. The data were expressed as means±SD, and differencesare considered to be significant when the P value was less than 0.05.The TEER test consisted of at least three independent experimentswith two transwell polycarbonate membrane culture dishes.

RESULTS

Effect of PBMCs on the permeability of HUVECs

The effect of PBMCs on the TEER of HUVECs wasexamined. HUVECs on a transwell membrane wereinfected with DV-2 at an m.o.i. of 0.5 to 0.0005 p.f.u. percell or treated with C6/36 supernatant or inactivated DV-2for 2 h. After washing, 56105 to 56102 PBMCs wereadded to DV-2-infected HUVECs and the TEER wasassessed. Addition of 56105 or 56104 PBMCs to HUVECsinfected with DV-2 at an m.o.i. of 0.5 p.f.u. per celldecreased the TEER, but addition of 56103 or fewerPBMCs did not (Tables 1 and 2). The results were similarusing PBMCs from donor B.

HUVECs infected with DV-2 at an m.o.i. of 0.5 p.f.u. percell did not demonstrate a decrease in the TEER or anincrease in albumin permeability compared with untreatedHUVECs unless PBMCs were added (Table 1). Addition ofPBMCs to untreated HUVECs or to HUVECs treated withC6/36 supernatant or inactivated DV-2 did not decreasethe TEER or increase the albumin permeability. The TEERof PBMCs alone was at the similar level to wells containingno cells (data not shown).

Changes in permeability were also assessed by the albuminpermeability assay. Addition of 56104 PBMCs to HUVECsinfected with DV-2 at an m.o.i. of 0.5 p.f.u. per cellincreased the albumin permeability across HUVECs(Fig. 1), but addition of PBMCs to HUVECs treated withC6/36 supernatant or inactivated DV-2 did not. Thus, theincrease in the albumin permeability across HUVECs wasconsistent with the decrease in the TEER. The increase inthe albumin permeability across HUVECs become moreprofound on days 2–5.

Effects of adherent and non-adherent PBMCs onthe permeability of HUVECs

PBMCs were separated into adherent and non-adherentfractions and used in the assays. The adherent and non-adherent fractions contained CD14-positive monocytes at94±4 % and 5±2 %, respectively. Both adherent and non-adherent fractions decreased the TEER of DV-2-infectedHUVECs. The extent of the decrease was more profoundwith the adherent fraction than with non-adherent fraction(Table 1).

B. E. Dewi, T. Takasaki and I. Kurane

644 Journal of General Virology 89


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Effect of the supernatant fluid from DV-2-infectedHUVECs and PBMCs on the permeability ofHUVECs

HUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u.per cell for 2 h, 56104 PBMCs were added and super-natants were collected at various time points. The collectedculture supernatant fluids were then added to HUVECs ontranswell membranes and the permeability was assessed for3 days. Culture supernatant fluids collected on days 1 and2 did not decrease the TEER, but those collected on days 3and 4 did (Table 2). The decrease in the TEER started 4 hafter addition of the supernatant fluids. Those collected ondays 5, 6 and 7 also decreased the TEER (data not shown).Supernatant fluids collected on days 3–7 from the culturecontaining HUVECs treated with inactivated DV-2 or C6/36 and PBMCs did not decrease the TEER (data notshown).

Effect of the level of DV-2 infection on thepermeability of HUVECs

To determine the relationship between the level of DV-2infection of HUVECs and the PBMC-induced decrease inthe TEER, HUVECs were infected with DV-2 at variousm.o.i. and then 56104 PBMCs were added. Infection ofHUVECs with DV-2 at an m.o.i. of 0.5 p.f.u. per celldecreased the TEER when PBMCs were added, butinfection at an m.o.i. of 0.05 p.f.u. per cell or less did not(Table 2). HUVECs treated with C6/36 supernatant or

treated with inactivated DV-2 did not decrease the TEERwhen 56104 PBMCs were added.

The percentage of DV-2 antigen-positive HUVECs wasexamined after infection at an m.o.i. of 0.5 p.f.u. per cell.DV-2 antigen-positive cells were detected as early as 1 dayafter infection (0.9±0.4 %), and the proportion of positivecells increased gradually until day 5 (23±3.1 %) (Fig. 2).DV-2 antigen was not detected in HUVECs treated withinactivated DV-2 or C6/36 cell culture supernatant.

Adherence of PBMCs to HUVECs infected withDV-2

Adherence of PBMCs to HUVECs was analysed. PBMCswere added to DV-2-infected or uninfected HUVECs andcultured for 1 day. Non-adherent PBMCs were washed outand the PBMCs that remained on the surface of HUVECswere counted. Large numbers of PBMCs were observed onDV-2-infected HUVECs (Fig. 3b), but only small numberswere observed on uninfected HUVECs (Fig. 3a). Thenumber of PBMCs adherent to the surface of DV-2-infectedHUVECs was 0.21±0.18 PBMCs per cell in DV-2-infectedHUVEC monolayers and 0.025±0.01 per cell in uninfectedmonolayers. When endothelial cells were pretreated withC6/36 cell supernatant or inactivated DV-2, the number ofPBMCs adherent to the surface of HUVECs was 0.042±0.02and 0.038±0.09 per cell, respectively. The results indicatethat DV-2 infection of HUVEC monolayers increased thenumber of PBMCs adherent to HUVECs.

Table 1. Decrease in the TEER of HUVECs induced by PBMCs

HUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell or treated with inactivated DV-2 or with C6/36 culture supernatant and PBMCs

were added to the HUVECs. The TEER was assessed at various times. The data represent means of two independent experiments±SD, each done in

duplicate. Statistical analysis was determined by Student’s t-test versus control HUVECs without any treatment. Values showing statistically

significant differences are underlined.

Treatment TEER (V cm”2) (mean±SD)

0 h 1 day 30 h 2 days 3 days 4 days 5 days 6 days 7 days

Control 9.98±0.47 12.21±0.33 12.71±0.47 14.85±1 19.31±0.23 19.14±0.23 18.81±0.23 17.82±0.7 15.84±0.99

DV-2 plus 56104

PBMCs

10.08±0.23 9.55±0.23* 9.24±0.23* 9.24±0.33* 8.58±0.47D 8.42±0.47D 8.22±0.66D 7.42±0.46D 6.77±0.23D

DV-2 plus 56104

adherent PBMCs

10.08±0.46 9.44±0.46* 9.17±0.07* 9.17±0.23* 8.4±0.0D 7.51±0.47D 5.61±0.23D 5.28±0.23D 4.12±0.7D

DV-2 plus 56104

non-adherent PBMCs

10.56±0.23 11.37±0.46* 11.7±0.23*11.54±0.23* 11.24±0.47D 10.37±0.0D 9.57±0.23D 9.07±0.47D 8.42±0.23D

TNF-a (1 mg ml21) 9.98±0.33 8.25±0.89* 7.59±0.47* 8.25±0.5D 7.01±0.7D 5.78±0.47D 4.95±1.25D 4.79±1.2D 3.96±0.66D

DV-2 (m.o.i. 0.5) 9.91±0.33 12.86±0.23 12.71±0.66 17.16±0.7 19.47±0.47 18.48±0.7 16.34±1.08 14.21±0.23* 12.4±1.16*

PBMCs (56104) 10.24±0.19 14.03±0 13.86±0.23 19.47±0.7 20.13±0 19.47±0.23 20.3±1.08 19.47±0.23 18.32±0.23

C6/36 plus 56104 PBMCs10.08±0.67 12.06±0 11.88±0.23 13.86±0.33 17.16±0.66 21.78±0.33 18.98±0.66 16.34±0.66 13.04±0.33*

Inactivated DV-2 plus

56104 PBMCs

9.91±0.33 12.08±0.33 11.72±0.46 14.52±0.33 16.5±0.66 21.62±0.66 20.3±0.54 18.15±0.4 13.37±0.5*

*P value ,0.05.

DP value ,0.01.




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Table 2. Comparison of the decrease in the TEER observed with various numbers of PBMCs, various m.o.i. of DV-2 and supernatantof DV-2-infected HUVECs and PBMCs

The TEER was assessed at various incubation times. Data are means of two independent experiments±SD, each done in duplicate. Statistical

analysis was determined by Student’s t-test versus control HUVECs without any treatment. Values showing statistically significant differences are

underlined.

Treatment TEER (V cm”2) (mean±SD)

0 h 1 day 30 h 2 days 3 days 4 days 5 days 6 days 7 days

Control 9.98±0.47 12.21±0.33 12.71±0.47 14.85±1 19.31±0.23 19.14±0.23 18.81±0.23 17.82±0.7 15.84±0.99

TNF-a (1 mg ml21) 9.98±0.33 8.25±0.89* 7.59±0.47* 8.25±0.5D 7.01±0.7D 5.78±0.47D 4.95±1.25D 4.79±1.2D 3.96±0.66D

DV-2 (m.o.i. 0.5)

+PBMCs (56105)

10.24±0.33 9.04±0.05* 8.91±0* 8.54±0.33D 8.05±0.33D 8.09±0D 6.59±0.33D 6.76±0.66D 5.12±0.33D

DV-2 (m.o.i. 0.5)

+PBMCs (56104)

10.08±0.23 9.55±0.23* 9.24±0.23* 9.24±0.33* 8.58±0.47D 8.42±0.47D 8.22±0.66D 7.42±0.46D 6.77±0.23D

DV-2 (m.o.i. 0.5)

+PBMCs (56103)

9.91±0.33 11.22±0.33 10.56±0.99 13.45±2.64 17.68±0.66 18.57±1.65 15.87±0.33 13.04±0* 10.4±0.33*

DV-2 (m.o.i. 0.5)

+PBMCs (56102)

10.24±0.33 12.54±0.33 12.54±0.33 18.83±0.99 19.31±0.33 19.82±0.33 17.86±0.23 15.53±0.33 14.39±0.33

DV-2 (m.o.i. 0.05)

+PBMCs (56104)

9.91±0.99 12.54±0.99 12.05±0.66 14.52±0.33 17.66±0.99 24.92±0.66 20.3±2.31 18.32±0.23 14.19±0.33

DV-2 (m.o.i. 0.005)

+PBMCs (56104)

9.91±0.33 13.2±0.99 13.7±0.33 16.83±0.99 20.63±1.32 26.89±0.33 23.1±0 16.83±2.31 14.36±2.64

DV-2 (m.o.i. 0.0005)

+PBMCs (56104)

10.24±0.66 13.2±0.33 13.53±0.66 17.16±1.98 20.46±0.33 27.39±0.33 24.26±2.64 19.47±0.66 17.16±0.33

DV-2 (m.o.i. 0.00005)

+PBMCs (56104)

9.99±0.12 12.87±0.47 13.2±0.7 17.41±0.82 20.46±0.23 26.32±0.82 23.02±0.11 18.77±1.75 15.26±1.28

Supernatant DV-2

+PBMCs (day 1)

9.08±0.33 12.21±0.99 12.72±0.46 14.85±0.33 19.3±0.66 19.1±0.66 18.8±0.54 17.85±0.4 15.8±0.5

Supernatant DV-2

+PBMCs (day 2)

9.31±0.47 11.89±0.33 12.34±0.46 13.56±0.33 16.98±0.66 18.62±0.66 17.9.3±0.54 17.15±0.4 14.37±0.66

Supernatant DV-2

+PBMCs (day 3)

9.08±0.33 8.35±0.33* 9.72±0.46* 8.15±0.66D 8.2±0.66D 8.54±0.83D 8.12±0.66D 8.15±0.33D 7.37±0.5D

Supernatant DV-2

+PBMCs (day 4)

9.05±0.66 8.3±0.5* 8.23±0.66* 8.09±0.5D 8.89±1.32D 7.62±0.66D 7.3±0.54D 8.15±0.4D 7.37±0.33D

*P value ,0.05.

DP value ,0.01.

Fig. 1. Clearance of labelled BSA acrosscontrol and treated HUVECs at variousincubation times. Addition of PBMCs toinfected HUVECs showed increased clear-ance of labelled BSA (P,0.05). A similarresult was also shown for HUVECs treatedwith 1 mg TNF-a ml”1.




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Morphological changes of HUVECs

The morphology of HUVECs was examined under a lightmicroscope 1 day after the addition of PBMCs. UntreatedHUVECs showed a cobblestone-like morphology, withtight cell–cell contact (Fig. 4a). Infection of HUVECs at anm.o.i. of 0.5 p.f.u. per cell did not change the morphologyof HUVECs (Fig. 4b). When HUVECs were infected withDV-2 at an m.o.i. of 0.5 p.f.u. per cell and PBMCs wereadded, HUVECs demonstrated a thinner and flatter,spindle-shaped morphology (Fig. 4e). This morphologicalchange was similar to that observed in TNF-a-treatedHUVECs (Fig. 4f). Addition of PBMCs to HUVECs treatedwith inactivated DV-2 or C6/36 did not change themorphology (Fig. 4c, d). Thus, the morphological changeswere concomitant with the decrease in the TEER andincreased albumin permeability of HUVECs (Table 1, Figs1 and 4).

Change in the level of VE-cadherin expression

VE-cadherin is a molecule that plays a critical role inmaintaining the integrity of vascular endothelial cells.Therefore, the expression of VE-cadherin on HUVECs wasexamined by immunofluorescence staining after DV-2infection and addition of PBMCs. Untreated HUVECsdemonstrated a tight apposition of adjacent cell mem-branes and strong membrane fluorescence at the periphery(Fig. 5a). When HUVECs were infected with DV-2 andPBMCs were added, HUVECs demonstrated intercellulargaps and VE-cadherin expression was decreased at theperiphery (Fig. 5e), similar to those treated with TNF-a

(Fig. 5f). There were multiple large intercellular gapsbetween adjacent cells (arrows in Figs 5e and f). WhenHUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u.per cell and no PBMCs were added (Fig. 5b), treated withinactivated DV-2 and PBMCs were added (Fig. 5c) ortreated with C6/36 cell supernatant and PBMCs were

added (Fig. 5d), HUVECs demonstrated a VE-cadherinstaining pattern similar to that of untreated cells.

Quantification of VE-cadherin by real-time RT-PCR

Levels of VE-cadherin mRNA were assessed by real-timeRT-PCR (Table 3). The levels of GAPDH were similaramong all the experimental groups, suggesting that equalamounts of mRNA were used in this study (Table 3). UsingVE-cadherin-specific primers and probe, untreatedHUVECs and those infected with DV-2 at an m.o.i. of0.5 p.f.u. per cell demonstrated positive signals at23.76±0.08 and 23.25±0.44 cycles, respectively. WhenHUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u.per cell and PBMCs were added, positive signal wasdeveloped at 26.23±0.18 cycles (P,0.05 compared withuntreated HUVECs). TNF-a-treated HUVECs demon-strated a positive signal at 26.89±0.04 cycles (P,0.01compared with untreated HUVECs). When HUVECs weretreated with C6/36 supernatant or inactivated DV-2 andPBMCs were added, positive signals were developed at

Fig. 3. Adherence of PBMCs to the surface of DV-2-infectedHUVECs after washing off the non-adherent PBMCs. (a) Numberof PBMCs adherent to the surface of DV-2-uninfected HUVECswas 0.015 per cell. (b) Number of PBMCs adherent to the surfaceof DV-2-infected HUVECs was 0.22 per cell. The arrow shows anadherent PBMC. Original magnification, �200.

Fig. 2. Percentages of DV-2 antigen-positive HUVECs. HUVECswere infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell. Positivecells were determined by indirect immunofluorescence assay atvarious incubation times.




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23.99±0.50 and 23.66±0.76 cycles, respectively. Theresults indicate that the amounts of VE-cadherin mRNAwere lower when HUVECs were infected with DV-2 and

PBMCs were added, and suggest that the decrease in theTEER is concomitant with the decrease in the level of VE-cadherin.

Fig. 4. Morphological changes of HUVECs 1 day after addition of 5�104 PBMCs, showing a control (a) and HUVECs treatedwith DV-2 at an m.o.i. of 0.5 p.f.u. per cell (b), inactivated DV-2 and PBMCs (c), C6/36 cell supernatant and PBMCs (d), DV-2at an m.o.i. of 0.5 p.f.u. per cell and PBMCs (e) and 1 mg TNF-a ml”1 (f). HUVECs were fixed using 10 % formaldehyde andstained with trypan blue.

Fig. 5. VE-cadherin expression on HUVECs 1 day after addition of 5�104 PBMCs, showing control HUVECs (a) and HUVECstreated with DV-2 at an m.o.i. of 0.5 p.f.u. per cell (b), inactivated DV-2 and PBMCs (c), C6/36 cell supernatant and PBMCs (d),DV-2 at an m.o.i. of 0.5 p.f.u. per cell and PBMCs (e) and 1 mg TNF-a ml”1 (f). The VE-cadherin staining pattern of controlHUVECs consisted of a continuous lining of cell borders with strong fluorescence. In contrast, treatment with DV-2 and PBMCsor TNF-a (e, f) led to a marked decrease in staining, with weak fluorescence.




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DISCUSSION

The pathological mechanisms of DHF/DSS are notcompletely understood. In order to elucidate thesemechanisms, interactions among endothelial cells,PBMCs and DV-2 were analysed in vitro using HUVECs.Addition of PBMCs to DV-2-infected HUVECs decreasedthe TEER. The decrease in the TEER was consistent withthe increased albumin permeability across HUVECs andconcomitant with morphological changes observed inHUVECs and the decreases in VE-cadherin expressionand VE-cadherin mRNA.

Infection of HUVECs with DV-2 at an m.o.i. of 0.5 p.f.u.per cell did not decrease the TEER unless PBMCs wereadded. It is possible that dengue virus mediates endothelialcell barrier function via an indirect route rather than directinfection of endothelial cells. In general, however, the tissuedamage observed in pathological studies is not very severecompared with the severity of illness (Bhamarapravati,1989). Furthermore, the lack of structural damage, theshort-lived nature of the plasma leakage syndrome and theremarkably rapid recovery of children with DSS all suggestthat the alteration of permeability is effected by a solublemediator (Innis, 1995).

Given the increased permeability of endothelial cells in thepresence of PBMCs, it is likely that increased permeabilityof HUVECs was in part due to the cytokines which wereproduced by DV-2-infected PBMCs. Dengue viruses canreplicate in monocyte-derived macrophages and mono-cyte-like cell lines (Kurane et al., 1990; O’Sullivan & Killen,1994) and induce cytokines (Bosch et al., 2002; Espina et al.,2003) and cytotoxic factors (Mukerjee et al., 1995; Shaioet al., 1995). Culture fluids from dengue virus-infectedperipheral blood monocytes activated endothelial cells, as

measured by upregulation of cell adhesion molecules(Anderson et al., 1997), and increased the permeability ofendothelial cells in vitro (Carr et al., 2003). It has also beenreported that cytokines play an important role in thepathogenesis of DHF (Rothman & Ennis, 1999), andplasma leakage in DHF patients is associated with theelevation of plasma levels of various cytokines (Green et al.,1999; Kurane et al., 1991).

There was a report that only the supernatant collected fromdengue virus-infected monocyte-macrophages at 72 hincreased permeability, and not that collected before 72 h(Carr et al., 2003), and similar results were observed in thepresent study. In this experiment, we found that the TEERwas decreased (P,0.05) on day 1 after the addition ofPBMCs. Furthermore, larger numbers of PBMCs attachedto the surface of DV-2-infected HUVEC monolayers afterwashing than to uninfected HUVECs. It is possible that thedecrease in the TEER before 48 h after addition of PBMCsis due to activation of PBMCs by adherence to the DV-2-infected endothelial cell monolayer. After infection withdengue virus, endothelial cells express adhesion molecules(Krishnamurti et al., 2002). PBMCs which attached toinfected endothelial cells are activated, and activatedPBMCs may produce cytokines that act locally and increaseplasma leakage. Activation of endothelial cells, in turn,might activate the adherent leukocytes (McIntyre et al.,1997; McIntyre, 2000) and produce cytokines, chemokinesand tissue factors in a contact-dependent manner (Monacoet al., 2002).

Addition of PBMCs to HUVECs infected with DV-2 at anm.o.i. of less than 0.5 p.f.u. per cell did not decrease theTEER. Although the underlying mechanism remains poorlyunderstood, it seems that the number of viruses isassociated with the severity of disease. Epidemiologicalstudies of DHF patients showed that DV-3 viraemia wasgreater in more severe clinical disease (Libraty et al., 2002).Previous studies examining DV-1 and DV-2 infectionshave reported higher circulation levels of replicating virus(Vaughn et al., 2000) or viral RNA copies (Murgue et al.,2000) in DHF than in DF patients. Addition of PBMCs tocontrol HUVECs or HUVECs pretreated with C6/36 cellssupernatant or pretreated with inactivated DV-2 did notdecrease the TEER. These results indicate that infection ofHUVECs is necessary for the decrease of the TEER in thisexperimental system.

We separated adherent and non-adherent cell fractionsfrom PBMCs and tested their effects on the decrease of theTEER. The majority of adherent PBMCs on plastic wellplates were determined to be monocytes by immunofluor-escence staining with anti-CD14 antibody. AdherentPBMCs decreased the TEER to a greater extent than non-adherent PBMCs. It was reported that adherent cells are themajor cell population that supports dengue virus replica-tion in vivo (Scott et al., 1980). Allport et al. (2000) alsoshowed that the adherence of monocytes on endothelialcells induces focal disruption of the VE-cadherin.

Table 3. Real-time quantitative RT-PCR analysis to determinethe level of VE-cadherin mRNA in control and treated HUVECs

Data are means±SD. Statistical analysis was done in comparison with

control HUVECs; values showing statistically significant differences

are underlined.

Treatment CT value (cycle)

GAPDH

detector

VE-cadherin

detector

Control HUVECs 28.41±0.10 23.76±0.08

DV-2+PBMCs (56104) 28.39±0.11 26.23±0.18*

Inactivated DV-2+PBMCs

(56104)

28.43±0.02 23.66±0.76

C6/36+PBMCs (56104) 28.52±0.04 23.99±0.50

DV-2 alone 29.03±0.24 23.25±0.44

PBMCs (56104) alone 28.47±0.05 23.12±0.57

TNF-a (1 mg ml21) 28.48±0.11 26.89±0.04D

*P value ,0.05.

DP value ,0.01.




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Consistent with these reports, the extent of the decreasewas more profound with adherent PBMCs than with non-adherent PBMCs in our study. We suggest that attachmentof PBMCs leads to production of cytokines that act locallyand increase the permeability of DV-2-infected HUVECs.

The morphology of HUVECs changed when HUVECs wereinfected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell andPBMCs were added. A similar result was found onHUVECs treated with TNF-a. It was reported that thelow TEER was associated with irregular cell shape, whenstudied morphologically (Tretiach et al., 2003), andalterations in the permeability of the vascular endothelium(Bucana et al., 1988). McKenzie & Ridley (2007) found thatTNF-a subsequently caused a progressive increase inpermeability and in stress fibre reorganization, cellelongation and intercellular gap formation over 8–24 h.Consistent with the increased permeability, occludin andJAM-A were removed from tight junctions and ZO-1 waspartially redistributed (McKenzie & Ridley, 2007). Similarresults were obtained in the present study. Morphologicalchanges were observed concomitantly with the decrease inthe TEER and increased permeability.

Additionally, we examined the levels of VE-cadherinmRNA using real-time RT-PCR. Only when PBMCs wereadded, HUVECs infected with DV-2 demonstrated lowerlevels of VE-cadherin mRNA. In immunofluorescenceassays, infection of HUVECs with DV-2 at an m.o.i. of0.5 p.f.u. per cell and addition of PBMCs resulted in amarked decrease in immunofluorescence for membrane-associated VE-cadherin. This treatment also led to thedevelopment of multiple intercellular gaps between adja-cent cells. Since VE-cadherin plays a crucial role in themaintenance of endothelial cell–cell contact and monolayerintegrity (Breviario et al., 1995; Del Maschio et al., 1996),the appearance of multiple intercellular gaps betweenadjacent cells probably indicates an increase in permeab-ility. These results are consistent with previous reportsusing TNF-a (Friedl et al., 2002; Hofmann et al., 2002) andthrombin (Rabiet et al., 1996).

The disassembly of the adherence-junction component, asindicated by the decrease in VE-cadherin expression,probably contributes to the increase in endothelialpermeability in this study. In addition to adherencejunctions, endothelial cells also possess tight junctions.Studies are also needed to understand the role of the tightjunction of endothelial cells in the pathogenesis of DHF.Furthermore, the relevance of the results in the presentstudy to the pathogenesis of DHF in vivo needs to beelucidated in future studies.

ACKNOWLEDGEMENTS

We thank Mr Takagi, Dr M. Ito, Dr S. Tajima, Mr A. Kotaki and MsR. Nerome, Laboratory of Vector-Borne Viruses, Department ofVirology 1, National Institute of Infectious Diseases, for theirtechnical help. This study was supported by a grant-in-aid (no.12877045) from the Ministry of Education, Sciences, Sports and

Culture, Japan, and a grant for Research on Emerging andReEmerging Infectious Diseases from the Ministry of Health, Laborand Welfare, Japan (H18-Shinkou-ippan-009).

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