dengue virus infection of human micro vascular

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Journal of Medical Virology 78:229242 (2006)

Dengue Virus Infection of Human MicrovascularEndothelial Cells From Different VascularBeds Promotes Both Common and SpecificFunctional Changes

Christophe N. Peyrefitte,1 Boris Pastorino,1 Georges E. Grau,2 J. Lou,2 Hugues Tolou,1

and Patricia Couissinier-Paris1*1Unite de virologie tropicale, Institut de Me decine Tropicale du Service de Sante des Arme es, Marseille, France2Unite des Rickettsies, CNRS UMR, Faculte de Medecine, IFR 48,Universite de la Mediterrane e, Marseille, France

Dengue shock syndrome (DSS), the major lifethreatening outcome of severe dengue disease,which occurs in some patients in the course ofdengue infection, is the consequence of plasmaleakage in the microvascular territories. Datafrom clinical and in vitro studies suggest that aninadequate immunological response is partlyresponsible for the pathophysiology of DSS, butfew is known concerning the consequences ofdirect infection of endothelial cells by denguevirus per se. In this study, an attempt was made to

study the response of two microvascular humancell lines originating, respectively, from liver anddermis to infection by a dengue type 2 virus, byanalyzing the virus-induced modulation of func-tional markers. It is shown that the two micro-vascular cell lines exhibit both common andspecific behaviors upon infection. In particular,LSEC and HMEC-1 replicate efficiently the low-passage virus and respond to infection by over-producing inflammatory mediators involved inthe cross talk with circulating immune cells.However, direct infection modulates differentlythe cell surface expression of molecules criticallyinvolved in the interactions between endothelialand inflammatory cells. ICAM-1 and HLA-I are upregulated as a consequence of infection in LSECwhereas direct infection results in downregula-tion of ICAM-1 in HMEC-1. The present resultsshow that infection of human microvascularcells by unadapted dengue virus results in bothcommonand specific activation patterns depend-ing likely on the tissue origin of the cells, thussuggesting that endothelia from different terri-tories may contribute differently to the patho-physiological events in the course of dengueinfection. J.Med. Virol. 78:229242, 2006. 2005 Wiley-Liss, Inc.

KEY WORDS: virus; flavivirus; endothelium;activation; plasma leakage

INTRODUCTION

Dengue viruses are arthropod-borne viruses belong-ing to the Flaviviridae family which are responsible forinfections in humans in all intertropical regions of theworld [Guzman et al., 2004]. Dengue infection presentsthrough a broad spectrum of clinical pictures of whichmild acute febrile illness is the more frequent. However,severe forms, that is dengue hemorrhage fever (DHF)and dengue shock syndrome (DSS), the major lifethreatening form of severe dengue disease, are reportedwith an increasing frequency [Guzman et al., 2004].Currently, only non-specific, symptomatic therapeuticsis available for the treatmentof patientspresenting withthese severe forms [Damonte et al., 2004].

The hemodynamic failure characterizing the dengueshock syndrome (DSS) is consecutive to a transientbut massive plasma leakage in the microvasculature[Srichaikul and Nimmannitya, 2000; Guzman andKouri, 2002]. Transitory endothelial dysfunctions, morethan cell destruction or irreversible alterations, likely

support the capillary leakage, as suggested by the rapid

Grant sponsor: Delegation Generale de lArmement (DGA);Grant number: 02 CO 013; Grant sponsor: Service de Sante des

Armees (SSA).

*Correspondence to: Dr. Patricia Couissinier-Paris, Institut deMedecine Tropicale du Service de Sante des Armees, BP 46, Parcdu Pharo, 13007 Marseille, France.E-mail: [email protected]

Accepted 30 September 2005

DOI 10.1002/jmv.20532

Published online in Wiley InterScience(www.interscience.wiley.com)

2005 WILEY-LISS, INC.


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reversibility of DSS in patients under resuscitation[Iyngkaran et al., 1995; Srichaikul and Nimmannitya,2000], and the absence of destruction of endothelial cellsin post-mortem biopsies from patients who died fromDSS [Bhamarapravati et al., 1967; Sahaphong et al.,1980]. Several evidences indicate that the immune

response to dengue virus plays a key role in thepathophysiological cascade leading to plasma leakage[Chen and Cosgriff, 2000; Lei et al., 2001; Guzman andKouri, 2002]. In particular, DHF and DSS have beenassociated with high levels of inflammatory cytokinesin the serum of patients [Iyngkaran et al., 1995; Greenet al., 1999; Gagnon et al., 2002; Liu et al., 2002; Suhartiet al., 2003]. Moreover, in vitro studies have shown thatsoluble mediators produced by monocytes or macro-phages infected by dengue virus in different conditions,activate endothelial cells in vitro [Anderson et al., 1997;Carr et al., 2003]. Other immune-mediated mechanismsinvolving cross-reactive auto-antibodies generated inresponse to the dengue NS1 protein, could also contri-bute to the pathogenesis of secondary dengue infectionby inducing limited apoptosis or cytolysis of endothelialcells [Lin et al., 2003].

The possibility that dengue virus infection may affectdirectly the functions of microvascular endothelial cellsis much debated. Indeed, no definitive conclusion can bedrawn from ex vivo studies due to the very limitednumber of histological data produced from post-mortembiopsies from DSS patients [Bhamarapravati et al.,1967; Sahaphong et al., 1980; Jessie et al., 2004]. In theabsence of relevant animal models, in vitro infection ofendothelial cells represent an alternative approach forthe identification of cell alterations that could be impli-

cated in the pathological process leading to DSS.Theability of dengueviruses to infect endothelial cells

in vitro has already been demonstrated by others usingdifferent sources of endothelial cells and viruses[Andrews et al., 1978; Bunyaratvej et al., 1997;

Avirutnan et al., 1998; Huang et al., 2000; Jessie et al.,2004; Talavera et al., 2004]. However, endothelial cellsfrom large vessels, used most often in those studies, thatis primary HUVEC or ECV304 cell line, exhibitimportant phenotypic and functional differences com-pared to endothelial cells from microvascular territorieswhere plasma leakage largely occurs [Bender et al.,1994; Mason et al., 1997; Lidington et al., 1999;Murakami et al., 2001; Kieda et al., 2002; Chi et al.,2003]. In particular, primary HUVEC have variable andunstable phenotype [Klein et al., 1995; Vermot-Des-rocheset al., 1995], whereas the endothelial originof thelarge vessel-derived ECV 304 cell line is highly con-troversial thus questioning its relevance as endothelialcell model [Brown et al., 2000; Drexler et al., 2002]. Inthis regard, those large vessels-derived endothelial cellsappear poorly relevant to the pathophysiology of dengueshock syndrome. On other hand, the diversity of virusesused in those previous studies, that is from wild-type tohighly adapted dengue viruses, may have introduced anelement of variability between the results previouslypublished, making their interpretation difficult.

The present study aimed at examining differentaspects of the response of two human microvascularendothelial cell lines to infection by a low-passage DEN-2 virus strain representative of the new Americangenotype associated with recent dengue outbreaks[Letmeyer et al., 1999]. The two cell lines studied, that

is the liver sinusoidal endothelial cell line LSEC and thedermal endothelial cell line HMEC-1, are endotheliarepresentative of tissues or organs known to be affectedin the course of dengue infection [Boonpucknavig et al.,1979; Desruelles et al., 1997; Mohan et al., 2000; Wahidet al., 2000; Pancharoen et al., 2002]. The study focusedon functional markers generally modulated in thecourse of endothelial activation, that is adhesion mole-cules and endothelial-derived inflammatory mediators[Krishnaswamy et al., 1999; Martinez-Mier et al., 2001;Galley and Webster, 2004], and more specifically onmarkers altered in the course of DSS [Hober et al., 1993;Raghupathy et al., 1998; Juffrie et al., 2001].

The present results show that direct infection of thetwo microvascular cells lines LSEC and HMEC-1 with awild-type DEN-2 virus, triggers activation in both celllines. However, infection has different effects on thosetwo types of endothelial cells with regard to criticalmarkers involved in the cross talk between endothelialand immune cells. These data suggest that vascularendothelia activated by direct infection could contributedifferentially to the pathogenesis of DSS.

MATERIALS AND METHODS

In Vitro Virus Propagation

All work with infectious virus was carried out in a bio

safety-level 3 laboratory. The DEN-2 virus strain usedin this study, DEN2/H/IMTSSA-MART/98-703 (Gen-Bank accession number AF 208496), was isolated in ourlaboratory from a patient exhibiting a classical denguefever syndrome. The viral genome was sequencedentirely [Tolou et al., 2000] and phylogenetic analysisshowed that this strain belonged to the new Americangenotype which originate from Southeast Asian geno-types [Letmeyer et al., 1999]. The batch of viralinoculum used in this study was prepared by twopassages in C6/36 cells (from Aedes albopictus; ATCCclone CRL 1660). Briefly, permissive insect cells weregrown at 288C in Leibowitzs L15 medium (Biowhittaker,

Verniers, Belgium) supplemented with 1% finalL-glutamin (Biowhittaker), 5% final fetal calf serum(FCS) (Biowhittaker) and 2% final tryptose phosphatebroth (Eurobio, Les Ulis, France). Infection was carriedout by seeding C6/36 cells in complete Leibowitzsmedium without FCS and inoculating them with thevirus at a multiplicity of infection (MOI) of 1, for 1 hr at288C. Complete medium containing 5% FCS was addedand cells were incubated further for 5 days. Cell culturesupernatants were collected and viral titers weredetermined by plaque assay on Vero cells according tothe dilution method previously reported [Luria et al.,1978]. Viral titres were estimated by the equation ofmaximum likelihood [Kleczkowski, 1968].

J. Med. Virol. DOI 10.1002/jmv

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LSEC and HMEC-1 Microvascular Cell Lines

LSEC and HMEC-1 cell lines have been establishedand characterized by Salmon et al. [2000] and by Adeset al. [1992], respectively. The LSEC line was estab-lished from primary liver sinusoidal endothelial cells bytransfectionusinga plasmid containing both the humantelomerase gene andthe SV40 large T sequence [Salmonet al., 2000]. The HMEC-1 line was established bytransfection of primary endothelial cells isolated fromhuman dermis using a plasmid encoding the SV40 largeT [Ades et al., 1992a]. Both microvascular cell linesexhibit endothelial properties from the primary cellsthey had been derived from, that is typical morphology,expression of von Willebrand factor, uptake of acety-lated LDL, expression of adhesion molecules, ability toform tube-like structures into Matrigel, and are stablethrough a high number of in vitro passages regardingendothelial properties [Ades et al., 1992; Salmon et al.,2000].

Culture of Microvascular Cell Lines

The LSEC and HMEC-1 lines were cultured in flasksor culture plates previously coated with 0.2% gelatin(Sigma Aldrich, Saint Quentin Fallavier, France) inPBS. LSEC were maintained in CMRL-1066 (Invitro-gen/Life technologies, Cergy Pontoise, France) supple-mented with 2 mM final L-glutamin (Cambrex) and 10%FCS (Cambrex, Verviers, Belgium) while HMEC-1 werecultured in EGM-2 MV medium (Cambrex) supplemen-ted with 2 mM L-glutamin, 10% FCS, and commercialsingle quots of recombinant human epidermal growthfactor and hydrocortisone.

Infection of Endothelial CellsWith Dengue Type 2 Virus

Despite their stabilityover a large numberof passages[Ades et al., 1992; Salmon et al., 2000], the HMEC-1 andLSEC lines were used at constant passage number 1for all experiments, that is passage 18 1 for HMEC-1and passage 21 1 for LSEC.

Briefly, the LSEC and HMEC-1 lines were seeded in6-well plates 48 hr before infection. LSEC were culturedin CMRL1066 medium supplemented with 2 mMglutamin and 10% heated FCS, while HMEC-1 weredeprivated of epidermal growth factor and hydrocorti-sone and maintained in EGM-2 MV medium (Cambrex)

supplemented with only 2 mM L-glutamin and 10%heatedFCS. Twodays after seeding, when cultures havereached confluency, cells were infected with super-natant of infected C6/36 cells at MOIs 1 and 0.1 for 2 hrat378C,ina5%CO2 atmosphere. Different control cellcultures were included: untreated endothelial cells(EC), EC incubated with supernatant from uninfectedC6/36, EC incubated with viral inoculum inactivated byheating at 808C for 30 min [Avirutnan et al., 1998] orwith corresponding heat-treated (808C, 30 min) super-natants from uninfected C6/36 cells. After 2 hr, theinoculum were removed and all the cell monolayersexcept the untreated EC control, were washed four

times with 8 ml per well of Dulbeccos PhosphateBuffered Saline (DPBS, Cambrex) supplemented with5% final FCS. Finally, 4 ml of fresh medium wasadded inallwellsand cultures were further incubated at 378C.Attimes indicated, supernatants and cells were harvestedfor analysis of viral and cellular parameters. The

supernatants were centrifuged (5 min, 3,000g, at208C), then either directly used for virus titration on

Vero cells as described before, or mixed (1 vol/2 vol) withlysis buffer (Roche) and frozen at208C for further viralRNA extraction. Cell pellets to be analyzed for thedetection of viral RNA negative strands were directlylysed in 300 ml of RLT-buffer (Qiagen, Courtaboeuf,France), then frozen at 208C until further RNAextraction.

Extraction of Viral RNA

Total dengue virus RNA was extracted from cellculture supernatants or cell pellets using the High Pure

Viral RNA kit (Roche Diagnostics, Meylan, France) orthe RNeasy kit (Qiagen), respectively. RNAs extractedfrom cell pellets were directly digested on column withDNaseI. Total RNAs obtained from either 200 ml ofsupernatants or from cell pellets were then subjected toRT-PCR as described thereafter.

Quantification of Positive Strand Viral RNAs

Quantification of viral RNAs was carried out asfollows: after extraction as described before, the RNAswere amplified using the Platinium quantitative RT-PCR Thermoscript One-Step System (Invitrogen-LifeTechnologies). Briefly, 2.5ml of the RNAs were amplified

in a 20 ml reaction mix containing 3.6 pM of each of thetwo PCR primers (F and R), 7 pM of dengue-specificfluorogenic probe [Warrilow et al., 2002], 10 ml of 2XThermoscript Reaction mix, 1 ml of RNAse OUT and0.5 ml of Thermoscript Plus/Platinium Taq Mix in aLightcycler thermocycler (Roche Diagnostics, Meylan,France). PCR reactions were performed according to thefollowing protocol: after initial reverse transcription at508C for 30min and 5 min denaturationat 958C, cDNAswere submitted to 45 amplification cycles with sequen-tial steps at 958C for 15 sec and 608C for 60 sec.

Indirect Immunofluorescence Staining of

Dengue-Infected CellsAfter being detached by brief exposure to a trypsin-

EDTA solution (Cambrex) and washed, endothelial cellswere seeded on 12-well fluorescence slides (VWRInternationnal, Fontenay-sous-Bois, France) and fixedwith acetone for 30 min at 208C. Fixed cells were thenrinsed once with PBS containing 0.05% Tween 20, oncewith PBSaloneand then incubated30 minat 378Cinthepresence of a solutionof BSA at 1%in PBS. A solution ofmouse monoclonal antibody specific for the E protein ofdengue virus (ab 9202, Abcam, Cambridge, UK) wasthen added for 1 hr at 378C. Cells were then washedtwice in PBS and incubated for 1 hr at 378C in the pre-


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sence of an Alexa fluor 488-conjugated goat anti-mouseIgG polyclonal antibody (Molecular Probes, The Nether-lands). After three washes in PBS, cells were finallycounterstained during 5 min at room temperature usinga solution of Evans blue in PBS. Slides were finallymounted to be examined under an Olympus BH-2

fluorescence microscope. The mean percentage of infec-ted cells was determined from at least three fields,and represented the number of fluorescent cells out ofcounterstained cells.

In Vitro Activation of Microvascular EndothelialCell Lines by Pro-Inflammatory Cytokines

Activation of LSEC andHMEC-1 by theinflammatorycytokine TNF was carried out initially to determine thebasal and the cytokine-activated states of the two celllines in our culture conditions. Briefly, endothelial cellsgrown at confluence in gelatin-coated 6-well plates werecultured in their respective culture medium supple-

mentedor notwith 20 ng/ml of recombinanthuman TNF(PeProtech, TEBU, France). Cultures were detachedat different times (24, 48, 72 hr) by brief exposure to acommercial solution of trypsin-EDTA (Cambrex),washed twice with ice-cold PBS containing 5% FCS(Cambrex), and used for cell surface phenotyping asdescribed below.

Similarly, the ability of endothelial cell lines tomodulate cell expressionof HLA-I antigens, a markerof interest in flavivirus infections [Kesson et al., 2002],was assessed by exposing HMEC-1 and LSEC lines tostimulation by recombinant human IFN-g (hIFN-g,PeProtech, TEBU, France) at 500 and 2,000 U/ml.HLA-I surface levels were analyzed by cytometry 3 daysafter addition of hIFN-g, a time corresponding to thepeak of expression in response to this cytokine.

Flow Cytometry Analysis of CellSurface Activation Markers

All antibodies used were tested previously at differentdilutions to determine the optimal concentration forthese assays. Preliminary experiments were also desig-ned to verify that the cell surface antigens studied wereinsensitive to the trypsin-EDTA treatment in the condi-tions used (data not shown).

Briefly, infected and control cells were harvested attimes indicated after infection. After detachment bybrief exposure of adherent cells to a commercial solutionof trypsin-EDTA, cells were washed twice with ice-coldPBS containing 5% FCS, and incubated at48C f o r 1 h rin the presence of FITC- or PE-conjugated mousemonoclonal antibodies directed against the human cellsurface antigens of interest, diluted in a stainingbuffer solution made of ice-cold PBS supplementedwith 5% of heat-inactivated human AB serum, 5% ofFCS and 0.01% of sodium azide. Antibodies used werespecific for ICAM-1 (BD Biosciences Pharmingen, LePont de Claix, France), VCAM-1 (eBioscience, Clinic-sciences, Montrouge, France), DC-SIGNR (R&D System,

Abingdon, UK) and HLA-I (Caltag laboratories, Tebu

international, Le Perray en Yvelines, France), respec-tively. Corresponding FITC- or PE- conjugated isotypiccontrol monoclonal antibodies (Caltag laboratories,Tebu international) were used as negative control inall immunophenotyping experiments. After two washesin ice-cold staining buffer, cells were fixed in a freshly

prepared solution of 0.5% paraformaldehyde (SigmaAldrich)in PBSand rapidly analyzedon a 4-colors FACSCalibur cytometer (Becton Dickinson, Pont de Claix,France). All data were processed using the BD Cell-QuestTM Pro software. Results are presented as meanfluorescence intensity (MFI) that corresponds to thegeometric mean value of fluorescence calculated for agiven cell surface marker.

Chemokine and Cytokine Quantitationby Sandwich ELISA

IL-1b, IL-6, IL-8, and RANTES present in super-natants fromdengue virus-infectedmicrovascularEC or

from control cultures were quantified using DuoSetR&D System ELISA kits, according to manufacturersprotocols. Supernatants were tested pure and 2 fold-diluted, in duplicate. Supernatants still exhibitingsaturating OD signals were tested further at higherdilutions.

Statistical Analysis

All viral and cellular parameters were assessed inthree independent experiments for the two studiedmicrovascular cell lines. For statistical comparisons ofthe two groups, unpaired Students t-test was used. A

P-value of


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Viral titers (or infectious doses, ID) in the supernatantsof infected HMEC-1 and LSEC ranged between 104 and105 ID/mlfrom day2 to 7 after infection(Fig. 2a,b),whileviral RNA copies were comprised between 105 and 106

copies/ml for infected cultures of HMEC-1 (Fig. 2a) andbetween 106 and 107 copies/ml for infected cultures ofLSEC (Fig. 2b). Thus, when related to the percentage ofinfectedcellsin each cell line, that is 1% forHMEC-1 and10% for LSEC, both endothelial cell lines exhibitedcomparable replication efficiency.

DEN-2 Infection Induces an IncreasedProduction of Inflammatory Cytokines and

Chemokines by LSEC and HMEC-1

IL-1b, IL-6, IL-8, and RANTES were quantified in theculture supernatants of infected cultures of LSEC andHMEC-1 since high levels of those mediators were

reported in the course of DSS in patients [Hober et al.,1993; Raghupathy et al., 1998; Juffrie et al., 2001;Suharti et al., 2003] or shown to be produced by dengue-infectedendothelialcellsin vitro [Avirutnan et al., 1998;Huang et al., 2000; Talavera et al., 2004].

IL-1b was undetectable in the supernatants ofinfected or mock control cultures of HMEC-1 and LSECat any time of infection (data not shown). As shown inFigure 3, IL-6 was produced constitutively and variablyby the two cell lines, but IL-6 levels increased signifi-cantly from day 4 after infection in supernatants ofinfected HMEC-1 (twofold increase; P< 104) (Fig. 3a),

and from day 2 in supernatants of infected LSECcultures (fourfold increase; P< 104) (Fig. 3b). IL-6levels were higher in the supernatants of cells infectedat MOI 1 compared to MOI 0.1-infected cells, butdifferences were statistically not significant.

Fig. 1. Infection rates of the microvascular cell lines HMEC-1 andLSEC as determined by indirect immunofluorescence staining ofinfected cells. a: Percentage of infected cells detected in cultures ofHMEC-1 and LSEC from day 1 to 7 after exposure to the non-adaptedDEN-2 virus strain. The percentage of infected microvascularendothelial cells after exposure to MOI 0.1 (opened diamond andopenedsquarefor HMEC-1and LSEC, respectively)or to MOI1 (closed

diamond and closed square for HMEC-1 and LSEC, respectively)indicated on the vertical axis, is the meanSD of at least threerepresentative fields.The resultspresented are representative of threeindependent experiments. b: Detection of dengue envelope proteinby indirect immunofluorescence in day 3-infected cultures of LSEC(upper micrograph) and HMEC-1 (lower micrograph) microvas-cular endothelial cell lines.

Fig. 2. Replication of the low-passage DEN-2 virus strain in themicrovascular cell lines HMEC-1and LSEC assessed by determinationof viral titers and quantification of viral RNA copies. Left ordinate:

Viral RNA copies/ml (continuous lines) were quantified by real-timeRT-PCR in the supernatants of infected endothelial cell at days 1, 2, 4,5, and 7 after infection as described in Materials and Methods. RNAcopy number presented correspond respectively to MOI 1- (closedsquares) and MOI 0.1-infected cells (closed diamonds). Right ordi-

nate: Viral infectious doses (ID/ml; dotted lines) were determinedby plaque assay titration on Vero cells at days 1, 2, 4, 5, and 7 afterinfection,as described in Materialsand Methods. Infectiousdosesweremeasured in culture supernatants of respectively MOI 1- (closedsquares) and MOI 0.1-infected cells (closed diamonds). All valuespresented are meansSD of values obtained in three independentexperiments.




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The chemokines IL-8 and RANTES were also quanti-fied in the cell culture supernatants of uninfected andinfected LSEC and HMEC-1. As shown in Figure 4, bothcell lines produced constitutively IL-8 (Fig. 4a) andRANTES (Fig. 4b). However, dengueinfection induced asignificant increase of IL-8 and RANTES production byboth LSEC (IL-8, 3.2-fold increase from day 2, P


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HLA-I cell surface levels appeared as soon asday 1 or 2 after infection, on HMEC-1 and LSEC,respectively.

As shown in Figure 6a,b, HLA-I MFI increased by2.35-fold (P


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Fig. 5. Phenotypic heterogeneity in the expression of ICAM-1, VCAM-1, and HLA-I on resting and cytokine- activated LSEC andHMEC-1. Cell surface expression of the adhesion molecules ICAM-1and VCAM-1, and of HLA-I antigens was assessed on HMEC-1 andLSEC before (resting state) and after 24 hr-activation by TNF, asdescribed in Materials and Methods. a: Cell surface expression ofICAM-1 on HMEC-1 (left graph) and LSEC (right graph). Greencurves represent the cell surface expression of ICAM-1 on unactivatedresting endothelial cells. Red curves represent the cell surfaceexpression of ICAM-1 on TNF-activated endothelial cells. b: Cellsurface expressionof VCAM-1on HMEC-1(leftgraph) andLSEC(right

graph). Green curves represent the cell surface expression of ICAM-1on unactivated endothelial cells. Red curves represent the cell surface

expression of VCAM-1 on TNF-activated endothelial cells. c: Expres-sion profileof HLA-I antigens on HMEC-1(left graph)and LSEC (rightgraph). Green curves represent the basal expression level of HLA-Iantigens on unactivated endothelial cells. Red and orange curvesrepresent thecell surface levelsof HLA-I antigens after 72 hr of cultureof HMEC-1 and LSEC in the presence of hIFN-g at respectively 500 U/ml(red curve)and 2,000 U/ml (orangecurve). Forall histograms, dottedlines represent the fluorescence intensity curves corresponding to thefixation of isotypic control antibodies for each condition. Only onerepresentative control curve is presented for each graph since the geo-metric valuesof fluorescenceof isotypic controlantibodies weresimilar

and control curves overlapped each other.

Fig. 6. Infection by the low-passage DEN-2 virus strain results inincreased expression of HLA-I and ICAM-1 on LSEC and decreasedexpression of ICAM-1 on HMEC-1. a, c, e: Fluorescence curvesrepresenting the cell surface expression of HLA-I (a) and ICAM-1 (b)on LSEC and the cell surface expression of ICAM-1 on HMEC-1 (c) arepresented for mock-treated cultures (green curve), MOI 0.1-infectedcultures (red curve) and MOI 1-infected cultures (orange curve).Results are representative of three independent experiments. Fluor-escencegraphs correspondingto isotypic controls have been subtractedfrom those of corresponding markers for each culture condition usingspecific functions included in the Cell Quest Pro software (Becton

DickinsonTM). b, d, f: For each histogram, white bars exhibit a basalvalue of 1 corresponding to the mean fluorescence intensity of thestudied antigen on mock-treated cells, relatively to its own value.Increase or decrease of mean fluorescence intensities of ICAM-1 andHLA-I antigens in response to infection is expressed as the ratiobetween the mean fluorescence intensity of studied antigens on MOI0.1-infected cells (sparse points) or MOI-1 infected cells (dense points)relatively to the mean fluorescence intensity of the same antigen onmock treated cells (white bars). Increase or decrease of meanfluorescence intensity of the studied antigens was analyzed at days 1,2, 4, 5, and 7 after infection.




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Fig. 6.




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levels observed on uninfected control cultures (data notshown).

Finally, the endothelial-restricted DC-SIGNR mole-cule remained undetectable at the surface of infectedand control cultures of LSEC and HMEC-1 in the wholestudy (data not shown).

DISCUSSION

Vascular endothelial cells are central effectors in thepathological process of vascular leakage leading to DSS.

The present study aimed at identifying phenotypicchanges induced in two human microvascular endothe-lial cell lines from distinct vascular beds, as a conse-quence of direct infection by a well-characterized andnon-adapted DEN-2 virus. The rationale for this studywas based on two observations: first, only partialfeatures of the response of endothelial cells to infectionby dengue virus have been addressed in previousstudies; second: interpretation and comparison betweendata reported in these studies have been stronglylimited by the diversity of the virus strains (differentserotypes, highly adapted virus, virus isolate frompatient) and of cellular models used (mostly primaryendothelial cells or cell line from large vessels).

The two endothelial cell lines studied, that is LSECand HMEC-1, were derived from liver sinusoidalendothelium [Salmon et al., 2000] and dermis [Adeset al., 1992; Xu et al., 1994], respectively, and haveretained most morphological and functional propertiesof the primary endothelial cells from which they havebeen derived. The availability of such lines allowed us toaddress the consequences of infection in endothelial

cells relevant to the pathophysiology of capillaryleakage. The virus chosen for the present study wascharacterized entirely in our laboratory and was shownto belong to the new American genotype encountered inrecent epidemics of dengue [Rico-Hesse et al., 1997;Tolou et al., 2000]. A short amplification was carried outin insect cells to produce the viral inoculum, in order tomaintain the virus as close as possible to the initialisolate, whereas evidences indicate that in vitro adapta-tion occurs mostly in mammalian cells [Lee et al., 1997;Thayan et al., 1997; Chen et al., 2003].

Infection studies using this low-passage DEN-2 strainshowed that 10% of LSEC and only 1% of HMEC-1 wereinfected. Previous studies reported higher infectionrates, that is 1025% of infected cells in cultures ofprimary endothelial cells from human umbilical vein(HUVEC) [Avirutnan et al., 1998; Warke et al., 2003],and close to 100 % of infected cells in studies using theECV304 cell line as cellular model of endothelial cells[Avirutnan et al., 1998; Bosch et al., 2002]. Severalfactors related both to cell and/or virus properties mayinfluence the efficiency of infection and explain suchdifferences. Both primary HUVEC or the ECV304 cellline often used as models in dengue infection studies[Anderson et al., 1997; Avirutnan et al., 1998; Huanget al., 2000; Bosch et al., 2002] are derived from largevessels, and exhibit functional and phenotypic proper-

ties very different from those of endothelial cellsassociated with microvascular territories [Benderet al., 1994; Mason et al., 1997; Lidington et al., 1999;Murakami et al., 2001; Kieda et al., 2002; Chi et al.,2003]. Differences in the expression pattern of cellsurface molecules important for virus adsorption, like

virus co-receptors, that is heparan sulfate [Chen et al.,1997; Germi et al., 2002; Lin et al., 2002], DC-SIGNR[Navarro-Sanchez et al., 2003; Tassaneetrithep et al.,2003], or other unindentified molecules, may influencethe efficiency of adsorption of a same dengue virus ondifferent types of cells [Diamond et al., 2000; Bielefeldt-Ohmann et al., 2001]. This may explain in particular the100%infection ratecurrently observed withthe ECV304cell line [Brown et al., 2000; Drexler et al., 2002] thesusceptibility of which to dengue infection depends onthe expression of three cell surface proteins of unknownidentity [Wei et al., 2003].

While heparan sulfate distribution was not investi-gated in this study, the expression of the endothelial-restricted C-type lectin DC-SIGNR [Bashirova et al.,2001; Pohlmann et al., 2001], was assessed. DC-SIGNRis an homolog of the dengue co-receptor DC-SIGN[Navarro-Sanchez et al., 2003; Tassaneetrithep et al.,2003] also described as an attachment factor for otherviruses [Pohlmann et al., 2001, 2003; Simmons et al.,2003; Marzi et al., 2004], and has been shown to beexpressed specifically on some types of endothelial cells[Bashirova et al., 2001]. In all infection experiments,DC-SIGNR remained undetectable at the surface ofuninfected and infected LSEC and HMEC-1. Since thismolecule was first reported on primary liver sinusoidalendothelial cells [Bashirova et al., 2001], its absence at

the surface of the liverendothelial LSEC line wasunexpected, suggesting that either DC-SIGNR has beenlost due to immortalization, or alternatively that itsexpression is dependent on in vivo microenvironment.Regarding the critical role of DC-SIGN in dengue virusinfection in some types of cells [Navarro-Sanchez et al.,2003; Tassaneetrithep et al., 2003], one can speculatethat its absence at the surface of LSEC and HMEC-1could explain, at least partly, the low percentages ofinfection observed for both LSEC and HMEC-1.

However, Talavera et al. [2004] have recently shownthat HMEC-1 cells could be infected up to 40% of totalcells in culture when exposed to a dengue 2 virus strainof unknown genotype, suggesting that virus entry maynot depend exclusively on DC-SIGNR in those cells. Themarked difference of infection levels observed for theendothelial cell line HMEC-1 in our experimentscompared to that of Talavera et al. [2004] may be dueto different factors of which differences of infectivityrelated to the virus genotype, adaptation of virus strainthrough multiple passages [Chen et al., 2003; Colognaand Rico-Hesse, 2003; Edgil et al., 2003], methodsemployed to prepare the viral batch used to infect cells.Other reasons, independent on the viral strain may alsoexplain at least partly those differences, of which cellculture conditions, infection methods, sensitivity of thedetection methods used.




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Despite the low rate of infection observed for bothLSEC and HMEC-1, both microvascular endothelial celllines replicate the low-passage DEN-2 strain efficientlyand at comparable levels. Surprisingly, the mean virusproduction, that is 105 I.D/ml estimated at 2 days ofinfection, is comparable to that reported for HUVEC

infected by the DEN 2 16681 laboratory strain, aprototype virus strain with high replication efficiency[Huang et al., 2000; Edgil et al., 2003]. This suggeststhat microvascular endothelial cells could act as animportant replication site during dengue virus infec-tion.

The ability of a virus to induce or not some cytopathiceffects in host cells is an important aspect of the hostcellvirus interaction. While actively and efficientlyreplicating the DEN-2 strain, infected cultures ofHMEC-1 andLSEC were devoidof detectable cytopathiceffect when compared to corresponding uninfectedcontrol cultures. Comparable observations have beenreported by others, respectively, on HUVEC or onHMEC-1 infected by dengue virus [Bunyaratvej et al.,1997; Talavera et al., 2004].

Microvascular endothelial cellsare an active interfacebetween blood immune cells and tissues. Many stimuliof which vasoactive compounds but also several patho-gens [Shen et al., 1997; Knight et al., 1999; Sundstromet al., 2001; Caruso et al., 2002; De Maula et al., 2002;Hippenstiel and Suttorp, 2003] induce endothelial cellsto change from a physiological state to a pro-inflamma-tory and pro-adhesive state. This is most often char-acterized by increased production of inflammatorysoluble mediators and by over-expression of cell surfacemolecules, that is adhesion molecules, HLA antigens,

and others, involved in the cross talk between endothe-lial cells and circulating immune cells.

Such effects could be observed as a consequence ofdirect exposure of LSEC and HMEC-1 to the DEN-2 lowpassage isolate. Both microvascular endothelial celllines over-produced IL-6, IL-8, and RANTES, whereasthis could not be attributed exclusively to infected cellssince cytokines were quantified in the total super-natants of cell cultures containing no more than 1 and10% of infected cells for HMEC-1 and LSEC lines,respectively. Intracellular staining of cytokines com-bined to cell surface staining of viral antigens shouldallow to define if only infected cells over-produce thosesoluble factors in response to dengue infection. How-ever, the increase of cytokines levels observed ininfected cultures of LSEC and HMEC-1 was comparableto that reported for dengue-infected HUVEC or ECV304[Huang et al., 2000; Bosch et al., 2002] that contain amuch higher percentage of infected cells. Differences inabsolute concentration of IL-6, IL-8, and RANTESproduced by HMEC-1 and LSEC, that is 5- to 30-foldlower concentrations between the former and the latter,might reflect the 10-fold difference of infection ratebetween LSEC and HMEC-1, or alternatively might bedue to functional differences between the two lines.

Increased production of IL-6, IL-8, and RANTESby LSEC and HMEC-1 in response to infection is in

agreement with results from previous studies carriedout using different dengue virus strains and types ofendothelial cells. All reported an over-production of IL-6[Huang et al., 2000], IL-8 [Avirutnan et al., 1998;Talavera et al., 2004], or RANTES [Avirutnan et al.,1998], thus suggesting that increased secretion of these

inflammatory mediators is a common feature of endo-thelial cells response to dengue viruses, whatever theorigin of viruses and the tissue-specificity of endothelialcells.

The increased secretion of pro-inflammatory media-tors by dengue-infected microvascular endothelial cellsshould be considered with respect to markers increasedin the course of systemic inflammation and particularlyin dengueshocksyndrome. High levelsof IL-6, oneof themost significant marker of severity in systemic inflam-mation [Reinhart et al., 2002] have been associated withthe most severe forms of dengue [Hober et al., 1993;

Juffrie et al., 2001]. High levels of the CXC chemokineIL-8 [Baggiolini et al., 1995] were also associated withsevere dengue infection [Raghupathy et al., 1998], andIL-8 has been shown to increase the permeability ofdengue-infected endothelial cells expressing adequateIL-8 receptors [Salcedo et al., 2000; Talavera et al.,2004], thus contributing potentially to the pathophysio-logical mechanisms of plasma leakage.

The increased secretion by dengue-infected endothe-lial cells of theCC chemokineRANTES mayenhancethelocal inflammatory response by facilitating the transmi-gration of inflammatory Th1-type lymphocytes to endo-thelium-associated tissues [Kawai et al., 1999].

Variations in the cell surface levels of adhesionmolecules and HLA antigens is another feature of

endothelial activation. Analysis were first carried outto characterize more precisely the phenotypic changesoccurring in HMEC-1 and LSEC lines in response tomediators known to activate endothelial cells like TNFor IFNg. The results show that resting and TNF-activated LSEC and HMEC-1 have distinct pattern ofexpression of adhesion molecules and HLA-I antigens.

Whereas LSEC exhibit higher constitutive levels ofICAM-1 compared to HMEC-1, they only slightly up-regulate ICAM-1 in response to TNF, compared toHMEC-1 which strongly over-express ICAM-1 in res-ponse to this cytokine. Differently, TNF-induces highlevels of VCAM-1 on LSEC but only low levels of VCAM-1 at the surface of the HMEC-1. Finally, LSEC exhibithigher constitutive levels of HLA-I than HMEC-1 whileboth cell lines respond similarly to stimulation by hIFN-g, that is about threefold increase in HLA-I meanfluorescence intensity. Those results underline thefunctional heterogeneity of the two microvascular endo-thelial cell lines studied, a critical parameter to considerwhen investigating the contribution of one or anothertype of vascular endothelium to a pathophysiologicalprocess [Bender et al., 1994; Murakami et al., 2001].

Interestingly, such a functional heterogeneity wasalso observed in the response of LSEC and HMEC-1 toinfection by the low-passage DEN-2 strain. Indeed,direct infection of LSEC resulted in an increased




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expression of ICAM-1 and HLA-I at the surface of thetotal cell population. Whereas more pronounced effectscould have been identified by looking specifically atinfected endothelial cells, this could not be done due tothe poor quality of double staining with antibodiesdirected to both studied markers and viral antigens.

The observed increased of ICAM-1 at the surface ofLSEC as a result of infection is an interesting featurethat differs from previous study on HUVEC infected bythehighly adapted Denguetype 2 strain 16681,showingno change in ICAM-1 cell surface expression in responseto dengue virus infection [Anderson et al., 1997].However, the virus-induced over-expression of ICAM-1and HLA-I observed on LSEC is comparable to thatreported on HUVEC infected with another flavivirus,that is West Nile virus [Shen et al., 1997], whereas inthis latter model, maximal upregulation of ICAM-1 wasobserved in the very early times of infection. Thuscommon signaling pathways may be involved in theupregulation of those molecules and particularly ofHLA-I antigens, during flavivirus infections [Kessonand King, 2001; Momburg et al., 2001; Cheng et al.,2004]. The functional consequences of increased expres-sion of ICAM-1 and HLA-I on infected LSEC could be ofimportance for the interactions of those cells withinflammatory immune cells, by contributing to enhancethe recruitment of circulating immune cells to the pro-inflammatory endothelium [Martinez-Mier et al., 2001].Over expression of HLA-I may interfere with thedevelopment of an early innate NK response [Lobigset al., 2003], while enhancing the cytotoxic responseof virus-specific CD8 T-cells towards infected cells, asshown for cells infected by West Nile virus [Kesson and

King, 2001], thus contributing to the immunopatholo-gical response. Despite limitations in extrapolating ourresults to in vivo cellular events, the fact that LSECrespond to dengue virus infection by acquiring featuresof activated endothelium, suggests that liver-associatedendothelial cells could play a role in the initiation or theamplification of the inflammatory cascade leading tovascular leakage in DSS.

Differently from LSEC, infection of HMEC-1 by thesame DEN-2 low-passage strain resulted in a decreasedexpression of ICAM-1, while HLA-I cell surface levelsremained unchanged compared to uninfected cells. Asfor LSEC, it is likely that double cell surface stainingallowing to focus our analysis on infected cells thatrepresentonly 1% of total HMEC-1 cultures, would haverevealed more pronounced effects of infection.

However, despite those limitations, the prolongeddecrease of ICAM-1 cell surface expression observed onHMEC-1 cells after exposure to dengue virus, may havefunctional consequences on the ability of HMEC-1to bind circulating leucocytes. Indeed, comparabledecrease of ICAM-1 cell surface levels in a differentmodel, was shown to limit the recruitment of inflamma-tory cells to this type of microvascular endothelial cells[Lindermann et al., 2000].

The present study aimed at examining the phenotypicchanges induced by direct exposure of human micro-

vascular endothelial cells from different vascular beds,to a non-adapted dengue type 2 virus. The results showthat microvascular endothelial cells from two distinctvascular beds, not only support viral replication but canbe activated through direct exposure to dengue virus,exhibiting some common but also some cell type-specific

responses to dengue infection, suggesting that theresponse of microvascular beds to dengue infectionmay differ from one territory to another one.

The observed changes should now be completed byfunctional studies to investigate if viral infectionmodifies the permeability or other cellular mechanismsrelated to vascular leakage, and by analysis of interac-tions between dengue-infected microvascular endothe-lial cells and immune cells in the context of dengueinfection.

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