FasL Fas pathway is involved in dengue virus induced apoptosis of the vascular endothelial cells

Journal of Medical Virology82:1392–1399(2010) FasL/Fas Pathway Is Involved in Dengue Virus Induced Apoptosis of the Vascular Endothelial CellsHongwu Liao,Juan Xu,and Junqi Huang*Department of Immunology,Zhongshan School of Medicine,Sun yet-sen University Key Laboratory of Tropical Diseases Control,Ministry of Education,Guangzhou,PR ChinaThe hallmark of the dengue hemorrhagic fever/ dengue shock syndrome is hematologic abnor-mality.The pathogenesis of dengue hemorrhagic fever/dengue shock syndrome remains unknown. Our work showed that the dengue virus serotype-2 induced apoptosis in human umbilical vein endo-thelial cells.Fas(CD95),Tumor Necrosis Factor receptors,and Tumor Necrosis Factor-related apoptosis-inducing ligand receptors are the most common death receptors,which can induce pared with the untreated human umbilical vein endothelial cells,Fas expression was increased both in the mRNA level and on the surface of infected human umbilical vein endo-thelial cells.FasL was expressed at similar levels on human umbilical vein endothelial cells over a course of dengue virus serotype-2infection,but the expression in mRNA level was increased in infected human umbilical vein endothelial cells.It is possible that there is soluble FasL secreted from human umbilical vein endothelial cells in the supernatant.Tumor Necrosis Factor-related apoptosis-inducing ligand receptor1and Tumor Necrosis Factor receptors1–2were constantly very low,whereas Tumor Necrosis Factor-related apoptosis-inducing ligand receptors 2–4decreased after dengue virus serotype-2 infection.This result suggested that dengue virus serotype-2may inhibit Tumor Necrosis Factor-related apoptosis-inducing ligand recep-tors-induced apoptosis.The apoptotic rates in human umbilical vein endothelial cells were decreased upon the addition of caspase family inhibitors.In addition,activated caspase8and caspase3were also observed by Western blot following dengue virus serotype-2infection. Thus,it is shown that the Fas/FasL pathway may participate in dengue virus-induced apoptosis of vascular endothelial cells in vitro.J.Med.Virol. 82:1392–1399,2010.ß2010Wiley-Liss,Inc. KEY WORDS:dengue virus;apoptosis;Fas/FasLINTRODUCTIONDengue virus,a member of the mosquito-borne flaviviruses,has been sub-grouped into four antigenic related serotypes,dengue virus types1,2,3,and4, which have spread in tropical and subtropical regions. Approximately 2.5billion people are at the risk of dengue virus infection[Guzman and Kouri,2002]. Primary infection with dengue virus usually results in mild dengue fever,which is characterized by fever, headache,muscle or joint pain,and rash.Secondary infection may lead to fatal dengue hemorrhagic fever and even dengue shock syndrome.Vascular leakage and hemorrhaging are significant and typical clinical man-ifestations in dengue hemorrhagic fever/dengue shock syndrome patients,suggesting that blood vessel dys-function plays an important role in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome [Andrews et al.,1978].Although there are many hypotheses,the mechanism of blood vessel dysfunction and vascular leakage is not well understood.Recently, accumulated evidence has shown that apoptosis induced by dengue virus infection may one of the causes of The authors declare that they have no competing interests.Hongwu Liao and Juan Xu contributed equally to this work.Grant sponsor:National Natural Science Foundation of China; Grant numbers:30872350,30400371,30571736;Grant sponsor: Natural Science Foundation of Guangdong Province;Grant number:81510008901000017;Grant sponsor:Guangdong Prov-ince Scientific Technology Project;Grant number: 2007B031516008;Grant sponsor:Guangdong Province Teamwork Project;Grant number:06201946;Grant sponsor:Guangzhou City Scientific Technology Project;Grant numbers:2006Z3-E4081,2008Z1-E221;Grant sponsor:National Development Project of High Technology Research(‘‘863’’);Grant number: 2007AA02Z415;Grant sponsor:National Development Project of Important Basic Research(‘‘973’’);Grant number:2007CB512404.*Correspondence to:Dr.Junqi Huang,Department of Immu-nology,Zhongshan School of Medicine,Sun Yat-sen University, 74#the2nd Zhongshan Road,Guangzhou510080,PR China.E-mail:junqi_huang@Accepted3March2010DOI10.1002/jmv.21815Published online in Wiley InterScience()ß2010WILEY-LISS,INC.pathogenesis[Despres et al.,1998;Marianneau et al., 1998;Jan et al.,2000].Viral infection can lead to apoptosis of host cells.In general,there are two apoptotic pathways:the death receptors pathway and the mitochondrion pathway. These two pathways can crossover and regulate apop-tosis coordinately.Death receptors,which are members of the Tumor Necrosis Factor receptor super family,can activate the caspase family,especially caspase8and caspase3,after combining with a homologous ligand and lead to apoptosis.Recently,more virus-induced apoptosis studies have focused on these death receptors, such as Fas,Tumor Necrosis Factor receptors,and receptors of Tumor Necrosis Factor-related apoptosis-inducing ligand,which have distinct structures and functions.Rivero et al.[2002]reported that Fas was involved in the pathogenesis of fulminant hepatitis. Some research[Serra et al.,1995;Almasan and Ashkenazi,2003]has confirmed the bingding of Tumor Necrosis Factor-related apoptosis-inducing ligand with Tumor Necrosis Factor-related apoptosis-inducing ligand receptor1or Tumor Necrosis Factor-related apoptosis-inducing ligand receptor2can induce cell apoptosis while the binding of Tumor Necrosis Factor-related apoptosis-inducing ligand with Tumor Necrosis Factor-related apoptosis-inducing ligand receptor3or Tumor Necrosis Factor-related apoptosis-inducing ligand receptor4does not induce apoptosis.They are decoy receptors.Matsuda et al.[2005]reported that Tumor Necrosis Factor-related apoptosis-inducing ligand/Tumor Necrosis Factor-related apoptosis-induc-ing ligand receptors may play a crucial role in the apoptosis of liver cells infected by dengue virus.Tumor Necrosis Factor receptors,including Tumor Necrosis Factor receptor1and Tumor Necrosis Factor receptor2, can bind Tumor Necrosis Factor-a,which is generated by activated macrophages and T cells and induces apoptosis.In this study,we will validate whether the death receptors pathway is involved in dengue virus induced human endothelial cell damage.Several in vitro studies have reported that endothelial cells were the target of dengue virus[Iyngkaran et al., 1995;Srichaikul and Nimmannitya,2000]and that cell apoptosis is directly caused by dengue virus[Avirutnan et al.,1998]or through nitric oxide after induction by anti-NS1in secondary dengue virus infection[Lin et al., 2002].However,little is known about the signal path-way of dengue virus-induced apoptosis.Lacking ideal animal models of dengue hemorrhagic fever/dengue shock syndrome,the importance of in vitro studies has become significantly evident.Therefore, primary human umbilical vein endothelial cells were chosen to investigate dengue virus serotype-2-induced apoptosis.Death receptors expression,caspases which are involved in apoptosis,and nitric oxide concentration in the medium were detected so that the precise mechanism of dengue hemorrhagic fever/dengue shock syndrome apoptosis can be better understood.In this study,after comparison infected human umbilical vein endothelial cells with untreated human umbilical vein endothelial cells,it was found that the Fas on human umbilical vein endothelial cells surface was increased while FasL was expressed constantly on human umbilical vein endothelial cells both before and after dengue virus serotype-2infection.Both Fas and FasL were increased in mRNA levels.Activated caspase 8and caspase3were observed in infected human umbilical vein endothelial cells and it was found that apoptosis of infected human umbilical vein endothelial cells was blocked by inhibitors of the caspase family to some extent,while nitric oxide did not increase.All of these results demonstrated that Fas/FasL pathway was involved in dengue virus induced endothelial cells apoptosis.MATERIALS AND METHODSExpansion and Titration of Dengue VirusSerotype-2The dengue virus serotype-2prototype New Guinea C strain was propagated in C6/36mosquito cells in Eagle’s minimal essential medium containing10%fetal bovine serum.After incubation for3or4days,the culture medium with virus was harvested when the cytopathic effect was obvious.Cell debris was removed by centrifugation,and the supernatant was aliquoted and stored atÀ808C until use.The virus was titrated by a plaque formation assay on C6/36cells.Cells were seeded at a concentration of1Â106/well in a24-well plate;serial dilutions of virus were added;and the mixtures were incubated at378C for5days.Plaques were visualized by staining with0.5%crystal violet. Virus concentrations are given as plaque-forming units (PFU)/ml.Human Umbilical Vein Endothelial CellsCultures and IdentificationPrimary human umbilical vein endothelial cells were isolated with trypsin-EDTA(0.25%)from human umbilical vein(from the First Affiliated Hospital,Sun Yat-sen University,Guangzhou)and grown in tissue cultureflasks that were pre-coated with2%gelatin. Cells were grown at378C with5%CO2in endothelial cell growth medium(SFM for primary cells and M199 for passaged cells)(Gibco,Carlsbad,CA)with20%fetal calf serum,100U/100m g/ml penicillin and streptomy-cin,and30m g/ml endothelial cell growth supplement (Upstate,Charlottesville,VA).Cells were identified by morphological characteristics and immunocytochemical staining with an antibody against factor VIII-related antigen(Lab Vision,Fremont,CA).Dengue Virus Serotype-2Infected Human Umbilical Vein Endothelial CellsSecond or third passage human umbilical vein endothelial cells were used for the experiments.Human umbilical vein endothelial cells were incubated with dengue virus serotype-2at different multiplicities of infection(m.o.i.)for2hr.Virus supernatants were thenJ.Med.Virol.DOI10.1002/jmvDengue Virus Causes Apoptosis Via Fas/FasL1393removed,and fresh growth medium with4%fetal bovine serum was added to each plate for further incubation. Apoptosis Assay With Annexin-v/7-aad Staining The adherent cells were removed by treatment with 0.25%trypsin-EDTA,pelleted by centrifugation at500g for10min,and washed twice in PBS.Cells were then incubated with0.1m g FITC-conjugated Annexin v (Jingmei,China)and0.2m g7-aad(7-amino-actinomycin D)(Jingmei)in100m l Ca2þbinding buffer(Jingmei)for 15min in the dark beforeflow cytometry analysis with Cell Quest software(BD Biosciences,San Diego,CA).Apoptosis Blockage AssayThe apoptosis blockage test was performed while the caspase family inhibitors were added(final concentra-tion of1m g/ml)to cells infected with dengue virus serotype-2.The caspase family inhibitors,including pancaspase inhibitor(Z-VAD-FMK,general caspase inhibitor as a positive control),caspase-3inhibitor (Z-DVED-FMK),caspase-6inhibitor(Z-VEID-FMK), caspase-8inhibitor(Z-IETD-FMK),caspase-9inhibitor (Z-LEHD-FMK),and a negative control(Z-FA-FMK), were purchased from Biovision.Then apoptosis assays were performed.Flow Cytometry Analysis of DeathReceptors and FasLUntreated and dengue virus serotype-2-infected human umbilical vein endothelial cells(m.o.i.¼4, infected48hr)were detached by trypsin and washed twice with PBS.Cell surface staining was performed using monoclonal antibodies to the death receptors (these mAbs were from R&D Systems,Minneapolis, MN).A total of106cells was incubated with0.25or0.5m g monoclonal antibodies against Tumor Necrosis Factor-related apoptosis-inducing ligand receptor1(TR1), Tumor Necrosis Factor-related apoptosis-inducing ligand receptor2(TR2),Tumor Necrosis Factor-related apoptosis-inducing ligand receptor3(TR3),Tumor Necrosis Factor-related apoptosis-inducing ligand receptor4(TR4),Tumor necrosis factor receptor1 (TNFR1),Tumor necrosis factor receptor2(TNFR2), Fas,or mouse IgG isotype for30min at2–88C in the dark.After incubation,the unconjugated antibody was removed by washing the cells twice with PBS and re-suspended the cells in200m l of PBS forfinalflow cytometry analysis.The same method was applied to examine FasL expression(mAb from Santa Cruz,CA).Quantitative Polymerase Chain Reaction Total RNA from infected and uninfected cells was extracted and transcribed into single-stranded cDNA by reversing buffer according to the manufacturer’s instructions(BD).The primers were designed by Primer Express2.0and were synthesized with the ABI 3900DNA synthesizer.Quantitative polymerase chain reactions were performed,and the data were analyzed by ABI7500automaticfluorescent quantitative poly-merase chain reaction.The primer sequences for the amplifications were:FAS forward primer:50-GGG CAT CTG GAC CCT CCT A-30,reverse primer:50-GGC ATT AAC ACT TTT GGA CGA TAA-30;FASL forward primer: 50-CAG TCC ACC CCC TGA AAA A-30,reverse primer: 50-CTT GAG TTG GAC TTG CCT GTT AA-30;and b-actin forward primer:50-GCA TGG GTC AGA AGG ATT CCT-30,reverse primer:50-TCG TCC CAG TTG GTG ACG-AT-30.Western Blot AnalysisAfter infection with dengue virus serotype-2with a m.o.i.of4for48hr,the cells were lysed in buffer containing62.5mM Tris/HCl(pH6.8),2%SDS,10% glycerol,6%2-mercaptoethanol,and0.01%bromophe-nol blue.Samples were collected by microcentrifugation for protein concentration assessment.Forty micrograms of protein per sample was analyzed by SDS-PAGE and electroblotted onto PVDF membranes(Amersham Bio-sciences,Piscataway,NJ).After blocking with5%non-fat dried milk,membranes were incubated with the primary antibody(Biovision)overnight at48C.Then a secondary peroxidase-conjugated antibody(KPL)was added at room temperature for1hr after washing in TBST buffer(0.1%Tween-20in Tris-buffered saline). Finally,the blots were developed using the enhanced chemiluminescence kit(PIERCE,Rockford,IL)and exposed to X-rayfilm.An mAb against actin(BIOS)was used as a protein loading control.RESULTSDengue Virus Serotype-2Infection Resulted in Human Umbilical Vein EndothelialCells ApoptosisStandard plaque-forming assay was used to confirm the concentration of dengue virus serotype-2and primary human umbilical vein endothelial cells have been identified by morphology and immunocyto-chemistry with antibody against factor VIII antigen.To investigate whether dengue virus serotype-2could induce human umbilical vein endothelial cells apopto-sis,an apoptosis assay was performed after the human umbilical vein endothelial cells were infected with different doses of dengue virus serotype-2(m.o.i.of 0.04,0.4,4,4,and40)for24,48,and72hr.As shown in Figure1,the percentage of apoptotic cells was increased along with the incubation time in different doses groups. There were about6%,7%,and17%apoptotic human umbilical vein endothelial cells in untreated group at24, 48,and72hr.There were about10%,21%,and28% apoptotic human umbilical vein endothelial cells in m.o.i.of four groups at24,48,and72hr.It was shown that human umbilical vein endothelial cells in untreated group remained active after48hr while there was obvious apoptosis in m.o.i.of four groups.According to these results,the experiment condition of m.o.i.of fourJ.Med.Virol.DOI10.1002/jmv1394Liao et al.dengue virus serotype-2at 48hr was used in the following experiments.Dengue Virus Serotype-2Induced DeathReceptor and Fas Expression To elucidate the function of death receptors on human umbilical vein endothelial cells undergoing dengue virus serotype-2-induced apoptosis,the expression of different death receptors (such as Tumor NecrosisFactor-related apoptosis-inducing ligand receptors 1–4,Tumor necrosis factor receptors 1–2,and Fas)was analyzed by flow cytometry before and after infection of m.o.i.of four dengue virus serotype-2at 48hr.Before dengue virus serotype-2infection,about 3%of human umbilical vein endothelial cells had Fas expression on the surface,while the percentage increased to 17.1%after dengue virus serotype-2infection (P <0.05).Less than 0.5%of the human umbilical vein endothelial cells expressed Tumor Necrosis Factor-related apoptosis-inducing ligand receptor 1or Tumor Necrosis Factor receptors 1–2before dengue virus serotype-2infection,and there was no significant change after dengue virus serotype-2infection.Prior to dengue virus serotype-2infection,about 23%,27.1%,and 12.3%of human umbilical vein endothelial cells expressed Tumor Necrosis Factor-related apoptosis-inducing ligand receptors 2,3,and 4respectively.However,the expression of these three death receptors decreased after dengue virus serotype-2infection (Fig.2A–C).These data indicated that Tumor Necrosis Factor-related apoptosis-inducing ligand/Tumor Necrosis Fac-tor-related apoptosis-inducing ligand receptors path-way was possibly less important than FasL/Fas pathway.Fas mRNA was then detected in the human umbilical vein endothelial cells and gained relative quantity data (target gene/parameter gene).The rela-tive quantity data in dengue virus 2group was about 4.6-fold higher than that of untreated group (Fig.2D).J.Med.Virol.DOI10.1002/jmvFig.1.Different concentrations of dengue virus serotype-2induced human umbilical vein endothelial cells apoptosis at different time.The percentages of apoptotic cells in untreated group were about 6%at 24hr,7%at 48hr and 17%at 72hr,while the percentages of apoptotic cells in dengue virus serotype-2infection group (m.o.i.¼4)were about 10%at 24hr,21%at 48hr,and 28%at 72hr.Fig. 2.Expression of death receptors on human umbilical vein endothelial cells.A :Expression of death receptors on untreated human umbilical vein endothelial cells was determined by flow cytometry.M1shows the percentage of positive cells compared with the background control using the isotype (shaded peak).B :Expression of death receptors on human umbilical vein endothelial cells infected with dengue virus serotype-2for 48hr (m.o.i.¼4).C :Compared to untreated control,the expression of Fas was greater while expression of Tumor Necrosis Factor-related apoptosis-inducing ligand receptors 2–4wasreduced after dengue virus serotype-2infection (N ¼3,P <0.05).There were no significant differences between control and treated group in the expressions of Tumor Necrosis Factor-related apoptosis-inducing ligand receptor 1and Tumor Necrosis Factor receptors 1–2.D :Fas was about 4.6-fold higher in the mRNA level in dengue virus serotype-2treated group than control group (N ¼3).Relative induction was calculated with qRT-PCR software (Applied Biosystems,Foster City,CA).Dengue Virus Causes Apoptosis Via Fas/FasL 1395FasL Was Expressed in Human UmbilicalVein Endothelial Cells FasL expression on the cell surface was examined by flow cytometry.The data showed that FasL was expressed on about 10%and 11.5%of human umbilical vein endothelial cells before and after dengue virus serotype-2infection respectively (Fig.3A).FasL mRNA levels were examined by quantitative polymerase chain reaction.The relative quantity data of dengue virus serotype-2groups was about 10-fold higher than that of untreated group.(Fig.3B).Dengue Virus Serotype-2InducedCaspase 8and 3ActivationTo examine which caspases were involved in apoptosis in human umbilical vein endothelial cells,caspase family inhibitors were added,and then an apoptosis assay was performed.The results were shown in Figure 4A.To some extent,the positive control andinhibitors of caspases 3,6,8,and 9lowered the percentage of apoptotic dengue virus serotype-2-infected human umbilical vein endothelial cells.These data indicated that caspases 8and 3were more important than caspases 6and 9.Subsequently,activated caspases 8and 3were detected by Western blot.Bands were seen at 26kDa (activated caspase 8)and 17/19kDa (activated caspase 3)in the human umbilical vein endothelial cells infected with dengue virus serotype-2,as shown in Figure 4B.Thus,the Fas-caspase 8-dependant pathway was involved in dengue virus serotype-2-induced human umbilical vein endothelial cells apoptosis.DISCUSSIONThe pathogenesis of dengue hemorrhagic fever/dengue shock syndrome has not been well understood.Vascular leakage is one of the hallmarks of dengue hemorrhagic fever/dengue shock syndrome.Recently,some reports have shown that apoptosis of the host cellsJ.Med.Virol.DOI10.1002/jmvFig.3.Expression of FasL in Human umbilical vein endothelial cells.A :FasL was expressed constantly on Human umbilical vein endothelial cells after dengue virus serotype-2infection (N ¼3,untreated control:9Æ1.2%;dengue virus serotype-2treated:11.5Æ1.0%,P >0.05).B :FasL was about 10-fold higher in the mRNA level in dengue virus serotype-2treated group than control group (N ¼3).Relative induction was calculated with qRT-PCR software (AppliedBiosystems).Fig.4.Caspase-dependent apoptosis.A :Apoptosis blockage assay.Dengue virus serotype-2infection represents dengue virus serotype-2infection at m.o.i.¼4after 48hr.Z-VAD-FMK is a general caspase inhibitor that inhibits all known caspases.Z-FA-FMK is a negative control for FMK inhibitors.Z-DEVD-FMK,Z-VEID-FMK,Z-IETD,and Z-LEHD are the inhibitors for caspases 3,6,8,and 9,respectively.B :An activated caspase 8band was observed at 26kDa,and activated caspase 3bands were observed at 17/19kDa after dengue virus serotype-2infection (m.o.i.¼4,48hr)by Western blotting.B-Actin was used as a internal control.1396Liao et al.may play an important role in dengue hemorrhagic fever [Despres et al.,1998;Marianneau et al.,1998;Jan et al., 2000].In this study,it was shown that dengue virus serotype-2caused apoptosis of human umbilical vein endothelial cells.Additionally,Fas expression was increased in dengue virus serotype-2-infected human umbilical vein endothelial cells both on the cell surface and in mRNA level,and FasL expression in mRNA level was also significantly increased.Activated caspase8 and caspase3were also observed by Western blot after dengue virus serotype-2infection.There were no significant changes in nitric oxide after infection in our study.Endothelial cells play an important role in both hemostasis and regulating the immune response to infection.It is thus logically assumed that hemorrhage or altered vascular permeability may result from endothelial cell damage.Recently,a report by Hsuen-chin clearly showed that intradermal inoculation of high titers of dengue virus predisposes endothelial cells to Tumor Necrosis Factor-a-induced cell death,which leads to hemorrhage[Chen et al.,2007].Cells undergo apoptosis through two major pathways:extrinsic death receptors pathway or intrinsic(mitochondrial)pathway. The death receptors pathway is evidently important and mediates apoptosis by viral infection.In recent years,the apoptosis mediated by CD95(Fas), Tumor Necrosis Factor receptors1–2,and Tumor Necrosis Factor-related apoptosis-inducing ligand receptors1–4was reported rochelle et al.[1998]reported that Epstein–Barr virus-induced apoptosis may be mediated by FasL.Clarke and Tyler [2007]reported the reovirus-induced apoptosis via Tumor Necrosis Factor-related apoptosis-inducing ligand,and Kim et al.[2007]reported that HIV could up-regulate the expression of Tumor Necrosis Factor-related apoptosis-inducing ligand of antigen-presenting cell and induce apoptosis.In our experiment,human umbilical vein endothelial cells were infected with dengue virus serotype-2of different titers,and cellular apoptosis was observed at different time points by usingflow cytometric analysis. It was found that dengue virus serotype-2could induce human umbilical vein endothelial cells apoptosis and that apoptotic rates increased with increasing time. Thus,fluorescence-labeled antibodies were used to mark cells that were infected with a m.o.i.of4dengue virus serotype-2for48hr.Then,the expression of death receptors was detected.The results demonstrated that dengue virus serotype-2infection promoted Fas expres-sion on human umbilical vein endothelial cells.Tumor Necrosis Factor-related apoptosis-inducing ligand receptor1and Tumor Necrosis Factor receptors1–2 were expressed constantly at low level,and Tumor Necrosis Factor-related apoptosis-inducing ligand receptors2–4decreased after dengue virus serotype-2 infection.Warke et al.[2008]demonstrated TRAIL inhibition of DV was apoptosis independent.Our results suggested the reduction of Tumor Necrosis Factor-related apoptosis-inducing ligand receptors2might be the reason Some investigators have reported that death receptors,such as Fas/CD95,undergo rapid internalization after triggering[Algeciras-Schimnich et al.,2002]and that Fas signaling enhances exocytosis after the initial wave of increased endocytosis[Reinehr and Haussinger,2008].Esposti also reported that the Fas enhances endocytic membrane traffic converging into the Golgi region[Degli Esposti et al.,2009].Many recent studies have examined the role of Fas-mediated cell death in the vasculature,and Fas/FasL-mediated apoptosis has been proposed to play a role in physio-logical and pathological cell turnover in the vasculature [Fukuo et al.,1997;Sata and Walsh,1998;Suhara et al.,1998].Fas is ubiquitously expressed,whereas the expression of FasL,a membrane-bound factor,is typically confined to inflammatory cells and tissues that routinely encounter inflammatory cells[Griffith et al., 1995;Sata et al.,2000].Owing to the greater Fas expression on human umbilical vein endothelial cells after dengue virus serotype-2infection,we next exam-ined FasL.Although there was no significant difference between untreated group and dengue virus serotype-2 infection group by usingflowing cytometry,FasL was increased in mRNA level.We believe that there is soluble FasL secreted from cells to supernatant.These results indicated FasL binding with Fas could induce human umbilical vein endothelial cells apoptosis. Apoptosis is an important mechanism maintaining growth,and its regulation contains a series of cascade reactions.Thus,how does dengue virus serotype-2 initiate caspase cascade reactions via the Fas/FasL pathway?Upon stimulation,FasL binding to Fas triggers Fas trimerization and recruits Fas-associated death domain protein,and then procaspase-8forms a death-inducing signaling complex.Procaspase-8oligo-merization drives its activation through self-cleavage, activating downstream effector caspases,such as cas-pase3,and leading to apoptosis.Caspase3is a very important downstream caspase.It plays a pivotal role in both death receptor pathway and mitochondrial path-way.Caspase inhibitors were added to dengue virus serotype-2-infected cells and the rate of apoptosis cells was detected.Experimental results showed that the extent of apoptosis decreased after the addition of general caspase inhibitors and specific inhibitors of caspases3,6,8,and9.Activated caspase8and caspase3 were observed in Western blot.Consequently,it was confirmed that dengue virus serotype-2can induce human umbilical vein endothelial cells apoptosis,and that the caspase cascade reaction mediated by Fas/FasL plays an important role in dengue virus serotype-2-induced apoptosis in human umbilical vein endothelial cells.However,because signal transduction pathways are very complex,further research is required to determine if there are several simultaneous signal transduction pathways of apoptosis and how exactly they function.Except for death receptor,nitric oxide can mediate apoptosis in different intracellular systems,although the precise mechanisms are not completely clear.LinJ.Med.Virol.DOI10.1002/jmvDengue Virus Causes Apoptosis Via Fas/FasL1397confirmed the nitric oxide-mediated impairment of endothelial cells using cross-reacting antibodies in dengue virus infection[Lin et al.,2002].Nitric oxide is produced by microphages that are activated by cytokines and microorganisms[Raddassi et al.,1993].Nitric oxide is also an important multifunctional molecular mediator of physiologic and pathologic processes,which may result in either initiation or suppression[Drexler,1999;Ward et al.,2000].Most researchers believe that nitric oxide canfight against virus by inhibiting its metabolic enzymes,forming oxygen-derived free radicals,inhibit-ing the duplication of virus,or preventing the synthesis of viral proteins.In the study by Lin et al.[1995], physiologically relevant concentrations of nitric oxide appeared ineffective in inducing apoptosis in human umbilical vein endothelial cells.In Luzm’s report[Espina et al.,2003],there was also no increase of nitric oxide in the course of dengue virus serotype-2-infected monocytes undergoing apoptosis.Valero reported[Valero et al., 2002]that the concentration of nitric oxide was greater in serum from dengue fever,patients than that in patients with dengue hemorrhagic fever and in normal individu-als;however,patients with dengue hemorrhagic fever and normal individuals had no apparent differences,and no age and sex differences were observed.Recently, Chareonsirisuthigul et al.[2007]reported that the there was no difference in the concentration of nitric oxide between dengue fever,patients and primarily infected patients,while there was a decrease in the nitric oxide concentration in secondarily infected patients.In our experiment,it was found that nitric oxide did not increase at12,24,48,or72hr in human umbilical vein endothelial cells infected by dengue virus serotype-2.All these conflicting in vivo and in vitro studies indicate that the function of nitric oxide is complicated.These are the important factors:different virus strains,different target cells,and different infection conditions(primarily or secondarily infected).In this study it was showed nitric oxide has no contribution to apoptosis in human umbilical vein endothelial cells during dengue virus serotype-2infection.In conclusion,it was confirmed that dengue virus serotype-2could induce apoptosis in human umbilical vein endothelial cells in vitro and that the caspase cascade reaction mediated by Fas/FasL plays a key role in this apoptosis.Further research is required to investigate the precise mechanism of impaired endo-thelial cells in dengue hemorrhagic fever/dengue shock syndrome.ACKNOWLEDGMENTSWe thank Dr.Z.L.Wang,from the First Affiliated Hospital,Sun Yat-sen University,Guangzhou,PR China,for providing human umbilical cords.REFERENCESAlgeciras-Schimnich A,Shen L,Barnhart BC,Murmann AE,Bur-khardt JK,Peter ME.2002.Molecular ordering of the initial signaling events of CD95.Mol Cell Biol22:207–220.Almasan A,Ashkenazi A.2003.Apo2L/TRAIL:Apoptosis signaling, biology,and potential for cancer therapy.Cytokine Growth Factor Rev14:337–348.Andrews BS,Theofilopoulos AN,Peters CJ,Loskutoff DJ,Brandt WE, Dixon FJ.1978.Replication of dengue and junin viruses in cultured rabbit and human endothelial cells.Infect Immun20:776–781. Avirutnan P,Malasit P,Seliger B,Bhakdi S,Husmann M.1998.Dengue virus infection of human endothelial cells leads to chemokine production,complement activation,and apoptosis.J Immunol161:6338–6346.Chareonsirisuthigul T,Kalayanarooj S,Ubol S.2007.Dengue virus (DENV)antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines,but suppresses anti-DENV free radical and pro-inflammatory cytokine production,in THP-1cells.J Gen Virol88:365–375.Chen HC,Hofman FM,Kung JT,Lin YD,Wu-Hsieh BA.2007.Both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage.J Virol81:5518–5526.Clarke P,Tyler KL.2007.Down-regulation of cFLIP following reovirus infection sensitizes human ovarian cancer cells to TRAIL-induced apoptosis.Apoptosis12:211–223.Degli Esposti M,Tour J,Ouasti S,Ivanova S,Matarrese P,Malorni W, Khosravi-Far R.2009.Fas death receptor enhances endocytic membrane traffic converging into the Golgi region.Mol Biol Cell 20:600–615.Despres P,Frenkiel MP,Ceccaldi PE,Duarte Dos Santos C,Deubel V.1998.Apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses.J Virol72: 823–829.Drexler H.1999.Nitric oxide synthases in the failing human heart:A doubled-edged sword?Circulation99:2972–2975.Espina LM,Valero NJ,Hernandez JM,Mosquera JA.2003.Increased apoptosis and expression of tumor necrosis factor-alpha caused by infection of cultured human monocytes with dengue virus.Am J Trop Med Hyg68:48–53.Fukuo K,Nakahashi T,Nomura S,Hata S,Suhara T,Shimizu M, Tamatani M,Morimoto S,Kitamura Y,Ogihara T.1997.Possible participation of Fas-mediated apoptosis in the mechanism of atherosclerosis.Gerontology43:35–42.Griffith TS,Brunner T,Fletcher SM,Green DR,Ferguson TA.1995.Fas ligand-induced apoptosis as a mechanism of immune privilege.Science270:1189–1192.Guzman MG,Kouri G.2002.Dengue:An ncet Infect Dis 2:33–42.Iyngkaran N,Yadav M,Sinniah M.1995.Augmented inflammatory cytokines in primary dengue infection progressing to shock.Singapore Med J36:218–221.Jan JT,Chen BH,Ma SH,Liu CI,Tsai HP,Wu HC,Jiang SY,Yang KD, Shaio MF.2000.Potential dengue virus-triggered apoptotic path-way in human neuroblastoma cells:Arachidonic acid,superoxide anion,and NF-kappaB are sequentially involved.J Virol74:8680–8691.Kim N,Dabrowska A,Jenner RG,Aldovini A.2007.Human and simian immunodeficiency virus-mediated upregulation of the apoptotic factor TRAIL occurs in antigen-presenting cells from AIDS-susceptible but not from AIDS-resistant species.J Virol81:7584–7597.Larochelle B,Flamand L,Gourde P,Beauchamp D,Gosselin J.1998.Epstein–Barr virus infects and induces apoptosis in human neutrophils.Blood92:291–299.Lin KT,Xue JY,Nomen M,Spur B,Wong PY.1995.Peroxynitrite-induced apoptosis in HL-60cells.J Biol Chem270:16487–16490. Lin CF,Lei HY,Shiau AL,Liu HS,Yeh TM,Chen SH,Liu CC,Chiu SC, Lin YS.2002.Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein1via production of nitric oxide.J Immunol169:657–664.Marianneau P,Flamand M,Deubel V,Despres P.1998.Apoptotic cell death in response to dengue virus infection:The pathogenesis of dengue haemorrhagic fever revisited.Clin Diagn Virol10:113–119.Matsuda T,Almasan A,Tomita M,Tamaki K,Saito M,Tadano M, Yagita H,Ohta T,Mori N.2005.Dengue virus-induced apoptosis in hepatic cells is partly mediated by Apo2ligand/tumour necrosis factor-related apoptosis-inducing ligand.J Gen Virol86:1055–1065.J.Med.Virol.DOI10.1002/jmv1398Liao et al.。