In vitro assessment of human endothelial cell permeability: effects of inflammatory cytokines and dengue virus infection

Journal of Virological Methods 121 (2004) 171–180

In vitro assessment of human endothelial cell permeability: effects of inflammatory cytokines and dengue virus infection Beti Ernawati Dewia,b,c , Tomohiko Takasakia , Ichiro Kuranea,c,∗ a

b

Laboratory of Vector-Borne Viruses, Department of Virology 1, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan Department of Microbiology, Medical Faculty, University of Indonesia, Jalan Pegangsaan Timur no. 16, Jakarta 10320, Indonesia c Department of Infection Biology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan Received 11 February 2004; received in revised form 21 June 2004; accepted 23 June 2004 Available online 6 August 2004

Abstract Electrical resistance across human umbilical vein endothelial cells (HUVECs) was measured using an electrical cell sensor system. The transendothelial electrical resistance (TEER) value was used to estimate the permeability through endothelial cells in vitro. Decrease in the TEER value was associated with increase in the passage of albumin through endothelial cells in the albumin permeability assay. The effects of cytokines and dengue virus infection on the permeability of HUVECs were examined by measuring the TEER value. Tumor necrosis factor alpha (TNF-␣) at 1 and 0.1 ␮g/ml decreased the TEER value, but TNF-␣ at lower dose did not. Interferon-gamma (IFN-␥) at 1 ␮g/ml also decreased the TEER value. In contrast, interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) or interferon-beta (IFN-␤) did not decrease the TEER value. The decrease in the TEER value was associated with the morphological changes of HUVECs. Dengue virus infection at a multiplicities of infection (m.o.i.) of 5 pfu/cell decreased the TEER value. Infection at an m.o.i. of 0.5 pfu/cell did not decrease the TEER value; however, addition of 0.01 ␮g/ml of TNF-␣ to these infected endothelial cells decreased the TEER value. The results suggest that TNF-␣ and dengue virus infection decrease synergistically the TEER value of endothelial cells. The TEER method is easy, reliable and can be applicable to further analysis of the increase in the permeability of endothelial cells in vitro induced by inflammatory cytokines and dengue virus infection. © 2004 Elsevier B.V. All rights reserved. Keywords: Endothelial cells; Permeability; Cytokine; Dengue virus

1. Introduction Vascular endothelial cells line the inner surface of blood vessels, and play an important role in vascular functions. Vascular endothelial cells function as a barrier between blood and interstitial compartments (Crone, 1986). Malfunction in the barrier properties of vascular endothelial cells leads to tissue edema (Peterson and Kirschbaum, 1998; Hanaoka et al., 2003). It is known that hormones, cytokines and neurotransmitters play an important role in the regulation of endothelial cell functions (Royall et al., 1992; Royall and Ischiropoulos, 1993; Rabiet et al., 1996; Galdiero et al., 1997). ∗ Corresponding author. Tel.: +81 3 5285 1169; fax: +81 3 5285 1169. E-mail address: [email protected] (I. Kurane).

0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.06.013

It has been reported that serum levels of vasoactive factors are elevated in patients with dengue hemorrhagic fever. These include interleukin-6 (IL-6) (Iyngkaran et al., 1995; Juffrie et al., 2001), tumor necrosis factor alpha (TNF-␣) (Iyngkaran et al., 1995; Hober et al., 1996; Bethell et al., 1998; Yadav et al., 1991; Azeredo et al., 2001; Suharti et al., 2002), interleukin10 (IL-10) (Green et al., 1999; Azeredo et al., 2001; Gagnon et al., 2002), IFN-gamma (IFN-␥) (Azeredo et al., 2001), interleukin-2 (IL-2) (Kurane et al., 1991; Hober et al., 1993; Green et al., 1999). The role of TNF-␣ in the increased permeability of vascular endothelial cells has been postulated (Brett et al., 1989; Munro et al., 1989; Royall et al., 1989; Shinjo et al., 1989; Goldblum and Sun, 1990; Koga et al., 1995; Blum et al., 1997; Jacobs and Levin, 2002). TNF-␣ induces the release of cytokines and chemokines (Mantovani et al., 1997), the

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production of enzymes, such as cyclooxygenases-2 (Mark et al., 2001) and nitric oxide synthesis (Freyer et al., 1999; Bove et al., 2001). TNF-␣ also increases the expression of adhesion molecules on endothelial cells (Kallmann et al., 2000; Madan et al., 2002; Dagia and Goetz, 2003; Javaid et al., 2003; Mo et al., 2003; Nakao et al., 2003; Sasaki et al., 2003). Endothelial cells can be infected with dengue virus in vitro (Andrews et al., 1978; Sahaphong et al., 1980; Killen and O’Sullivan, 1993). Dengue virus infection alters cytokine production, and expression of adhesion molecules on endothelial cells (Bunyaratvej et al., 1997; Avirutnan et al., 1998; Diamond et al., 2000; Huang et al., 2003). Although endothelial cells are not infected massively, there is some evidence of dengue virus infection of endothelial cell in vivo (Hall et al., 1991; Gubler and Zaki, 1998). It is possible that low levels of dengue virus infection of endothelial cells may provide profound effect locally, along with other factors such as cytokines and peripheral blood mononuclear cells. Furthermore, dengue virus infection of endothelial cells induces cytokine production and alters adhesion molecule expression. It is, thus, important to address these questions in a reliable in vitro experimental system. In order to address the effect of dengue virus and cytokines on endothelial cell functions, a method for assessing the permeability of human umbilical vein endothelial cells (HUVECs) was established. Electrical resistance across HUVECs was measured using the electrical cell sensor system, Endohm chambers with WPI’s EVOM resistance meter. This method provided reproducible resistance value of HUVECs. Then, the effect of dengue virus infection and inflammatory cytokines on endothelial cell permeability we examined using the established method. The result demonstrated synergistic effect of TNF-␣ and dengue virus infection on the decrease in the TEER value, suggesting increased permeability.

2. Materials and methods 2.1. Endothelial cells Primary human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (San Diego, CA, catalogue no. CC-2517). HUVECs were propagated and maintained in endothelial cell basal medium-2 (EBM-2) supplemented with human recombinant epidermal growth factor (hEGF), human fibroblast growth factor-basic with heparin (hFGF-B), vascular endothelial growth factor (VEGF), ascorbic acid, hydrocortisone, human recombinant insulin-like growth factor (long R3-IGF-1), heparin, gentamicin, amphotercin and 2% fetal bovine serum (FBS). The cells were cultured at 37 ◦ C in humidified air containing 5% CO2 . The cells were used at three to five passages in the present study, because the expression of the membrane receptors could be lowered at high cell passages (Schmid et al., 1995). HUVECs were passaged when they reached 90% confluency, using 0.25% trypsin in EDTA. Trypsin was neutral-

ized by trypsin neutralizing solution (Clonetics, San Diego, CA). After washing twice with HBSS (Clonetics), HUVECs were seeded on a fibronectin (Sigma, 25 ␮g/ml)-coated, 24well transwell polycarbonate membrane tissue culture dish (6.5 mm diameter, 3.0 ␮m pore size, Corning Costar, Cambridge). The HUVECs were seeded at a density of 1 × 105 cells per well and placed into lower chamber containing 600 ␮l of EBM-2 complete medium. In transendothelial electrical resistance (TEER) and transendothelial albumin permeability assays, HUVECs were used after 24 h of culture to attain HUVECs adhered on the transwell polycarbonate membrane. HUVECs were seeded on slide chambers in dengue virus infection studies. 2.2. Cytokines Cytokines used in the present study were recombinant human TNF-␣ with a titer of 2 × 107 units/mg, IL-2, IL-6 with a titer of 1 × 107 units/mg, IL-8, IL-10 with a titer of 5 × 105 units/mg, interferon-beta (IFN-␤) with a titer of 2 × 107 units/mg, and IFN-␥ with a titer of 2 × 107 units/mg. All the cytokines were purchased from PeproTech EC Ltd., London and reconstituted according to the manufacture’s instructions. 2.3. Dengue virus type 2 Dengue virus type 2 (DV-2), the New Guinea C strain, was used in the study. DV-2 was propagated in C6/36 cells at 28 ◦ C for 7 days. The culture supernatant was harvested and centrifuged at 900 × g for 5 min and then filtered through the syringe driven millex GV with 0.22 um filter unit (Millipore Co., Bedford, MA, USA). DV-2 was stored at −70 ◦ C until use. The titer was determined by plaque assay using Vero cells (Yamada et al., 2002). Briefly, a 10-fold serial dilution of DV2 was inoculated onto Vero cell monolayer in duplicate wells. Absorption was carried out at 37 ◦ C in 5% CO2 for 2 h with agitation at 30 min interval. Methylcellulose overlay medium was added and infected Vero cells were incubated at 37 ◦ C in 5% CO2 for 7–9 days. Plaque numbers were counted after methylene blue staining. 2.4. Infection of HUVECs with dengue virus HUVECs were infected with DV-2 at multiplicities of infection (m.o.i.) of 5–0.005 plaque forming unit (pfu)/cell. Briefly, medium was removed from the upper transwell polycarbonate membrane culture dish and HUVECs were infected by adding 50 ␮l of serially diluted DV-2. Adsorption was carried out at 37 ◦ C in 5% CO2 for 2 h. Cells were washed with PBS(−) (Wako Pure Chem Industries Ltd., USA) and 100 ␮l of HUVECs culture medium was added. Controls were set up using uninfected C6/36 cell culture supernatant or heatinactivated DV-2 at m.o.i of 5 or 0.5 pfu/cell (Putnak et al., 1991).

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2.5. Indirect immunofluorescence assay (IFA) HUVECs were stained for DV-2 antigen by indirect immunofluorescence assay. HUVECs on the slide chamber were washed in PBS(−), and air-dried under laminar air flow for 20 min. Cells were fixed in acetone at −20 ◦ C for 20 min and rinsed in PBS. Cells were blocked with block ace (Yukijirushinyugyo Co., Japan) to reduce non-specific binding at 37 ◦ C for 60 min. After washing with PBS(−) three times for 3 min, cells were incubated with 1:250 diluted hyperimmune mouse ascitis fluids to DV-2 (Brandt et al., 1967) at 37 ◦ C for 60 min. Cells were washed as described earlier. The cells were then treated with 1:500 diluted FITC-conjugated sheep anti-mouse IgG (Cappel, USA) in PBS(−) with 5% block ace at 37 ◦ C for 60 min. Slide was covered with Syva microtech mounting fluid that contain PBS with glycerol (Boehring diagnostic Inc. Co., CA, USA) and observed under a fluorescence microscope. 2.6. Transendothelial electrical resistance (TEER) Electrical resistance across HUVECs was measured using the electrical cells sensor system, the Endohm chamber (Millicell-ERS, Millipore, World Precisions, Saratoga, FL, USA). The Endohm chamber is a resistance measurement device that gives value of membrane resistance. The Endohm chambers with WPI’s EVOM resistance meter enabled the reproducible measurement of the resistance of endothelial cells in a transwell polycarbonate membrane culture dish. The bottom and the top of each chamber contain a pair of concentric electrodes: a voltage-sensing silver/silver chloride pellet in the center and annular current electrode made of silver and coated with gray colored silver chloride. The upper electrode reads the voltage relative to the bottom one. The value included the resistance of the inter-electrode solution and blank membrane; therefore, transwell polycarbonate membrane culture dish without cells was always used as the blank. TEER was measured at various incubation time after addition of serial dilutions of cytokines or DV-2 infection. The TEER value was first measured at 24 h after seeding and this time point was defined as 0 h. Every test was done in duplicate and repeated at least three times. Treatment with cytokines was done by adding 1 ␮l of serially diluted cytokines (100–0.001 ␮g/ml) into transwell polycarbonate membrane culture dishes. Added cytokines were kept in the culture during the observation period. The upper and lower chamber media were not changed until the end of experiment. HUVECs without treatment was used as control. The TEER value was calculated by the formula: [the average resistance of experimental wells − the average resistance of blank wells] × 0.33(the area of the transwell membrane). 2.7. Transendothelial albumin permeability The transendothelial albumin permeability test was carried out as reported previously with modifications (Bonner

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and O’Sullivan, 1998; Carr et al., 2003). Confluent monolayer of HUVECs were prepared on 24-well transwell polycarbonate membranes as described above. HUVECs were washed twice with PBS(−), and placed into the new lower chamber containing 600 ␮l of PBS(−). One hundred microliters of stained bovine serum albumin was added to the upper chamber of transwell polycarbonate membrane culture dish which contained HUVECs monolayer. The culture plate was incubated at 37 ◦ C in 5% CO2 for 30 min. The upper chambers were removed and the absorbance of the solution in the lower chamber was measured at 595 nm. Results were expressed as the amount of BSA detected in the lower chamber after 30 min of incubation. 2.8. Morphology of HUVECs HUVECs were seeded in 96-well flat bottom microplate (Nunc, Denmark) and treated with cytokines or infected with DV-2. At 24 and 48 h, when the control and the experimental wells showed significantly different TEER values, HUVECs were fixed with 10% formaldehyde in PBS at room temperature for 30 min and stained with trypan blue (Gibco BRL, Life Technology, NY, USA). After air-dried, the morphology of HUVECs were observed under a light microscope. 2.9. Viability of HUVECs The viability of HUVECs was assessed as reported previously (Liu et al., 1995) with slight modifications. At 24 and 48 h, when the control and the experimental wells showed significantly different TEER values, the viability of HUVECs was assessed. After washed with PBS(−), 100 ␮l of 0.2% trypan blue was added to 96-well plates containing HUVECs. After 15 min at 37 ◦ C in 5% CO2 , trypan blue was poured out. HUVECs were observed and percent of viable cells were counted under a light microscope. 2.10. Statistics The Student’s t-test was used to determine the significance of differences among the experimental groups. The difference was considered to be significant when P-value was equal to or lower than 0.05.

3. Results 3.1. Assessment of electrical resistance of HUVECs HUVECs were seeded at 1 × 105 cells/well on fibronectincoated polycarbonate transwell membrane. The electrical resistance was assessed after 24 h of incubation, when the cells formed confluent monolayer. Under the regular experimental condition, the TEER value was 10– 12  cm2 .

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Fig. 1. Dose-dependent decrease in the TEER value of HUVECs induced by TNF-␣. HUVECs were treated with various concentration of TNF-␣. The TEER value was measured at various incubation time. The data show the average of two independent experiment ± S.D., each done in duplicate.

3.2. Decrease in TEER of HUVECs by TNF-␣

3.3. Comparison of the effects of cytokines on TEER

The TEER assay was validated using a known permeabilizing agent, TNF-␣. HUVECs grown on transwell membranes were incubated with TNF-␣ at 1–0.0001 ␮g/ml. TNF-␣ decreased the TEER value in a dose-dependent manner (Fig. 1). The TEER value started to decrease at 2 h after addition of TNF-␣ at 1 ␮g/ml. The effect become more profound as time passed (P < 0.05). TNF-␣ at 0.1 ␮g/ml also induced gradual decrease in the TEER value, but the level of decrease was not as marked as 1 ␮g/ml. Interestingly, the TEER value of HUVECs treated with 0.1 ␮g/ml of TNF-␣ recovered after 3 days. TNF-␣ at 0.01 ␮g/ml or lower did not demonstrated significant effect on the TEER value.

It has been reported that plasma leakage in dengue haemorrhagic fever is associated with elevated levels of plasma cytokines. Therefore, the effect was examined of cytokines which have been reported to be elevated in these patients, on the TEER of HUVECs. Effects of IL-2, IL-6, IL-8, IL10, IFN-␤, and IFN-␥ were examined at serial dilutions from 1 to 0.0001 ␮g/ml (Fig. 2). IFN-␥ at 1 ␮g/ml decreased the TEER value compared to the control between days 1 and 7 (P < 0.05), but IFN-␥ at 0.1 ␮g/ml and lower did not (data not shown). IL-2, IL-6, IL-8, IL-10, or IFN␤ did not decrease the TEER value during the test period.

Fig. 2. Comparison of the effect of cytokines on the TEER value of HUVECs from 2 h to 7 days of treatment. The data show the average ± S.D. of two independent experiments, each done in duplicate.

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3.5. Synergistic effect of dengue virus infection and TNF-␣ Addition of TNF-␣ at 0.01 ␮g/ml to the HUVECs which were infected with DV-2 at an m.o.i. of 0.5 pfu/cell decreased the TEER value of HUVECs (Fig. 5). DV-2 infection alone at an m.o.i. of 0.5 pfu/cell or addition of 0.01 ␮g/ml of TNF ␣ alone did not decrease the TEER value. The addition of TNF␣ at 0.01 ␮g/ml to HUVECs previously treated with C6/36 cell supernatant or heat-inactivated DV-2 did not decreased the TEER value. These results suggest that dengue virusinfected and uninfected HUVECs responded differently to the low concentration of TNF-␣. Fig. 3. Decrease in the TEER value of HUVECs after infection with dengue virus. HUVECs were infected with DV-2 at various multiplicities of infection. The supernatant of uninfected C6/36 cells and inactivated DV-2 were used as control.

3.4. Effect of dengue virus infection on the TEER HUVECs were infected with DV-2 at m.o.i. of 5–0.0005 pfu/cell, and the TEER was examined at various time points after infection (Fig. 3). Infection with DV-2 at an m.o.i. of 5 pfu/cell decreased the TEER value (P < 0.05). Infection with DV-2 at an m.o.i. of 0.5 pfu/cell and lower did not decrease the TEER value. It is assumed that the early decrease in the TEER value was not due to cell death, because there were not significant differences in viability and number of adherent cells between infected and uninfected HUVECs during tested period (data not shown). Fig. 4 shows the percentage of DV-2 antigen-positive HUVECs after infection at m.o.i. of 5 and 0.5 pfu/cell as revealed by indirect immunofluorescence staining. The percentage of DV-2 antigen-positive cells reached the maximum on day 5, when infected at an m.o.i. of 5 pfu/cell. The percentage of DV-2 antigen-positive cells after infection at an m.o.i. of 0.5 pfu/cell was not as high as after infection at an m.o.i. of 5 pfu/cell (Fig. 4). No antigen-positive cells were detected on HUVECs after treatment with heat-inactivated DV-2 or C6/36 cell culture supernatant.

3.6. Morphological changes in HUVECs The morphology of HUVECs changed after treatment with 1 ␮g/ml of TNF-␣, infection with DV-2 at an m.o.i. of 5 pfu/cell, or infection with DV-2 at an m.o.i. of 0.5 pfu/cell and TNF-␣ treatment at 0.01 ␮g/ml (Fig. 6). HUVECs without any treatment, those infected with DV-2 at an m.o.i. of 0.5 pfu/cell, those treated with TNF-␣ at 0.01 ␮g/ml showed a round, cobblestone-like morphology with tight cell–cell contact (Fig. 6A, E and F). After treatment with 1 ␮g/ml of TNF␣, infection with DV-2 at an m.o.i. of 5 pfu/cell, infection with DV-2 at an m.o.i. of 0.5 pfu/cell and TNF-␣ treatment at 0.01 ␮g/ml, the cells showed a thinner and flatter spindle shape (Fig. 6B–D). The morphological change was concomitant with the decrease in the TEER value and in transendothelial albumin permeability (Figs. 1, 3 and 5). 3.7. Correlation between TEER with transendothelial albumin permeability In order to confirm that the decrease in the TEER value reflected the increase in the permeability of HUVECs monolayer, the permeability of the cells were also examined by albumin permeability method and compared with the TEER value (Fig. 7). A linear regression was demonstrated between the TEER value and the BSA permeability across HUVECs

Fig. 4. Percentages of DV-2 antigen-positive HUVECs. HUVECs were infected with DV-2 at m.o.i. of 5 and 0.5 pfu/cell. DV-2 antigen-positive cells were determined by immunofluorescence assay.

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Fig. 5. Effect of the addition of TNF-␣ at 0.01 ␮g/ml on the TEER value of HUVECs infected with DV-2. Treatment with 0.01 ␮g/ml of TNF-␣ alone and infection with DV-2 at m.o.i. of 0.5 pfu/cell alone were used as controls. Addition of TNF-␣ at 0.01 ␮g/ml to the HUVECs which were infected with DV-2 at an m.o.i. of 0.5 pfu/cell decreased the TEER of HUVECs.

(R2 = 0.8144). The results indicate that the TEER value reflected the permeability of HUVECs.

4. Discussion In the present study, a method for assessing transendothelial electrical resistance (TEER) was established, using the Endohm chamber. The electrical cell sensor system, the Endohm chamber, provided reproducible values in transwell

polycarbonate membrane culture dish. The decrease in the TEER value was consistent with the increase in albumin permeability through HUVECs. Therefore, the TEER value was used as that which indicates the level of permeability. The morphology of HUVECs changed after treatment with 1 ␮g/ml of TNF-␣, infection with DV-2 at an m.o.i. of 5 pfu/cell, or infection with DV-2 at an m.o.i. of 0.5 pfu/cell and treatment with 0.01 ␮g/ml of TNF-␣. Morphological changes were concomitant with the decrease in the TEER value and the permeability of HUVECs. Decrease in the

Fig. 6. Morphological change of HUVECs at 48 h after infection with DV-2 or treatment with TNF-␣: (A) control HUVECs; (B) treated with TNF-␣ at 1 ␮g/ml; (C) infected with DV-2 at an m.o.i. of 5 pfu/cell; (D) infected with dengue virus at an m.o.i. of 0.5 pfu/cell and treated with 0.01 ␮g/ml of TNF-␣; (E) infected with dengue virus at an m.o.i. of 0.5 pfu/cell; (F) treated with 0.01 ␮g/ml of TNF-␣.

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Fig. 7. Comparison between the TEER value and transendothelial albumin permeability. A linear regression was demonstrated (R2 = 0.8144).

TEER value at early stage was not due to massive cell death, since there were no significant differences in the viability and number between infected and uninfected cells. It was reported that the low TEER value was associated with irregular cell shape, when studied morphologically (Tretiach et al., 2003), and the alterations in the permeability of the vascular endothelium (Bucana et al., 1988). Similar results were obtained in the present study. Morphological changed were observed concomitantly with the decrease in the TEER value. Thus, light microscopic observation may also provide supporting evidence for the increase in vascular permeability. It has been reported that TNF-␣ increases the transit of macromolecules across vascular endothelium (Brett et al., 1989; Goldblum and Sun, 1990; Burke-Gaffney and Keenan, 1993; Sedgwick et al., 2002). We demonstrated that TNF␣ decreased the TEER of HUVECs. This result confirmed that the method for assessing the TEER in the present study reflected the changes in endothelial cell functions. TNF-␣ activates various vascular cellular components and results in dysfunction of endothelial cells (Ferro et al., 2000; Petrache et al., 2001), and also induce apoptosis of endothelial cells (Robaye et al., 1991; Polunovsky et al., 1994; Karsan et al., 1996; Slowik et al., 1997; Petrache et al., 1999). It has been reported that plasma leakage in DHF is associated with the elevation of plasma levels of various cytokines. We examined the effect of cytokines on the permeability of endothelial cells. IL-2, IL-6, IL-8, IL-10, IFN-␤ and IFN-␥ were tested. It was reported that IL-2 (Bucana et al., 1988; Cotran et al., 1988; Downie et al., 1992; Ehringer et al., 1998; Rodella et al., 2001), IL-6 (Maruo et al., 1992; De Vries et al., 1996; Desai et al., 2002), and IL-8 (Biffl et al., 1995) increased permeability of endothelial cells. In the present study, TNF-␣ and IFN-␥ increased the permeability, but others did not. These differences may suggest unequal susceptibility of endothelial cells, depending on the vascular bed origins, and emphasize the importance in the selection of types and origins of endothelial cells used in experiments (Tan et al., 2001; Komori et al., 2002; Wang et al., 2002; Ahmed-Choudhury et al., 2003; De Staercke et al., 2003; Elherik et al., 2003).

The results showed that HUVECs infected with dengue virus at an m.o.i. of 5 pfu/cell demonstrated decrease in the TEER value and associated with albumin permeability through HUVECs. Dengue virus infection may induce production of vasoactive substances and results in increased vascular permeability. Interestingly, dengue virus-infected and uninfected HUVECs demonstrated different responses to low concentrations of TNF-␣ which alone did not decrease the TEER value. Addition of 0.01 ␮g/ml of TNF-␣ to HUVECs infected with dengue virus at an m.o.i. of 0.5 pfu/cell increased the permeability. We suggest that there is a synergistic effect between dengue virus infection and TNF-␣ in the elevation of permeability of HUVECs. In conclusion, a reliable method was developed for assessing the permeability of endothelial cells. This system effectively measured the permeability change induced by TNF-␣ and dengue virus infection. Furthermore, there was a synergistic effect between dengue virus infection and TNF-␣. More precise analysis on the role of cytokines and dengue viruses in the induction of DHF is needed, and the analysis can be undertaken in part using this system. Acknowledgements We thank Dr. M. Ito, Dr. S. Tajima, and Ms. R. Nerome, Laboratory of Vector-Borne Viruses, Department of Virology 1, National Institute of Infectious Diseases, for their technical help. This study was supported by the grant-in-aid (No. 12877045) from Ministry of Education, Sciences, Sports and Culture, Japan and the grant for Research on Emerging and Re-Emerging Infectious Diseases from Ministry of Health, Labour and Welfare, Japan. References Ahmed-Choudhury, J., Russell, C.L., Randhawa, S., Young, L.S., Adams, D.H., Afford, S.C., Choudhury, J.A., 2003. Differential induction of nuclear factor-kappaB and activator protein-1 activity after CD40 ligation is associated with primary human hepatocyte apoptosis or

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intrahepatic endothelial cell proliferation. Mol. Biol. Cell 14 (4), 1334–1345. Andrews, B.S., Theofilopoulos, A.N., Peters, C.J., Loskutoff, D.J., Brandt, W.E., Dixon, F.J., 1978. Replication of dengue and junin viruses in cultured rabbit and human endothelial cells. Infect. Immun. 20 (3), 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. Immunol. 161 (11), 6338–6346. Azeredo, E.L., Zagne, S.M., Santiago, M.A., Gouvea, A.S., Santana, A.A., Neves-Souza, P.C., Nogueira, R.M., Miagostovich, M.P., Kubelka, C.F., 2001. Characterisation of lymphocyte response and cytokine patterns in patients with dengue fever. Immunobiology 204 (4), 494–507. Bethell, D.B., Flobbe, K., Cao, X.T., Day, N.P., Pham, T.P., Buurman, W.A., Cardosa, M.J., White, N.J., Kwiatko, 1998. Pathophysiologic and prognostic role of cytokines in dengue hemorrhagic fever. J. Infect. Dis. 177 (3), 778–782. Biffl, W.L., Moore, E.E., Moore, F.A., Carl, V.S., Franciose, R.J., Banerjee, A., 1995. Interleukin-8 increases endothelial permeability independent of neutrophils. J. Trauma 39 (1), 98–103. Blum, M.S., Toninelli, E., Anderson, J.M., Balda, M.S., Zhou, J., O’Donnell, L., Pardi, R., Bender, J.R., 1997. Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines. Am. J. Physiol. 273 (1 Part 2), 286–294. Bonner, S.M., O’Sullivan, M.A., 1998. Endothelial cell monolayers as a model system to investigate dengue shock syndrome. J. Virol. Methods 71 (2), 159–167. Bove, K., Neumann, P., Gertzberg, N., Johnson, A., 2001. Role of ecNOSderived NO in mediating TNF-induced endothelial barrier dysfunction. Am. J. Physiol. Lung Cell. Mol. Physiol. 280 (5), 914–922. Brandt, W.E., Buescher, E.L., Hetrick, F.M., 1967. Production and characterization of arbovirus antibody in mouse ascitic fluid. Am. J. Trop. Med. Hyg. 16, 339–347. Brett, J., Gerlach, H., Nawroth, P., Steinberg, S., Godman, G., Stern, D., 1989. Tumor necrosis factor/cachectin increases permeability of endothelial cell monolayers by a mechanism involving regulatory G proteins. J. Exp. Med. 169 (6), 1977–1991. Bucana, C.D., Trial, J., Papp, A.C., Wu, K.K., 1988. Bovine aorta endothelial cell incubation with interleukin 2: morphological changes correlate with enhanced vascular permeability. Scanning Microsc. 2 (3), 1559–1566. Bunyaratvej, A., Butthep, P., Yoksan, S., Bhamarapravati, N., 1997. Dengue viruses induce cell proliferation and morphological changes of endothelial cells. Southeast Asian J. Trop. Med. Public Health 28 (Suppl. 3), 32–37. Burke-Gaffney, A., Keenan, A.K., 1993. Modulation of human endothelial cell permeability by combinations of the cytokines interleukin1alpha/beta, tumor necrosis factor-alpha and interferon-gamma. Immunopharmacology 25 (1), 1–9. Carr, J.M., Hocking, H., Bunting, K., Wright, P.J., Davidson, A., Gamble, J., Burrell, C.J., Li, P., 2003. Supernatants from dengue virus type-2 infected macrophages induce permeability changes in endothelial cell monolayers. J. Med. Virol. 69 (4), 521–528. Cotran, R.S., Pober, J.S., Gimbrone Jr., M.A., Springer, T.A., Wiebke, E.A., Gaspari, A.A., Rosenberg, S.A., Lotze, M.T., 1988. Endothelial activation during interleukin 2 immunotherapy. A possible mechanism for the vascular leak syndrome. J. Immunol. 140 (6), 1883–1888. Crone, C., 1986. Modulation of solute permeability in microvascular endothelium. Fed. Proc. 45 (2), 77–83. Dagia, N.M., Goetz, D.J., 2003. A Proteasome inhibitor reduces concurrent, sequential and long term IL-1{beta} and TNF-{alpha} induced endothelial cell adhesion molecule expression and resultant adhesion. Am. J. Physiol. Cell Physiol. 285 (4), 813–822. Desai, T.R., Leeper, N.J., Hynes, K.L., Gewertz, B.L., 2002. Interleukin-6 causes endothelial barrier dysfunction via the protein kinase C pathway. J. Surg. Res. 104 (2), 118–123.

De Staercke, C., Phillips, D.J., Hooper, W.C., 2003. Differential responses of human umbilical and coronary artery endothelial cells to apoptosis. Endothelium 10 (2), 71–78. De Vries, H.E., Blom-Roosemalen, M.C., van Oosten, M., de Boer, A.G., van Berkel, T.J., Breimer, D.D., Kuiper, J., 1996. The influence of cytokines on the integrity of the blood–brain barrier in vitro. J. Neuroimmunol. 64 (1), 37–43. Diamond, M.S., Edgil, D., Roberts, T.G., Lu, B., Harris, E., 2000. Infection of human cells by dengue virus is modulated by different cell types and viral strains. J. Virol. 74, 7814–7823. Downie, G.H., Ryan, U.S., Hayes, B.A., Friedman, M., 1992. Interleukin2 directly increases albumin permeability of bovine and human vascular endothelium in vitro. Am. J. Respir. Cell Mol. Biol. 7 (1), 58–65. Ehringer, W.D., Edwards, M.J., Wintergerst, K.A., Cox, A., Miller, F.N., 1998. An increase in endothelial intracellular calcium and F-actin precedes the extravasation of interleukin-2-activated lymphocytes. Microcirculation 5 (1), 71–80. Elherik, K.E., Khan, F., Belch, J.J., 2003. Differences in endothelial function and vascular reactivity between Scottish and Arabic populations. Scot. Med. J. 48 (3), 85–87. Ferro, T., Neumann, P., Gertzberg, N., Clements, R., Johnson, A., 2000. Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha. Am. J. Physiol. Lung Cell. Mol. Physiol. 278 (6), 1107–1117. Freyer, D., Manz, R., Ziegenhorn, A., Weih, M., Angstwurm, K., Docke, W.D., Meisel, A., Schumann, R.R., Schonfelder, G., Dirnagl, U., Weber, J.R., 1999. Cerebral endothelial cells release TNF-alpha after stimulation with cell walls of Streptococcus pneumoniae and regulate inducible nitric oxide synthase and ICAM-1 expression via autocrine loops. J. Immunol. 163 (8), 4308–4314. Gagnon, S.J., Mori, M., Kurane, I., Green, S., Vaughn, D.W., Kalayanarooj, S., Suntayakorn, S., Ennis, F.A., Rothman, A.L., 2002. Cytokine gene expression and protein production in peripheral blood mononuclear cells of children with acute dengue virus infections. J. Med. Virol. 67 (1), 41–46. Galdiero, M., de l’Ero, G.C., Marcatili, A., 1997. Cytokine and adhesion molecule expression in human monocytes and endothelial cells stimulated with bacterial heat shock proteins. Infect. Immun. 65 (2), 699–707. Goldblum, S.E., Sun, W.L., 1990. Tumor necrosis factor-alpha augments pulmonary arterial transendothelial albumin flux in vitro. Am. J. Physiol. 258 (2 Part 1), 57–67. Green, S., Vaughn, D.W., Kalayanarooj, S., Nimmannitya, S., Suntayakorn, S., Nisalak, A., Rothman, A.L., Ennis, F.A., 1999. Elevated plasma interleukin-10 levels in acute dengue correlate with disease severity. J. Med. Virol. 59 (3), 329–334. Gubler, D.J., Zaki, S.R., 1998. Dengue and other viral hemorrhagic fevers. In: Nelson, A.M., Horsburgh Jr., C.R. (Eds.), Pathology of Emerging Infections. Part 2. ASM Press, Washington, DC, pp. 43–71. Hall, W.C., Crowell, T.P., Watts, D.M., Barros, V.L.R., Kruger, H., Pinheiro, F., Peters, C.J., 1991. Demonstration of yellow fever and dengue antigens in formalin-fixed paraffin-embedded human liver by immunohistochemical analysis. Am. J. Trop. Med. Hyg. 45 (4), 408–417. Hanaoka, M., Droma, Y., Naramoto, A., Honda, T., Kobayashi, T., Kubo, K., 2003. Vascular endothelial growth factor in patients with highaltitude pulmonary edema. J. Appl. Physiol. 94 (5), 1836–1840. Hober, D., Delannoy, A.S., Benyoucef, S., De Groote, D., Wattre, P., 1996. High levels of sTNFR p75 and TNF alpha in dengue-infected patients. Microbiol. Immunol. 40 (8), 569–573. Hober, D., Poli, L., Roblin, B., Gestas, P., Chungue, E., Granic, G., Imbert, P., Pecarere, J.L., Vergez-Pascal, R., Wattre, P., 1993. Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL6), and interleukin-1beta (IL-1beta) in dengue-infected patients. Am. J. Trop. Med. Hyg. 48 (3), 324–331. Huang, Y.H., Lei, H.Y., Liu, H.S., Lin, Y.S., Chen, S.H., Liu, C.C., Yeh, T.M., 2003. Tissue plasminogen activator induced by dengue virus infection of human endothelial cells. J. Med. Virol. 70 (4), 610–616.

B.E. Dewi et al. / Journal of Virological Methods 121 (2004) 171–180 Iyngkaran, N., Yadav, M., Sinniah, M., 1995. Augmented inflammatory cytokines in primary dengue infection progressing to shock. Singapore Med. J. 36 (2), 218–221. Jacobs, M., Levin, M., 2002. An improved endothelial barrier model to investigate dengue haemorrhagic fever. J. Virol. Methods 104, 173–185. Javaid, K., Rahman, A., Anwar, K.N., Frey, R.S., Minshall, R.D., Malik, A.B., 2003. Tumor necrosis factor-alpha induces early-onset endothelial adhesivity by protein kinase Czeta-dependent activation of intercellular adhesion molecule-1. Circ. Res. 92 (10), 1089–1097. Juffrie, M., Meer, G.M., Hack, C.E., Haasnoot, K., Sutaryo, Veerman, A.J., Thijs, L.G., 2001. Inflammatory mediators in dengue virus infection in children: interleukin-6 and its relation to C-reactive protein and secretory phospholipase A2. Am. J. Trop. Med. Hyg. 65 (1), 70–75. Kallmann, B.A., Hummel, V., Lindenlaub, T., Ruprecht, K., Toyka, K.V., Rieckmann, P., 2000. Cytokine-induced modulation of cellular adhesion to human cerebral endothelial cells is mediated by soluble vascular cell adhesion molecule-1. Brain 123 (Part 4), 687–697. Karsan, A., Yee, E., Harlan, J.M., 1996. Endothelial cell death induced by tumor necrosis factor-alpha is inhibited by the Bcl-2 family member, A1. J. Biol. Chem. 271 (44), 27201–27204. Killen, H., O’Sullivan, M.A., 1993. Detection of dengue virus by in situ hybridization. J. Virol. Methods 41 (2), 135–146. Koga, S., Morris, S., Ogawa, S., Liao, H., Bilezikian, J.P., Chen, G., Thompson, W.J., Ashikaga, T., Brett, J., Stern, D.M., et al., 1995. TNF modulates endothelial properties by decreasing cAMP. Am. J. Physiol. 268 (5 Part 1), 1104–1113. Komori, K., Inoguchi, H., Kume, M., Shoji, T., Furuyama, T., 2002. Differences in endothelial function and morphologic modulation between canine autogenous venous and arterial grafts: endothelium and intimal thickening. Surgery 131 (Suppl. 1), 249–255. Kurane, I., Innis, B.L., Nimmannitya, S., Nisalak, A., Meager, A., Janus, J., Ennis, F.A., 1991. Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue. J. Clin. Invest. 88 (5), 1473–1480. Liu, H., Chen, J., Feng, C., Zheng, H., 1995. A study on the viability of corneal endothelium in rabbit by dual staining with trypan blue and alizanin red S. Yan Ke Xue Bao 11 (2), 69. Madan, B., Singh, I., Kumar, A., Prasad, A.K., Raj, H.G., Parmar, V.S., Ghosh, B., 2002. Xanthones as inhibitors of microsomal lipid peroxidation and TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs). Bioorg. Med. Chem. 10 (11), 3431–3436. Mantovani, A., Sozzani, S., Introna, M., 1997. Endothelial activation by cytokines. Ann. N. Y. Acad. Sci. 832, 93–116. Mark, K.S., Trickler, W.J., Miller, D.W., 2001. Tumor necrosis factoralpha induces cyclooxygenase-2 expression and prostaglandin release in brain microvessel endothelial cells. J. Pharmacol. Exp. Ther. 297 (3), 1051–1058. Maruo, N., Morita, I., Shirao, M., Murota, S., 1992. IL-6 increases endothelial permeability in vitro. Endocrinology 131 (2), 710–714. Mo, S.J., Son, E.W., Rhee, D.K., Pyo, S., 2003. Modulation of TNF-alphainduced ICAM-1 expression, NO and H2 O2 production by alginate, allicin and ascorbic acid in human endothelial cells. Arch. Pharm. Res. 26 (3), 244–251. Munro, J.M., Pober, J.S., Cotran, R.S., 1989. Tumor necrosis factor and interferon-gamma induce distinct patterns of endothelial activation and associated leukocyte accumulation in skin of Papio anubis. Am. J. Pathol. 135 (1), 121–133. Nakao, S., Kuwano, T., Ishibashi, T., Kuwano, M., Ono, M., 2003. Synergistic effect of TNF-alpha in soluble VCAM-1-induced angiogenesis through alpha 4 integrins. J. Immunol. 170 (11), 5704– 5711. Peterson, M.W., Kirschbaum, J., 1998. Asbestos-induced lung epithelial permeability: potential role of nonoxidant pathways. Am. J. Physiol. 275 (2 Part 1), 262–268.

179

Petrache, I., Choi, M.E., Otterbein, L.E., Chin, B.Y., Mantell, L.L., Horowitz, S., Choi, A.M., 1999. Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells. Am. J. Physiol. 277 (3 Part 1), 589–595. Petrache, I., Verin, A.D., Crow, M.T., Birukova, A., Liu, F., Garcia, J.G., 2001. Differential effect of MLC kinase in TNF-alpha-induced endothelial cell apoptosis and barrier dysfunction. Am. J. Physiol. Lung Cell. Mol. Physiol. 280 (6), 1168–1178. Polunovsky, V.A., Wendt, C.H., Ingbar, D.H., Peterson, M.S., Bitterman, P.B., 1994. Induction of endothelial cell apoptosis by TNF alpha: modulation by inhibitors of protein synthesis. Exp. Cell Res. 214 (2), 584–594. Putnak, R., Feighny, R., Burrous, J., Cochran, M., Hackett, C., Smith, G., Hoke, C., 1991. Dengue-1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicits virus-neutralizing antibody in mice and protects them from virus challenge. Am. J. Trop. Med. Hyg. 45 (2), 159–167. Rabiet, M.J., Plantier, J.L., Rival, Y., Genoux, Y., Lampugnani, M.G., Dejana, E., 1996. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler. Thromb. Vasc. Biol. 16 (3), 488–496. Robaye, B., Mosselmans, R., Fiers, W., Dumont, J.E., Galand, P., 1991. Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro. Am. J. Pathol. 138 (2), 447– 453. Rodella, L., Zamai, L., Rezzani, R., Artico, M., Peri, G., Falconi, M., Facchini, A., Pelusi, G., Vitale, M., 2001. Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells. Br. J. Haematol. 115 (2), 442– 450. Royall, J.A., Berkow, R.L., Beckman, J.S., Cunningham, M.K., Matalon, S., Freeman, B.A, 1989. Tumor necrosis factor and interleukin 1alpha increase vascular endothelial permeability. Am. J. Physiol. 257 (6 Part 1), 399–410. Royall, J.A., Gwin, P.D., Parks, D.A., Freeman, B.A., 1992. Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha. Arch. Biochem. Biophys. 294 (2), 686–694. Royall, J.A., Ischiropoulos, H., 1993. Evaluation of 2 ,7 -dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch. Biochem. Biophys. 302 (2), 348–355. Sahaphong, S., Riengrojpitak, S., Bhamarapravati, N., Chirachariyavej, T., 1980. Electron microscopic study of the vascular endothelial cell in dengue hemorrhagic fever. Southeast Asian J. Trop. Med. Public Health 11 (2), 194–204. Sasaki, M., Ostanin, D., Elrod, J.W., Oshima, T., Jordan, P., Itoh, M., Joh, T., Minagar, A., Alexander, J.S., 2003. TNF-alpha-induced endothelial cell adhesion molecule expression is cytochrome P-450 monooxygenase dependent. Am. J. Physiol. Cell Physiol. 284 (2), 422– 428. Schmid, E.F., Binder, K., Grell, M., Scheurich, P., Pfizenmaier, K., 1995. Both tumor necrosis factor receptors, TNFR60 and TNFR80, are involved in signaling endothelial tissue factor expression by juxtacrine tumor necrosis factor alpha. Blood 86 (5), 1836–1841. Sedgwick, J.B., Menon, I., Gern, J.E., Busse, W.W., 2002. Effects of inflammatory cytokines on the permeability of human lung microvascular endothelial cell monolayers and differential eosinophil transmigration. J. Allergy Clin. Immunol. 110 (5), 752–756. Shinjo, K., Tsuda, S., Hayami, T., Asahi, T., Kawaharada, H., 1989. Increase in permeability of human endothelial cell monolayer by recombinant human lymphotoxin. Biochem. Biophys. Res. Commun. 162 (3), 1431–1437. Slowik, M.R., Min, W., Ardito, T., Karsan, A., Kashgarian, M., Pober, J.S., 1997. Evidence that tumor necrosis factor triggers apoptosis in human endothelial cells by interleukin-1-converting enzyme-like protease-dependent and -independent pathways. Lab. Invest. 77 (3), 257–267.

180

B.E. Dewi et al. / Journal of Virological Methods 121 (2004) 171–180

Suharti, C., van Gorp, E.C., Setiati, T.E., Dolmans, W.M., Djokomoeljanto, R.J., Hack, C.E., Ten, C.H., van der Meer, J.W., 2002. The role of cytokines in activation of coagulation and fibrinolysis in dengue shock syndrome. Thromb. Haemost. 87 (1), 42–46. Tan, K.H., Dobbie, M.S., Felix, R.A., Barrand, M.A., Hurst, R.D., 2001. A comparison of the induction of immortalized endothelial cell impermeability by astrocytes. Neuroreport 12 (7), 1329–1334. Tretiach, M., van Driel, D., Gillies, M.C., 2003. Transendothelial electrical resistance of bovine retinal capillary endothelial cells is influenced by cell growth patterns: an ultrastructural study. Clin. Exp. Ophthalmol. 31 (4), 348–353.

Wang, Q., Pfeiffer, G.R., Stevens, T., Doerschuk, C.M., 2002. Lung microvascular and arterial endothelial cells differ in their responses to intercellular adhesion molecule-1 ligation. Am. J. Respir. Crit. Care Med. 166 (6), 872–877. Yadav, M., Kamath, K.R., Iyngkaran, N., Sinniah, M., 1991. Dengue haemorrhagic fever and dengue shock syndrome: are they tumour necrosis factor-mediated disorders? FEMS Microbiol. Immunol. 4 (1), 45–49. Yamada, K., Takasaki, T., Nawa, M., Kurane, I., 2002. Virus isolation as one of the diagnostic methods for dengue virus infection. J. Clin. Virol. 24, 203–209.