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1 Laboratory of Vector-Borne Viruses, Department of Virology 1, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
2 Department of Microbiology, Medical Faculty, University of Indonesia, Jalan Pegangsaan Timur no. 16, Jakarta 10320, Indonesia
3 Department of Infection Biology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan
Correspondence
Ichiro Kurane
kurane{at}nih.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Dengue viruses usually cause a mild to severe febrile illness, dengue fever (DF), but may cause dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) in some cases (Gubler, 1998; Halstead, 1979). It is estimated that up to 100 million individuals are infected with dengue viruses, with 500 000 cases of DHF and 25 000 deaths annually (Gubler, 1998). It has been suggested that antibody-dependent enhancement of dengue virus infection contributes to the pathogenesis of DHF (Halstead, 1979, 1988). It has also been proposed that the severity of the disease is related to the virulence of the dengue virus strain (Leitmeyer et al., 1999; Rico-Hesse et al., 1997).
Although the mechanism of pathogenesis of DHF has not been completely elucidated, it is apparent that profound plasma leakage is one of the characteristic physiological features. Due to the lack of a good animal model, endothelial cells have been used as a surrogate to elucidate the pathogenesis of dengue virus interactions. Endothelial cells can be infected with dengue virus in vitro (Bunyaratvej et al., 1997; Huang et al., 2003; Killen & O'Sullivan, 1993). Dengue virus infection induces cytokine production (Avirutnan et al., 1998; Bosch et al., 2002) and alters the expression of adhesion molecules on endothelial cells (Krishnamurti et al., 2002). Although endothelial cells are not massively infected, there is evidence of dengue virus infection of endothelial cells in vivo (Gubler & Zaki, 1998; Hall et al., 1991; Jessie et al., 2004). It is possible that low levels of dengue virus infection of endothelial cells may induce profound effects locally, along with other factors such as cytokines and peripheral blood mononuclear cells (PBMCs).
Endothelial cells form a continuous cell layer along the wall of blood vessels and control the movement of solutes and fluid from the vascular space to the tissue (Tretiach et al., 2003). Two main types of junctional structures, adherence and tight junctions, are involved in cell–cell contact. The adherence junction is a multimolecular complex composed predominantly of transmembrane proteins termed cadherins (Navarro et al., 1995; Steinberg & McNutt, 1999) and is a major contributor to the maintenance of monolayer integrity (Allport et al., 2000; Dejana et al., 1999; Del Maschio et al., 1996). Vascular endothelial cells express a unique member of the cadherin family, vascular endothelial cadherin (VE-cadherin). VE-cadherin is a novel member of the cadherin family and is selectively expressed at intercellular boundaries of all types of endothelial cells, but is not expressed by any other cell types (Dejana et al., 2001; Lampugnani et al., 1992). VE-cadherin plays an important role in the maintenance of endothelial monolayer integrity. Blocking of VE-cadherin functions with specific antibodies results in increased vascular permeability (Corada et al., 2001; Hordijk et al., 1999).
Activation of endothelial cells facilitates the adherence of leukocytes to endothelial cells (McIntyre et al., 1985, 1997). Moreover, activated endothelial cells, in turn, activate adherent cells (McIntyre et al., 1985, 1997). Therefore, we hypothesized that adherence of leukocytes to endothelial cells infected with dengue virus might change the functions of endothelial cells and increase permeability. In the present study, we attempted to determine the effect of PBMCs on the permeability of dengue virus-infected human umbilical vein endothelial cells (HUVECs). A transendothelial electrical resistance (TEER) assay and albumin permeability assay were used to assess the permeability of HUVECs in vitro (Dewi et al., 2004). In addition, we examined the relationship between the permeability of endothelial cells and VE-cadherin expression.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Separation of adherent and non-adherent PBMCs.
Adherent PBMCs were isolated as described by Malkovsky et al. (1987). One million PBMCs in 1 ml RPMI supplemented with 10 % FBS were added to 12-well tissue-culture plates (Nunc). After 2 h of incubation at 37 °C in 5 % CO2, non-adherent cells were removed by pipetting and plates were washed twice with RPMI medium without FBS. Adherent PBMCs in the wells were removed by gentle scraping with a rubber cell scraper. Adherent and non-adherent PBMCs were washed and resuspended at a concentration of 1x106 cells ml–1 in RPMI supplemented with 10 % FBS. Fifty microlitres non-adherent or adherent PBMCs at a concentration of 1x106 cells ml–1 was added to HUVECs on transwell membranes in the experiments. The percentage of monocytes in fractionated PBMCs was determined by immunofluorescence with FITC-conjugated, anti-CD14 antibody (Biomedia). Anti-CD14 antibody reacts with the 55 kDa single-chain membrane glycoprotein that is expressed primarily on monocytes and macrophages.
Infection of HUVECs with dengue virus.
HUVECs were purchased from Clonetics (catalogue no. CC-2517) and propagated in supplemented endothelial cell basal medium-2 (Dewi et al., 2004). HUVECs were infected with dengue-2 virus (DV-2) New Guinea C strain on transwell membranes at an m.o.i. of 0.5 p.f.u. per cell. Briefly, medium in the upper transwell polycarbonate membrane culture dish was removed and the cells were infected by adding 50 µl DV-2 per well. Cells were incubated for 2 h at 37 °C in 5 % CO2, and washed with PBS without CaCl2 and MgCl2 [PBS (–)]. Culture supernatant fluid from uninfected C6/36 cells and heat-inactivated DV-2 (Putnak et al., 1991) were used as controls. HUVECs were also infected with DV-2 on slide chambers for determining the percentage of DV-2 antigen-positive cells and for immunofluorescence staining of VE-cadherin.
Effect of PBMCs and culture supernatant fluids from DV-2-infected HUVECs and PBMCs on the permeability of HUVECs.
HUVECs were infected with DV-2 for 2 h, as described above, and 50 µl of 106 to 102 PBMCs ml–1 was added to the infected HUVECs. In other experiments, HUVECs on transwell membranes were washed and 100 µl culture supernatant fluid from DV-2-infected HUVECs and PBMCs were added. After various incubation times, the TEER was assessed. Culture supernatant fluids from uninfected HUVECs treated with PBMCs were used as controls. Human TNF-
(1 µg ml–1) with a titre of 2x107 U mg–1 was used as a positive control (Dewi et al., 2004).
Transendothelial electrical resistance (TEER).
The TEER of HUVECs was measured using the electrical cell sensor system Endohm chamber (Millicell-ERS; Millipore) (World Precision Instruments), as reported previously (Dewi et al., 2004). The TEER was first measured at 24 h after seeding, and this time point was defined as 0 h. The TEER was measured at various incubation time points after addition of PBMCs or culture supernatant fluids from DV-2-infected HUVECs and PBMCs. The TEER included the resistance of the interelectrode solution and blank membrane; therefore, a transwell polycarbonate membrane culture dish without cells was always included as the blank. Every test was done in duplicate and repeated at least three times. The mean and SD were calculated from two independent experiments with two transwell culture dishes per group.
Transendothelial albumin permeability.
Transendothelial albumin permeability on transwell polycarbonate membrane culture dishes was also examined using trypan blue-bound BSA as described by Bonner & O'Sullivan (1998) with a minor modification (Dewi et al., 2004). Two transwell culture dishes per group were washed with PBS and 100 µl trypan blue-bound BSA was added to the upper chamber of each transwell polycarbonate membrane culture dish containing HUVEC monolayers (Dewi et al., 2004). The amount of trypan blue-bound BSA detected in the lower chamber was considered to represent the level of HUVEC permeability.
Counting the PBMCs attached to HUVECs.
In order to determine whether dengue virus infection increases the number of PBMCs adherent to HUVECs, HUVEC-attached PBMCs were counted. HUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell for 2 h. PBMCs were added to HUVECs and incubated for 24 h. Cells were washed twice with PBS (–), fixed with methanol at 37 °C for 30 min and washed twice with PBS (–). Cells were then stained with Giemsa for 30 min at room temperature and washed once with water and once with 30 % methanol in PBS (–). After air drying, the PBMCs on HUVECs were counted in five fields for each treatment with a magnification of x200 under a light microscope.
Morphology of HUVECs.
HUVECs were seeded into 96-well flat-bottomed microplates (Nunc) and infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell. PBMCs were added and incubated for 24 h. HUVECs were then fixed and stained as reported previously (Dewi et al., 2004). After air drying, the morphology of HUVECs was observed under a light microscope with magnification of x200.
Indirect immunofluorescence staining.
PBMCs and HUVECs were stained for DV-2 antigen by indirect immunofluorescence staining as described previously (Dewi et al., 2004). Cells were incubated with 1 : 250-diluted hyperimmune mouse ascites fluid to DV-2 (Brandt et al., 1967) at 37 °C for 60 min. After washing, the cells were reacted with 1 : 500-diluted FITC-conjugated sheep anti-mouse IgG (Cappel) in PBS containing 5 % Block Ace (AbD Serotec) at 37 °C for 60 min. The slide was washed and covered with Syva microtech mounting fluid which contained PBS with glycerol (Boehring Diagnostic Inc.). Cells were observed under a fluorescence microscope.
Immunofluorescence staining of VE-cadherin.
HUVECs on slide chambers were washed with PBS and air-dried for 20 min. Cells were fixed in acetone at –20 °C for 20 min and rinsed in PBS. Cells were treated with Block Ace at 37 °C for 60 min. After washing three times for 3 min with PBS, cells were incubated with 1 : 500-diluted, FITC-labelled antibody to VE-cadherin (Bender MedSystems) in PBS containing 5 % Block Ace at 37 °C for 60 min. Slides were washed and covered with Syva microtech mounting fluid containing phosphate buffer with glycerol (Boehring Diagnostic Inc.). Cells were observed under a fluorescence microscope at x400 magnification.
RNA isolation from HUVECs.
HUVECs on 12-well plates (Nunc) were lysed with 1 ml Isogen (Wako Nippon Gene) and 200 µl chloroform was then added. RNA isolation was done according to the manufacturer's instructions (Wako Nippon Gene). The RNA was dissolved in 20 µl ultrapure DNase-free, RNase-free distilled water (Invitrogen Life Technologies) and then treated with DNase I (Invitrogen Life Technologies) at room temperature for 15 min to remove contaminating DNA. The RNA concentration was measured using a spectrophotometer and adjusted to 100 µg ml–1 and used for real-time RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to confirm the use of equal amounts of HUVEC mRNA.
Real-time RT-PCR.
TaqMan primers and probes for detection of the VE-cadherin gene were prepared according to the sequence of GenBank accession no. NM_001795
[GenBank]
and those for the GAPDH gene used GenBank accession no. BC004109
[GenBank]
. Primers used for amplification of VE-cadherin were forward primer 5'-TCACCTGGTCGCCAATCC-3' and reverse primers 5'-AGGCCACATCTTGGGTTCCT-3'. The probe specific for VE-cadherin was 5'-TCTCTTTCTTTTCTCTGTCTACTCCTTATCCCTTGGTT-3'. Primers used for amplification of GAPDH were forward primer 5'-GAAATCCCATCACCATCTTCCA-3' and reverse primer 5'-CCAGCATCGCCCCACTT-3'. The probe specific for GAPDH was 5'-AGCGAGATCCCTCC-3'.
The ABI PRISM 7000 sequence detection system (Applied Biosystems) was used for one-step RT-PCR and hybridization. Aliquots (5–10 ng) of purified RNA were used as the template in 25 µl total reaction volumes. The reagents were added to the TaqMan one-step RT-PCR master mix reagents (PE Applied Biosystems). The real-time RT-PCR program was: initial incubation for 30 min at 48 °C for reverse transcription and denaturation at 95 °C for 10 min followed by 45 cycles of 95 °C for 15 s and 57 °C for 1 min. All tests were performed in duplicate.
Statistical analysis.
The significance of differences was determined by Student's t-tests compared with control HUVECs without treatment. The data were expressed as means±SD, and differences are considered to be significant when the P value was less than 0.05. The TEER test consisted of at least three independent experiments with two transwell polycarbonate membrane culture dishes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and 2). The results were similar using PBMCs from donor B.
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). Addition of PBMCs to untreated HUVECs or to HUVECs treated with C6/36 supernatant or inactivated DV-2 did not decrease the TEER or increase the albumin permeability. The TEER of PBMCs alone was at the similar level to wells containing no cells (data not shown).
Changes in permeability were also assessed by the albumin permeability assay. Addition of 5x104 PBMCs to HUVECs infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell increased the albumin permeability across HUVECs (Fig. 1), but addition of PBMCs to HUVECs treated with C6/36 supernatant or inactivated DV-2 did not. Thus, the increase in the albumin permeability across HUVECs was consistent with the decrease in the TEER. The increase in the albumin permeability across HUVECs become more profound on days 2–5.
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).
Effect of the supernatant fluid from DV-2-infected HUVECs and PBMCs on the permeability of HUVECs
HUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell for 2 h, 5x104 PBMCs were added and supernatants were collected at various time points. The collected culture supernatant fluids were then added to HUVECs on transwell membranes and the permeability was assessed for 3 days. Culture supernatant fluids collected on days 1 and 2 did not decrease the TEER, but those collected on days 3 and 4 did (Table 2). The decrease in the TEER started 4 h after addition of the supernatant fluids. Those collected on days 5, 6 and 7 also decreased the TEER (data not shown). Supernatant fluids collected on days 3–7 from the culture containing HUVECs treated with inactivated DV-2 or C6/36 and PBMCs did not decrease the TEER (data not shown).
Effect of the level of DV-2 infection on the permeability of HUVECs
To determine the relationship between the level of DV-2 infection of HUVECs and the PBMC-induced decrease in the TEER, HUVECs were infected with DV-2 at various m.o.i. and then 5x104 PBMCs were added. Infection of HUVECs with DV-2 at an m.o.i. of 0.5 p.f.u. per cell decreased the TEER when PBMCs were added, but infection at an m.o.i. of 0.05 p.f.u. per cell or less did not (Table 2). HUVECs treated with C6/36 supernatant or treated with inactivated DV-2 did not decrease the TEER when 5x104 PBMCs were added.
The percentage of DV-2 antigen-positive HUVECs was examined after infection at an m.o.i. of 0.5 p.f.u. per cell. DV-2 antigen-positive cells were detected as early as 1 day after infection (0.9±0.4 %), and the proportion of positive cells increased gradually until day 5 (23±3.1 %) (Fig. 2). DV-2 antigen was not detected in HUVECs treated with inactivated DV-2 or C6/36 cell culture supernatant.
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), but only small numbers were observed on uninfected HUVECs (Fig. 3a). The number of PBMCs adherent to the surface of DV-2-infected HUVECs was 0.21±0.18 PBMCs per cell in DV-2-infected HUVEC monolayers and 0.025±0.01 per cell in uninfected monolayers. When endothelial cells were pretreated with C6/36 cell supernatant or inactivated DV-2, the number of PBMCs adherent to the surface of HUVECs was 0.042±0.02 and 0.038±0.09 per cell, respectively. The results indicate that DV-2 infection of HUVEC monolayers increased the number of PBMCs adherent to HUVECs.
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). Infection of HUVECs at an m.o.i. of 0.5 p.f.u. per cell did not change the morphology of HUVECs (Fig. 4b). When HUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell and PBMCs were added, HUVECs demonstrated a thinner and flatter, spindle-shaped morphology (Fig. 4e). This morphological change was similar to that observed in TNF--treated HUVECs (Fig. 4f). Addition of PBMCs to HUVECs treated with inactivated DV-2 or C6/36 did not change the morphology (Fig. 4c, d). Thus, the morphological changes were concomitant with the decrease in the TEER and increased albumin permeability of HUVECs (Table 1, Figs 1 and 4).
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). When HUVECs were infected with DV-2 and PBMCs were added, HUVECs demonstrated intercellular gaps and VE-cadherin expression was decreased at the periphery (Fig. 5e), similar to those treated with TNF- (Fig. 5f). There were multiple large intercellular gaps between adjacent cells (arrows in Figs 5e and f). When HUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell and no PBMCs were added (Fig. 5b), treated with inactivated DV-2 and PBMCs were added (Fig. 5c) or treated with C6/36 cell supernatant and PBMCs were added (Fig. 5d), HUVECs demonstrated a VE-cadherin staining pattern similar to that of untreated cells.
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). The levels of GAPDH were similar among all the experimental groups, suggesting that equal amounts of mRNA were used in this study (Table 3). Using VE-cadherin-specific primers and probe, untreated HUVECs and those infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell demonstrated positive signals at 23.76±0.08 and 23.25±0.44 cycles, respectively. When HUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell and PBMCs were added, positive signal was developed at 26.23±0.18 cycles (P<0.05 compared with untreated HUVECs). TNF--treated HUVECs demonstrated a positive signal at 26.89±0.04 cycles (P<0.01 compared with untreated HUVECs). When HUVECs were treated with C6/36 supernatant or inactivated DV-2 and PBMCs were added, positive signals were developed at 23.99±0.50 and 23.66±0.76 cycles, respectively. The results indicate that the amounts of VE-cadherin mRNA were lower when HUVECs were infected with DV-2 and PBMCs were added, and suggest that the decrease in the TEER is concomitant with the decrease in the level of VE-cadherin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Infection of HUVECs with DV-2 at an m.o.i. of 0.5 p.f.u. per cell did not decrease the TEER unless PBMCs were added. It is possible that dengue virus mediates endothelial cell barrier function via an indirect route rather than direct infection of endothelial cells. In general, however, the tissue damage observed in pathological studies is not very severe compared with the severity of illness (Bhamarapravati, 1989). Furthermore, the lack of structural damage, the short-lived nature of the plasma leakage syndrome and the remarkably rapid recovery of children with DSS all suggest that the alteration of permeability is effected by a soluble mediator (Innis, 1995).
Given the increased permeability of endothelial cells in the presence of PBMCs, it is likely that increased permeability of HUVECs was in part due to the cytokines which were produced by DV-2-infected PBMCs. Dengue viruses can replicate in monocyte-derived macrophages and monocyte-like cell lines (Kurane et al., 1990; O'Sullivan & Killen, 1994) and induce cytokines (Bosch et al., 2002; Espina et al., 2003) and cytotoxic factors (Mukerjee et al., 1995; Shaio et al., 1995). Culture fluids from dengue virus-infected peripheral blood monocytes activated endothelial cells, as measured by upregulation of cell adhesion molecules (Anderson et al., 1997), and increased the permeability of endothelial cells in vitro (Carr et al., 2003). It has also been reported that cytokines play an important role in the pathogenesis of DHF (Rothman & Ennis, 1999), and plasma leakage in DHF patients is associated with the elevation of plasma levels of various cytokines (Green et al., 1999; Kurane et al., 1991).
There was a report that only the supernatant collected from dengue virus-infected monocyte-macrophages at 72 h increased permeability, and not that collected before 72 h (Carr et al., 2003), and similar results were observed in the present study. In this experiment, we found that the TEER was decreased (P<0.05) on day 1 after the addition of PBMCs. Furthermore, larger numbers of PBMCs attached to the surface of DV-2-infected HUVEC monolayers after washing than to uninfected HUVECs. It is possible that the decrease in the TEER before 48 h after addition of PBMCs is due to activation of PBMCs by adherence to the DV-2-infected endothelial cell monolayer. After infection with dengue virus, endothelial cells express adhesion molecules (Krishnamurti et al., 2002). PBMCs which attached to infected endothelial cells are activated, and activated PBMCs may produce cytokines that act locally and increase plasma leakage. Activation of endothelial cells, in turn, might activate the adherent leukocytes (McIntyre et al., 1997; McIntyre, 2000) and produce cytokines, chemokines and tissue factors in a contact-dependent manner (Monaco et al., 2002).
Addition of PBMCs to HUVECs infected with DV-2 at an m.o.i. of less than 0.5 p.f.u. per cell did not decrease the TEER. Although the underlying mechanism remains poorly understood, it seems that the number of viruses is associated with the severity of disease. Epidemiological studies of DHF patients showed that DV-3 viraemia was greater in more severe clinical disease (Libraty et al., 2002). Previous studies examining DV-1 and DV-2 infections have reported higher circulation levels of replicating virus (Vaughn et al., 2000) or viral RNA copies (Murgue et al., 2000) in DHF than in DF patients. Addition of PBMCs to control HUVECs or HUVECs pretreated with C6/36 cells supernatant or pretreated with inactivated DV-2 did not decrease the TEER. These results indicate that infection of HUVECs is necessary for the decrease of the TEER in this experimental system.
We separated adherent and non-adherent cell fractions from PBMCs and tested their effects on the decrease of the TEER. The majority of adherent PBMCs on plastic well plates were determined to be monocytes by immunofluorescence staining with anti-CD14 antibody. Adherent PBMCs decreased the TEER to a greater extent than non-adherent PBMCs. It was reported that adherent cells are the major cell population that supports dengue virus replication in vivo (Scott et al., 1980). Allport et al. (2000) also showed that the adherence of monocytes on endothelial cells induces focal disruption of the VE-cadherin. Consistent with these reports, the extent of the decrease was more profound with adherent PBMCs than with non-adherent PBMCs in our study. We suggest that attachment of PBMCs leads to production of cytokines that act locally and increase the permeability of DV-2-infected HUVECs.
The morphology of HUVECs changed when HUVECs were infected with DV-2 at an m.o.i. of 0.5 p.f.u. per cell and PBMCs were added. A similar result was found on HUVECs treated with TNF-. It was reported that the low TEER was associated with irregular cell shape, when studied morphologically (Tretiach et al., 2003), and alterations in the permeability of the vascular endothelium (Bucana et al., 1988). McKenzie & Ridley (2007) found that TNF- subsequently caused a progressive increase in permeability and in stress fibre reorganization, cell elongation and intercellular gap formation over 8–24 h. Consistent with the increased permeability, occludin and JAM-A were removed from tight junctions and ZO-1 was partially redistributed (McKenzie & Ridley, 2007). Similar results were obtained in the present study. Morphological changes were observed concomitantly with the decrease in the TEER and increased permeability.
Additionally, we examined the levels of VE-cadherin mRNA using real-time RT-PCR. Only when PBMCs were added, HUVECs infected with DV-2 demonstrated lower levels of VE-cadherin mRNA. In immunofluorescence assays, infection of HUVECs with DV-2 at an m.o.i. of 0.5 p.f.u. per cell and addition of PBMCs resulted in a marked decrease in immunofluorescence for membrane-associated VE-cadherin. This treatment also led to the development of multiple intercellular gaps between adjacent cells. Since VE-cadherin plays a crucial role in the maintenance of endothelial cell–cell contact and monolayer integrity (Breviario et al., 1995; Del Maschio et al., 1996), the appearance of multiple intercellular gaps between adjacent cells probably indicates an increase in permeability. These results are consistent with previous reports using TNF- (Friedl et al., 2002; Hofmann et al., 2002) and thrombin (Rabiet et al., 1996).
The disassembly of the adherence-junction component, as indicated by the decrease in VE-cadherin expression, probably contributes to the increase in endothelial permeability in this study. In addition to adherence junctions, endothelial cells also possess tight junctions. Studies are also needed to understand the role of the tight junction of endothelial cells in the pathogenesis of DHF. Furthermore, the relevance of the results in the present study to the pathogenesis of DHF in vivo needs to be elucidated in future studies.
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ACKNOWLEDGEMENTS |
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Received 6 August 2007;
accepted 30 October 2007.
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