1 Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 14-740, México, DF 07360, Mexico
2 Departamento de Investigaciones Inmunológicas, Instituto de Diagnóstico y Referencia Epidemiológicos, SSA, México, DF, Mexico
Correspondence
Isaura Meza
imeza{at}mail.cinvestav.mx
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ABSTRACT |
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Present address: Escuela Superior de Medicina del Instituto Politécnico Nacional, Plan de San Luis y Diaz Mirón, DF 11340, Mexico.
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INTRODUCTION |
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Analysis of dengue virusendothelium interaction has been approached using human endothelial cells from primary cultures of umbilical cord (HUVEC) or cell lines derived from it (Andrews et al., 1978; Chen et al., 1996
; Bosch et al., 2002
). In these in vitro models it has been shown that the viral E protein binds to the endothelial cell surface in the absence of Fc receptors (Chen et al., 1996
) and that infected cells produce high levels of IL8 as well as IL6 (Bosch et al., 2002
; Huang et al., 2000
). E protein also binds to other cell lines such as Vero, Chinese hamster ovary and glia (Chen et al., 1996
).
The natural target for Dengue virus would be microvascular endothelial cells in several tissues where plasma leakage is believed to occur (Krishnamurti et al., 2001; Jacobs & Levin, 2002
). Because the specific response to virus penetration in these cells is still poorly understood, we have utilized human dermal microvascular endothelial cells (HMEC-1) to investigate the mechanisms by which the infection could induce cellular permeability changes. Compared with big-vessel endothelial cells (e.g. HUVEC), microvascular endothelial cells such as HMEC-1 could provide a suitable model to study dengue virus interaction, because this line constitutes a homogeneous population and forms stable monolayers that retain morphological and functional characteristics of normal human microvascular endothelia (Ades et al., 1992
; Xu et al., 1994
). Furthermore, these cells have well-organized tight junctions (TJ) and allow vectorial transport (Blum et al., 1997
; Kielbassa et al., 1998
). The TJ complex regulates permeability in epithelial and endothelial layers, acting as a selective barrier to passage of ions and other molecules (Anderson, 2001
; Dejana et al., 2000
). Several proteins participate in the organization and function of TJ. Importantly, their localization and interactions are modulated by association with the actin cytoskeleton, in particular the actin ring tethered to the plasma membrane on the apical side (Meza et al., 1980
; Madara, 1991
; Rao et al., 2002
). An increase in cellular permeability could result from alterations in the sealing capacity of TJ, caused by modifications to the TJ complex interactions (Cereijido et al., 1989
).
We report here that HMEC-1 can be infected by Dengue 2 virus (D2V) and that confluent monolayers formed by these cells show alterations in permeability at 48 h post-infection (p.i.), as well as actin cytoskeleton rearrangements and displacement of occludin from the TJ complex. These changes occur concomitantly with increases in the thickness of stress fibres and focal adhesions in uninfected cells in the same monolayer. Culture medium from monolayers, recovered 48 h p.i., also induced a significant increase in permeability and reorganization of actin-containing structures, including phosphorylation of tyrosines in proteins forming focal adhesions. These effects were closely reproduced by addition of IL8 to uninfected monolayers and were partially inhibited by neutralizing antibodies to IL8. Genistein, a specific inhibitor of phosphotyrosine kinases, inhibited the effects of the infected-cell culture medium. These data show that D2V infection of HMEC-1 monolayers induced an increase in endothelial permeability eliciting cytoskeleton rearrangements that facilitate TJ disorganization. Although studies in vitro cannot be extrapolated to the patient, our data suggest that endothelial alterations could occur in vivo after D2V infection. Mechanisms activated by secreted cytokines could balance the virus-induced alterations and possibly the secretion of anti-inflammatory cytokines by cells of the immune system, to regain tissue homeostasis and eliminate the virus (Chaturvedi et al., 1999). In the case of DHF, severe permeability alterations could be caused by similar mechanisms but inadequate T-cell activation, after a secondary infection, would contribute to the observed pathology, as proposed recently by Mongkolsapaya et al. (2003)
.
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METHODS |
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Preparation of virus stocks, titration and inactivation.
D2V was donated by B. Briseño (Institute for Diagnosis and Epidemiologic Reference, Mexico City). The sample was isolated from a Mexican patient who developed DF. Monolayers of C6/36 HT were inoculated with the virus and incubated at 34 °C for up to 12 passages. After the last passage, cells were lysed by freezethaw cycles and stored in aliquots at 70 °C. Virus was titrated by the plaque assay using BHK-21 cells. Briefly, a tenfold serial dilution of the virus was added to BHK-21 monolayers cultured in 24-well plates at 2·5x105 cells ml1 and these were incubated at 37 °C for 4 h. After this time, 0·5 ml MEM containing 10 % FBS and 3 % (w/v) carboxymethylcellulose was added to each well. After 5 days incubation at 37 °C, plaques were visualized by staining with naphthol blue black. Virus concentrations are given as p.f.u. ml1. Virus inactivation was performed as indicated by Anderson et al. (1997) by exposure to short-wave UV-light irradiation from a germicidal lamp for 10 min. The inactivation of virus infectivity was verified by plaque assays in BHK-21 cells.
Cell infection.
Confluent monolayers of HMEC-1 were trypsinized and resuspended in growth medium. Cells were seeded on glass coverslips (3·5x105 cells ml1) for immunofluorescence assays and on collagen-coated 12 mm Millicell filter inserts with a 0·4 µm pore size (Millipore) for permeability assays. After 72 h, the culture medium was removed from confluent monolayers and active or UV-inactivated virus was added at 1 p.f.u. per cell and incubated at 37 °C for 90 min. After this time, the inoculum was removed and fresh growth medium was added. Infected cells were analysed at 12, 24, 48, 72 and 96 h p.i.
Detection of viral proteins E and NS1.
Virus-infected HMEC-1 monolayers grown on glass coverslips were fixed in 3·7 % formaldehyde for 20 min and were then permeabilized for 5 min with 0·05 % Triton X-100 in PBS. After rinsing with PBS, cells were blocked with PBS/2 % BSA for 30 min and exposed for 1 h at 37 °C to mAbs to D2V E protein (donated by the Pedro Kourí Institute, Havana, Cuba) or NS1 protein (donated by A. Falconar, London School of Hygiene & Tropical Medicine, U.K; Falconar & Young, 1991) at 1 : 10 and 1 : 50 dilutions, respectively. After washing with PBS, cells were incubated with FITC-conjugated anti-mouse IgG at 1 : 200 dilution (Molecular Probes) for 1 h at 37 °C. Coverslips were mounted with VectaShield H-1000 (Vector Laboratories). Stained cells were analysed with a Zeiss epifluorescence microscope using a 63x Planapo objective.
Flow cytometry analysis.
At 12, 24, 48, 72 and 96 h p.i., D2V-infected and control cells were harvested from six-well culture plates. For flow-cytometry determination of infection, the harvested cells were washed twice with MCDB131 and then fixed with 3·7 % formaldehyde in PBS for 20 min at room temperature. After rinsing with PBS, cells were permeabilized with 0·05 % Triton X-100 in PBS and incubated with the mAb to the D2V E protein at 1 : 50 dilution for 30 min at room temperature. Cells were washed twice with PBS and incubated with FITC-conjugated anti-mouse IgG for 30 min at room temperature. After rinsing with PBS, cells were analysed in a fluorescence-activated cell sorter (Becton Dickinson).
Permeability measurements.
Permeability was determined by measuring the paracellular passage of different sizes of fluorescein isothiocyanate dextran (FITCdextran) (Sigma-Aldrich) and [3H]mannitol (NEN Life Science Products). Cells were grown to confluency on filter inserts and infected at 1 p.f.u. per cell. [3H]Mannitol and 4, 70 and 500 kDa FITCdextran were dissolved in the medium and in Ringer's buffer (115 mM NaCl, 25 mM NaHCO3, 5 mM K2HPO3, 2 mM MgSO4, 1 mM CaCl2 and 2 mM L-glutamine), respectively. To measure flux in the apical to the basolateral direction, the tracer solution [1·25 µCi ml1 (46·25 kBq ml1) for [3H]mannitol or 10 µg ml1 for the FITCdextran of different sizes] was loaded on the apical side of the monolayer and cells were incubated for 1 h at 37 °C. After this period, the tracer concentration in the basolateral compartment was measured. Concentrations of [3H]mannitol were determined in a -scintillation counter. The FITCdextran concentration was determined using a spectrofluorometer at an excitation wavelength of 492 nm and emission at 520 nm.
Localization of actin filaments, occludin, vinculin and tyrosine-phosphorylated proteins.
HMEC-1 were grown to confluency on glass coverslips and were fixed with 3·7 % formaldehyde. The cells were washed with PBS, treated with 0·05 % Triton X-100 for 3 min, blocked with PBS/2 % BSA for 30 min and exposed for 1 h to antibodies against occludin (Zymed Laboratories), vinculin (Sigma; UV4505) or phosphotyrosine (Upstate Biotechnology). All the antibodies were utilized at 1 : 100 dilution. Monolayers were rinsed with PBS and incubated with FITC-tagged secondary antibody (IgG antimouse, 1 : 200 dilution) After rinsing with PBS, rhodaminephalloidin (Molecular Probes) was used to stain actin-containing structures as indicated by the manufacturer. Coverslips were then washed with PBS, mounted and analysed as described for localization of viral proteins.
Cytokine determination.
IL8 was measured in culture medium from D2V-infected monolayers or from inactivated virus-inoculated monolayers by microELISA (Quantikine; R&D Systems). The minimum detectable concentration for IL8 by this assay was 10 ng ml1. TNF- activity was tested in a bioassay in which exposure to 10 ng TNF-
ml1 induced cell death in more than 50 % of L929 cells within 12 h (Evans, 2000
).
Culture medium from D2V-infected and inactivated-virus-inoculated cells.
HMEC-1 were grown to confluency in 24-well tissue culture plates and were inoculated with active D2V or UV-inactivated D2V at 1 p.f.u. per cell. The inoculum was removed after 90 min and fresh medium was added. At 48 h p.i., culture medium was collected and exposed to UV light to inactivate contaminating viral particles, as described above. Medium was stored at 4 °C under sterile conditions and used within 1 week. Confluent monolayers were grown on inserts or glass coverslips and were treated with these culture media for 2 h. The flux of [3H]mannitol was measured in the monolayers grown on the filter inserts and cells on glass coverslips were stained for actin, vinculin and phosphotyrosines, as described above.
Effect of IL8.
Recombinant IL8 (Quantine Systems) was added to control confluent monolayers at concentrations of 100200 pg ml1 dissolved in MCDB131. Cells were treated for 2 h at 37 °C and permeability measurements, actin, vinculin and phosphotyrosine staining were evaluated as described above.
Inhibition of IL8 effect.
Culture media from D2V-infected cells and from inactivated D2V-inoculated cells were mixed with antibodies against IL8 (Santa Cruz Biotechnology) or with irrelevant antibodies at a final concentration of 5 µg ml1 and incubated overnight at 4 °C. Confluent monolayers were grown on inserts or glass coverslips and were treated with these culture media for 2 h. Permeability measurements and cell staining were done as described above.
Inhibition of protein phosphorylation and actin reorganization.
Confluent HMEC-1 monolayers were grown on coverslips, serum-starved for 12 h and pretreated with 60 µg genistein ml1 for 1 h (Sigma-Aldrich). The cells were then exposed to virus-infected cell-culture medium or culture medium from cells inoculated with inactivated D2V. After 2 h, cells were fixed and stained to visualize actin and phosphotyrosines.
Statistics.
Data are expressed as mean±standard error of three independent experiments. The statistical significance was assessed by Student's t-test.
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RESULTS |
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Participation of phosphotyrosine kinases in actin rearrangements induced by D2V-infected cell-culture medium
The cellular mechanisms by which IL8 could be inducing changes in the actin cytoskeleton were explored considering the reported role of cytokines in signalling pathways, and in particular of IL8 as an effector for Rho and Rac GTPases (Schraufstatter et al., 2001). Therefore, the participation of phosphotyrosine kinases (PTK) in the modification of stress fibres and focal adhesions in monolayers treated with D2V-infected cell-culture medium or IL8 was analysed. A strong positive signal for phosphotyrosines was found in the numerous focal adhesions of cells treated with the D2V-infected cell-culture medium (Fig. 8
c), which also showed thick and abundant stress fibres (Fig. 8a
). Reduction in stress fibres and focal adhesion was observed when cells were pretreated for 1 h with genistein, before addition of culture medium from infected monolayers (Fig. 8b, d
). Identical results were obtained in monolayers treated with IL8 after 1 h preincubation with genistein (data not shown). These results indicate that phosphorylation of tyrosines is necessary in proteins participating in actin reorganization at the basal level of the cells, suggesting that IL8 present in culture medium from virus-infected monolayers could induce their phosphorylation.
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DISCUSSION |
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The HMEC-1 line, derived from human dermis microvasculature, retains morphological, biochemical and functional characteristics of primary microvascular endothelial cultures and forms TJ complexes that maintain a stable barrier function. Therefore, these cells offer an in vitro model with closer similarity to the tissue that seems to be the main target of pathogens such as Dengue virus (Ades et al., 1992; Xu et al., 1994
; Kielbassa et al., 1998
). We report here permissiveness of HMEC-1 monolayers to productive infection by D2V. At 48 h p.i., and before any evident cytopathic effect, permeability changes were detected for small molecules such as [3H]mannitol, suggesting a slight modification of the TJ function as a permeability barrier. The barrier function to macromolecules was lost at later times of infection (72 h), although cytopathic effects, manifested as cell rounding and detachment from the substrate, were evident only after several days.
The barrier function of endothelium depends on structural integrity and it is regulated by multiple factors (Lum & Malik, 1996; Blum et al., 1997
). In HMEC-1 monolayers, discontinuity in the localization of the transmembrane TJ protein occludin at the TJ complex region was observed 48 h p.i. with D2V. Loss of occludin continuity at the cell periphery coincided with permeability to mannitol and, at later times of infection, with a progressive increase in the permeability to larger molecules. Other viruses and some bacteria have been reported to alter the organization of TJ in host cells, but the mechanisms utilized by these pathogens are not yet fully understood (Cudmore et al., 1997
; Obert et al., 2000
; Amieva et al., 2003
).
An important modulator of endothelial and epithelial permeability is the actin cytoskeleton through its interaction with TJ complex components (Meza et al., 1980; Madara, 1991
; Lum & Malik, 1996
; Blum et al., 1997
). In dengue virus-infected HMEC-1, actin fibres forming the peripheral ring at the apical side were rapidly disorganized and possibly fragmented as actin aggregates were observed in the cytoplasm of infected cells. However, virus entry and replication did not drastically affect the sealing of TJ. Later, when viral particles were assembled and released, more dramatic modifications of the cytoskeleton coincided with displacement of occludin from the membrane and its concentration into aggregates and a significant increase in permeability. At the same time, thickening of stress fibres and focal adhesions was observed in uninfected cells neighbouring infected ones. The structural modifications of the cytoskeleton in uninfected cells cannot be explained by a direct effect of the virus as seems to be the case for infected cells but, rather, is interpreted as the result of a secondary effect of viral infection. It is possible that rearrangement of actin-containing structures into thicker stress fibres and focal adhesions will provide better attachment to the substrate to maintain the integrity of a monolayer threatened by viral infection.
It has been reported that cytokines modulate endothelial permeability and generate subtle changes in actin cytoskeleton organization and in the proteins forming intercellular junctions (Blum et al., 1997; Jiang et al., 1999
; Schraufstatter et al., 2001
). It is also known that HUVEC infected by Dengue virus release IL8 and other cytokines (Avirutnan et al., 1998
; Bosch et al., 2002
). However, reports of cell infection with Dengue virus have not addressed the role of cytokines on the structural reorganization of the actin cytoskeleton and its possible relationship with permeability changes. We report here that D2V-infected HMEC-1 cultures release IL8 and that addition of culture medium obtained 48 h p.i., or addition of IL8 to control monolayers, reproduced the modifications in permeability and reorganization of the cytoskeleton observed in cells infected with the virus for approximately the same periods of time. The partial inhibition of both permeability and actin reorganization by antibodies against IL8 corroborated the participation of this cytokine in the structural and functional modifications produced by virus-infected cell culture medium. However, the changes observed cannot be attributed to IL8 alone, because infected-cell culture medium showed stronger effects and antibodies to IL8 did not completely reverse its effects. Identification of other components in the culture medium of infected monolayers will be necessary before drawing definitive conclusions.
Our results also show that actin reorganization induced by viral infection was sensitive to inhibitors of PTK. This finding is very suggestive of a role for IL8, and possibly other cytokines released by the infected endothelial cells, as activators of PTK signalling pathways, which, by phosphorylation of specific targets, could induce structural reorganization of TJ and actin (Lum & Malik, 1996; Schraufstatter et al., 2001
). Previous reports regarding the disruption of TJ caused by elevated levels of tyrosine phosphorylation of proteins at the intercellular junction complex support this idea (Collares-Buzato et al., 1998
; van Nieuw Amerongen et al., 1998
). Furthermore, cell retraction as a consequence of TJ disarray and detachment from neighbours could also be regulated by PTK phosphorylation of actin-binding and TJ-associated proteins (Collares-Buzato et al., 1998
; Chen et al., 2002
). Activation of Rho family GTPases, which are the main participants in stress fibre formation and vinculin association with actin, could also be the consequence of higher levels of IL8 and other cytokines (Lum & Malik, 1996
; Imamura et al., 1998
). Very recently, it has been reported that IL8 induced tyrosine phosphorylation of the focal adhesion kinase (FAK) and modified its association with vinculin in focal adhesions (Feniger-Barish et al., 2003
).
In summary, utilizing HMEC-1 monolayers as a model, we have shown that dengue virus entry and assembly can proceed in cells from the microvasculature. Virus infection can be correlated with TJ and actin cytoskeleton functional and structural alterations that lead to important increments in transendothelial permeability. Furthermore, virus infection elicits the release of IL8, which induces further structural modifications that may be important for dynamic changes in the monolayer. While studies in vitro with Dengue virus cannot be extrapolated to what occurs in the infected patient, our studies suggest that, in vivo, similar structural changes and release of cytokines by endothelial and other cells infected by the virus could elicit permeability alterations but, at the same time, activate mechanisms to maintain homeostasis and balance inflammatory responses to get rid of the virus and damaged cells (Carr et al., 2003). In more severe forms of the infection, such as DHF, the host organism may not be able to balance the effects of the virus and the inflammatory responses by primed lymphocytes and regain homeostasis, leading to drastic pathological manifestations, as recently suggested by Mongkolsapaya et al. (2003)
.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Amieva, M. R., Vogelmann, R., Covacci, A., Tompkins, L. S., Nelson, W. J. & Falkow, S. (2003). Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA. Science 300, 14301434.
Anderson, J. M. (2001). Molecular structure of tight junctions and their role in epithelial transport. News Physiol Sci 16, 126130.
Anderson, R., Wang, S., Osiowy, C. & Issekutz, A. C. (1997). Activation of endothelial cells via antibody-enhanced dengue virus infection of peripheral blood monocytes. J Virol 71, 42264232.[Abstract]
Andrews, B. S., Theophilopoulos, A. N., Peters, C. J., Loskutoff, D. J., Brandt, W. E. & Dixon, F. J. (1978). Replication of dengue and junin viruses in culture rabbit and human endothelial cells. Infect Immun 20, 776781.[Medline]
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, 63386346.
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, H286294.[Medline]
Bonner, S. M. & O'Sullivan, M. A. (1998). Endothelial cell monolayers as a model system to investigate dengue shock syndrome. J Virol Methods 71, 159167.[CrossRef][Medline]
Bosch, I., Xhaja, K., Estevez, L., Raines, G., Melichar, H., Warke, R. V., Fournier, M. V., Ennis, F. A. & Rothman, A. L. (2002). Increase production of interleukin-8 in primary human monocytes and in human epithelial and endothelial cell lines after dengue virus challenge. J Virol 76, 55885597.
Carr, J. M., Hocking, H., Bunting, K. P., Wright, 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, 521528.[CrossRef][Medline]
Cereijido, M., Gonzalez-Mariscal, L. & Contreras, G. (1989). Tight junction: barrier between higher organisms and environment. News Physiol Sci 4, 7275.
Chambers, T. J., Hahn, C. S., Galler, R. & Rice, C. M. (1990). Flavivirus genome and organization, expression, and replication. Annu Rev Microbiol 44, 649688.[CrossRef][Medline]
Chaturvedi, U. C., Elbishbishi, E. A., Agarwal, R., Raghupathy, R., Nagar, R., Tandon, R., Pacsa, A. S., Younis, O. I. & Azizieh, F. (1999). Sequential production of cytokines by dengue virus-infected human peripheral blood leukocyte cultures. J Med Virol 59, 335340.[CrossRef][Medline]
Chen, Y., Maguire, T. & Marks, R. M. (1996). Demonstration of binding of dengue virus envelope protein to target cells. J Virol 70, 87658772.[Abstract]
Chen, B. H., Tzen, J. T., Bresnick, A. R. & Chen, H. C. (2002). Roles of Rho-associated kinase and myosin light chain kinase in morphological and migratory defects of focal adhesion kinase-null cells. J Biol Chem 277, 3385733863.
Collares-Buzato, C. B., Jepson, M. A., Simmons, N. L. & Hirst, B. H. (1998). Increased tyrosine phosphorylation causes redistribution of adherens junction and tight junction proteins and perturbs paracellular barrier function in MDCK epithelia. Eur J Cell Biol 76, 8592.[Medline]
Cudmore, S., Reckmann, I. & Way, M. (1997). Viral manipulations of the actin cytoskeleton. Trends Microbiol 5, 142148.[CrossRef][Medline]
Dejana, E., Lampugnani, M. J., Martinez-Estrada, O. & Bazzoni, G. (2000). The molecular organization of endothelial junctions and their functional role in vascular morphogenesis and permeability. Int J Dev Biol 44, 743748.[Medline]
Del Vecchio, P. J., Siflinger-Birnboim, A., Belloni, P. N., Holleran, L. A., Lum, H. & Malik, A. B. (1992). Culture and characterization of pulmonary microvascular endothelial cells. In Vitro Cell Dev Biol 28A, 711715.[Medline]
Evans, T. J. (2000). Bioassay for tumor necrosis factor-alpha and -beta. Mol Biotechnol 15, 243248.[CrossRef][Medline]
Falconar, A. K. I. & Young, P. R. (1991). Production of dimer-specific and dengue virus group cross-reactive mouse monoclonal antibodies to the dengue 2 virus non-structural glycoprotein NS1. J Gen Virol 72, 961965.[Abstract]
Feniger-Barish, R., Yron, I., Meshel, T., Matityahu, E. & Ben-Baruch, A. (2003). IL-8-induced migratory responses through CXCR1 and CXCR2: association with phosphorylation and cellular redistribution of focal adhesion kinase. Biochemistry 42, 28742876.[CrossRef][Medline]
Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita, S. A. & Tsukita, S. H. (1993). Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123, 17771788.[Abstract]
Huang, Y. H., Lei, H. Y., Liu, H. S., Lin, Y. S., Liu, C. C. & Yeh, T. M. (2000). Dengue virus infects human endothelial cells and induces IL-6 and IL-8 production. Am J Trop Med Hyg 63, 7175.
Imamura, H., Takaishi, K., Nakano, K., Kodama, A., Oishi, H., Shiozaki, H., Monden, M., Sasaki, T. & Takai, Y. (1998). Rho and Rab small G proteins coordinately reorganize stress fibers and focal adhesions in MDCK cells. Mol Biol Cell 9, 25612575.
Jacobs, M. & Levin, M. (2002). An improved endothelial barrier model to investigate dengue haemorrhagic fever. J Virol Methods 104, 173185.[CrossRef][Medline]
Jiang, W. G., Martin, T. A., Matsumoto, K., Nakamura, T. & Mansel, R. E. (1999). Hepatocyte growth factor/scatter factor decreases the expression of occludin and transendothelial resistence (TER) and increases paracellular permeability in human vascular endothelial cells. J Cell Physiol 181, 319329.[CrossRef][Medline]
Kielbassa, K., Schmitz, C. & Gerke, V. (1998). Disruption of endothelial microfilaments selectively reduces the transendothelial migration of monocytes. Exp Cell Res 243, 129141.[CrossRef][Medline]
Krishnamurti, C., Kalayanarooj, S., Cutting, M. A. & 9 other authors (2001). Mechanisms of hemorrhage in dengue without circulatory collapse. Am J Trop Med Hyg 65, 840847.
Lum, H. & Malik, A. B. (1996). Mechanisms of increased endothelial permeability. Can J Physiol Pharmacol 74, 787800.[CrossRef][Medline]
Madara, J. (1991). Relationships between the tight junctions and the cytoskeleton. In Tight Junctions, pp. 105119. Edited by M. Cereijido. Boca Raton, FL: CRC Press.
McCarthy, K. M., Skare, I. B., Stankewich, M. C., Furuse, M., Tsukita, S., Rogers, R. A., Lynch, R. D. & Schneeberger, E. E. (1996). Occludin is a functional component of the tight junction. J Cell Sci 109, 22872298.
Meza, I., Ibarra, G., Sabanero, M., Martínez-Palomo, A. & Cereijido, M. (1980). Occluding junctions and cytoskeletal components in a cultured transporting epithelium. J Cell Biol 87, 746754.
Mongkolsapaya, J., Dejnirattisai, W., Xu, X. N. & 11 other authors (2003). Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat Med 9, 921927.[CrossRef][Medline]
Obert, G., Peiffer, I. & Servin, A. L. (2000). Rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal Caco-2 cell monolayers. J Virol 74, 46454651.
Raghupathy, R., Chaturvedi, U. C., Al-Sayer, H. & 10 other authors (1998). Elevated levels of IL-8 in dengue hemorrhagic fever. J Med Virol 56, 280285.[CrossRef][Medline]
Ramos, C., Sánchez, G., Pando, R. H., Baquera, J., Hernández, D., Mota, J., Ramos, J., Flores, A. & Llausás, E. (1998). Dengue virus in the brain of a fatal case of hemorrhagic dengue fever. J Neurovirol 4, 465468.[Medline]
Rao, R. K., Basuroy, S., Rao, V. U., Karnaky, K. J., Jr & Gupta, A. (2002). Tyrosine phosphorylation and dissociation of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the cytoskeleton by oxidative stress. Biochem J 368, 471481.[CrossRef][Medline]
Rigau-Pérez, J. G., Clark, G. G., Gubler, D. J., Reiter, P., Sanders, E. J. & Vorndam, A. V. (1998). Dengue and dengue haemorrhagic fever. Lancet 352, 971977.[CrossRef][Medline]
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, 194204.[Medline]
Schraufstatter, I. U., Chung, J. & Burger, M. (2001). IL-8 activates endothelial cell CXCR1 and CXCR2 through Rho and Rac signaling pathways. Am J Physiol Lung Cell Mol Physiol 280, L1094L1103.
van Nieuw Amerongen, G. P., Draijer, R., Vermeer, M. A. & van Hinsberg, V. W. M. (1998). Transient and prolonged increase in endothelial permeability induced by histamine and thrombin. Circ Res 83, 11151123.
Xu, Y., Swerlick, R. A., Sepp, N., Bosse, D., Ades, E. W. & Lawley, T. J. (1994). Characterization of expression and modulation of cell adhesion molecules on an immortalized human dermal microvascular endothelial cell line (HMEC-1). J Invest Dermatol 102, 833837.[Abstract]
Received 13 September 2003;
accepted 19 January 2004.