Hindawi Publishing Corporation
Advances in Virology
Volume 2012, Article ID 840654, 8 pages
doi:10.1155/2012/840654
Review Article
Roles for Endothelial Cells in Dengue Virus Infection
Nadine A. Dalrymple and Erich R. Mackow
Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY 11794-5222, USA
Correspondence should be addressed to Erich R. Mackow, erich.mackow@stonybrook.edu
Received 20 January 2012; Accepted 19 July 2012
Academic Editor: Sujan Shresta
Copyright © 2012 N. A. Dalrymple and E. R. Mackow. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Dengue viruses cause two severe diseases that alter vascular fluid barrier functions, dengue hemorrhagic fever (DHF) and dengue
shock syndrome (DSS). The endothelium is the primary fluid barrier of the vasculature and ultimately the effects of dengue
virus infection that cause capillary leakage impact endothelial cell (EC) barrier functions. The ability of dengue virus to infect the
endothelium provides a direct means for dengue to alter capillary permeability, permit virus replication, and induce responses that
recruit immune cells to the endothelium. Recent studies focused on dengue virus infection of primary ECs have demonstrated that
ECs are efficiently infected, rapidly produce viral progeny, and elicit immune enhancing cytokine responses that may contribute to
pathogenesis. Furthermore, infected ECs have also been implicated in enhancing viremia and immunopathogenesis within murine
dengue disease models. Thus dengue-infected ECs have the potential to directly contribute to immune enhancement, capillary
permeability, viremia, and immune targeting of the endothelium. These effects implicate responses of the infected endothelium
in dengue pathogenesis and rationalize therapeutic targeting of the endothelium and EC responses as a means of reducing the
severity of dengue virus disease.
1. Introduction
Dengue viruses are transmitted by mosquitoes and infect
∼50 million people annually with an additional 2.5 billion
people at risk living in tropical areas [1–3]. Expanding
mosquito habitats are increasing the range of dengue virus
outbreaks and the occurrence of severe diseases with 5–30%
mortality rates: dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [1–3]. The majority of patients
are asymptomatic or display mild symptoms of dengue
fever (DF) which include rapid onset of fever, viremia,
headache, pain, and rash [4]. Patients with DHF and DSS
display symptoms of DF in addition to increased edema,
hemorrhage, thrombocytopenia, and shock [1–3]. Although
patient progression to DHF and DSS is not fully understood
[3, 5], antibody-dependent enhancement (ADE) of dengue
infection increases the potential for DSS and DHF [3, 6,
7]. There are four dengue virus serotypes (types 1–4) and
infection by one serotype predisposes individuals to more
severe disease following a subsequent infection by a different
dengue serotype. The circulation of serotype-specific crossreactive antibodies or preexisting maternal antibodies may
contribute to progression to DHF/DSS by facilitating viral
infection of immune cells and eliciting cytokine and chemotactic immune responses. In a murine antibody dependent
enhancement model of dengue disease it was observed that a
dramatic increase in infected hepatic endothelial cells (ECs)
coincides with the onset of severe disease [8] and suggests
a role for the endothelium in an immune-enhanced disease
process during dengue infection.
The major target tissues for dengue virus infection have
been difficult to determine but virus has been isolated from
human blood, lymph node, bone marrow, liver, heart, and
spleen [9–14]. Blood samples are more easily obtained from
dengue patients than tissues and yield a wide array of
information about cytokine responses elicited by dengue
virus infection [1–3, 14–18]. While many of these cytokines
are present in DF patients, the majority of them are increased
during DHF. Overall, DHF responses include greater cytokine production, T- and B-cell activation, complement
activation, and T-cell apoptosis [3]. Complement pathway
activation and elevated levels of complement proteins C3,
C3a, and C5a are significant in that they can direct opsonization, chemotaxis of mast and other immune cells, and direct
2
the localized release of the vascular permeability factor histamine from mast cells [17, 19–23]. Importantly, cytokines
and complement factor responses all act on the endothelium
and alter normal fluid barrier functions of ECs.
The ability of dengue virus to infect immune, dendritic,
and endothelial cells fosters a role for immune responses to
act on the endothelium and increase capillary permeability
[5, 24–29]. However, the redundant nature of capillary
barrier functions suggests that permeability is likely to
be multifactorial in nature with many factors working in
concert to modulate EC responses and permeabilize the
endothelium. Dengue infected ECs are observed in DHF/DSS
patient autopsy samples and in murine dengue virus disease
models [8, 9, 14, 30]. This suggests that dengue infected ECs
may also contribute directly to pathogenesis by increasing
viremia, secreting cytokines, modulating complement pathways, or transforming the endothelium into an immunologic
target of cellular and humoral immune responses.
Plasma constituents contain factors secreted by an estimated ∼1013 ECs present in the body, and autopsy samples
and murine dengue disease models clearly demonstrate that
vascular ECs are infected [8, 9, 30, 31]. The endothelium
is the primary fluid barrier of the vasculature and dengue
virus-induced responses resulting in edema or hemorrhagic
disease ultimately cause changes in EC permeability. Unique
EC receptors, adherens junctions, and signaling pathways
respond to cytokines, permeability factors, immune complexes, clotting factors, and platelets, normally acting in concert to control vascular leakage [5, 32–36]. Virally induced
changes in endothelial or immune cell responses have the
potential to alter this orchestrated balance with pathologic
consequences [5, 32–35]. However, very little is known about
the role of dengue virus-infected ECs in disease or the
kinetics, timing, and replication of dengue viruses within
patient ECs. The inability to kinetically study the endothelium in dengue patients and the relative ease of assessing
blood components has resulted in a focus on immune cells
instead of ECs. Yet, the endothelium is the ultimate target of
permeabilizing responses. Here, we discuss studies of dengue
infected ECs and the potential for the dengue infected
endothelium to contribute to dengue pathogenesis.
2. Human Responses to Dengue Virus Infection
DHF and DSS are severe manifestations of dengue virus
infection that result in increased vascular permeability,
hemorrhage, and shock [3]. The presence of preexisting
antibodies to dengue virus predisposes patients to severe
disease following infection by a second dengue serotype
[3]. A myriad of responses are associated with dengue
infection that may contribute to disease, but the pathogenic
mechanisms that result in DHF and DSS remain ambiguous
[1–3, 26]. One common element of the dengue disease
process is that enhanced immune responses increase vascular
permeability by acting on the endothelium. Although it
is clear that immune cells and their responses contribute
to pathogenesis, the endothelium, which regulates vascular
leakage, has not been considered a significant component of
DHF and DSS [2, 5, 34, 35, 37].
Advances in Virology
2.1. Patient Studies. The ability of dengue virus to infect
human ECs has been documented in autopsy samples of
the heart, liver, and lung [9, 14]. In a postmortem study,
Jessie et al. reported dengue virus antigen in sinusoidal ECs
in the liver as well as macrophages, lymphoid cells in the
spleen, the vascular endothelium of the lung, monocytes
within the blood, and kidney tubules [9]. Salgado et al. also
demonstrated the presence of viral antigen in endothelial
cells within the heart and small myocardial vessels of a
patient postmortem [14]. Depressed myocardial function has
been associated with hemorrhagic forms of dengue virus
infection [38]. Although no dengue virus RNA was detected
in these cells by in situ hybridization, viral antigen uptake was
also not confirmed. Additionally, no morphological damage
to the endothelium was observed that might explain vascular
leakage through disruption of the endothelium. However, the
presence of circulating ECs, EC markers (VCAM and ICAM),
and increased von Willebrand factor antigen and procoagulants, specifically in DHF patients, has been reported in
other cases [39, 40]. Nevertheless, autopsy samples do not
take into account contributions of dengue virus infection of
ECs at earlier time points that may contribute to viremia
and immune enhancing responses. Kinetic analysis of the
endothelium in patients is invasive and has not been
addressed. In general, little clinical data has been obtained
between viral inoculation and onset of fever and viremia
[3]. Thus, findings that ECs are infected with dengue have
been marginalized since immune enhancing responses are
presumed to be derived solely from immune cells. However,
a variety of mechanisms exist for ECs to elicit immune
enhancing cytokine and complement responses that recruit
immune cells to the endothelium or directly alter barrier
functions of EC adherens junctions [3–5, 41]. Finding that
ECs are infected in patients suggests a direct means by which
the infected endothelium may be altered by dengue virus.
Additionally, preexisting antibodies may target DV antigen within infected ECs, further contributing to immuneenhanced permeability deficits observed in DHF and DSS.
2.2. Patient Responses to Infection and Markers of DHF/DSS.
A hallmark of severe dengue disease is the presence of
elevated levels of cytokines and chemokines including IP10, ITAC, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, IL-13, TNFα,
IFNα, IFNγ, MIF, RANTES, histamine, and complement
proteins C3, C3a, and C5a within blood and tissues [1–3, 14–
18]. In patients, complement activation and an increase in
complement protein products correlate with the severity of
disease [42–45]. In a study by Avirutnan et al., C5b-9 complexes, complement-activated membrane-attack complexes,
and C3a were formed on dengue-infected ECs in the presence
of antibody—containing immune serum, though they did
not direct complement-mediated cell lysis [26]. C3a is an
anaphylatoxin that recruits mast cells and directs histamine
release that locally increases vascular permeability [17, 19–
22]. Elevated C3a, C5a, and histamine have been associated
with severe permeability deficits in dengue virus patients
and in the development of DHF and DSS [2, 3, 17, 26, 42].
Their presence in the blood of patients with severe dengue
disease is significant since these anaphylatoxins direct lysis
Advances in Virology
of infected cells and mast cell degranulation, leading to
histamine release.
Importantly, cytokines, chemokines, and complementactivating factors can all be secreted by and act on the
endothelium, influencing EC regulation of fluid barrier
function and vascular leakage [5, 32–35]. The ability of
dengue virus to infect the endothelium intimates that additional mechanisms could contribute to vascular permeability
deficits through both direct- and immune-enhanced disease
processes. Dengue infected ECs may elicit chemokine and
cytokine responses that further activate or recruit immune
cells to the endothelium and preexisting dengue antibodies
may target viral proteins displayed on infected endothelium.
Recent analysis of primary EC transcriptional responses indicates that dengue virus strongly induces secretion of immune
cell activating cytokines, chemokines, and complement
factors that are likely to contribute to an immune enhanced
disease process [46]. Since permeability is ultimately the
result of responses that act on the endothelium, dengue
infected ECs are key elements in DSS and DHF that must
be considered more fully within animal and in vitro models.
2.3. Roles of NS1 and Cross-Reactive NS1 Antibody. Dengue
proteins may also play critical roles in enhancing DV
pathogenesis within ECs through a variety of mechanisms.
In particular, the NS1 protein is uniquely expressed in three
forms: cytosolic, cell-surfaced expressed, and secreted [47–
49]. Soluble secreted NS1 is both highly abundant and
highly antigenic [50]. Likewise, NS1 antibodies are also
present in high quantities and have been shown to bind the
surface of platelets and ECs [51]. Because ECs are in steady
contact with blood, they are susceptible to enhanced immune
cell targeting promoted by adherent cross-reactive NS1
antibodies [51–53]. This targeting, as well as intracellular
signaling triggered by direct NS1 antibody binding, may
contribute to tissue-specific endothelial dysfunction and
vascular leakage through EC activation [5, 51–53]. However,
despite the abundance of NS1 antibodies that could promote
leakage, vascular permeability is transient and additional
contributions of NS1 have been poorly explored within
humans and mouse models. Secreted NS1 can also bind
cellular heparan sulfate E present on primary liver and lung
ECs [54] and, along with the cell surface form of NS1, may
further recruit circulating antibodies and immune factors to
dengue infected ECs [55, 56].
As a secreted protein, the dengue NS1 protein modulates
classical complement activation by binding to the C4b binding protein, thereby inactivating C4b [57]. Thus, cell-surface
expressed NS1 on ECs could serve as a platform for C4b
inactivation and antagonize classical complement activation
pathways. Secreted NS1 may similarly attenuate complement
activation by binding C1s/proC1s and C4 in a complex
that reduces C4 levels required for complement pathway
activation [58]. Together, NS1 and NS1 antibodies form a
potent combination within DHF and DSS patients capable
of eliciting or regulating immune and complement responses
that act on the endothelium and contribute to dengue pathogenesis [59]. Curiously, the alternative complement pathway
activator complement factor B, transcriptionally induced in
3
dengue-infected endothelial cells [46], may induce C3a- and
C5a-directed chemotaxis and histamine release by bypassing
NS1 complement regulation. Antibody targeting of factor D,
which activates factor B through cleavage, inhibits complement and leukocyte activation in nonhuman primates and
several therapeutics have been developed that antagonize C3a
and C5a receptor binding [60–63]. These advances suggest
that the alternative pathway may be a new potential target
for therapeutically reducing the severity of DHF and DSS
diseases. Additional barrier stabilizing effectors that target
the endothelium may also be considered as a means of
therapeutically reducing vascular leakage and inflammation
that contribute to dengue pathogenesis [64, 65].
3. In Vivo Animal Models of
Dengue Virus Infection
3.1. Mouse Models of Dengue Virus Infection. Progress in
understanding dengue pathogenesis has been hampered by
the lack of suitable mouse models that replicate human
cellular tropism and disease symptoms. In normal mice,
dengue infection results in limb paralysis and little mortality
[31]. Recently mouse-adapted dengue strains that mimic
aspects of severe human disease in interferon (IFN) receptor
knockout AG129 mice have been used as a dengue virus
animal model [8, 31, 66–68]. Organ damage, hemorrhage,
vascular leakage, viremia, and elevated cytokine levels analogous to that in humans are observed following dengue
infection of AG129 mice [66, 67]. In one study, high titer
inoculation of mice initiated TNFα-induced apoptosis of
ECs, leading to vascular leakage [69]. However, IFN defective
murine models further complicate our understanding of
the dengue disease process since they do not fully mimic
human responses to infection and lack IFN responses that
limit dengue spread and induce EC proliferation and repair.
Despite these limitations, current models have provided new
insight into dengue virus pathogenesis and allow for kinetic
studies of dengue virus infection of ECs.
Vascular leakage occurs in AG129 mice infected with
mouse-adapted dengue strains and several studies have
isolated murine-infected ECs within the spleen and liver
[8, 30]. In support of a role for infected ECs in mediating
severe dengue disease, Zellweger et al. recently reported that
in the presence of subneutralizing levels of dengue-specific
antibodies (ADE-mediated infection), a large percentage of
infected liver sinusoidal ECs (LSECs) were detected and
correlated directly with disease severity [8]. No evidence for
ADE-mediated infection of ECs exists in vitro [70], although
liver sinusoidal cells reportedly express Fcγ receptors that
may contribute to immune enhanced infection of liver ECs
[71]. In the same study, infection of mucosal macrophages
was not enhanced by the presence of dengue antibodies and
occurred after detection of infected LSECs, suggesting that
increased viral loads from LSEC infection, but not ADE,
enhanced immune cell infection [8]. Although a mechanism
for this occurrence has yet to be determined, these findings
give importance to the role of ECs in mediating dengue
pathogenesis in the mouse animal model. Therefore, in
addition to increasing viremia, the ability of dengue virus
4
to infect ECs in vivo may provide a means for infection to
alter capillary permeability and induce cytokine responses
from ECs that recruit immune cells and contribute to dengue
pathogenesis.
3.2. Mouse Model Responses Influenced by IFN. Since IFN
plays a significant role in the regulation of viral spread and
the growth and repair of the endothelium [3, 72–76], it is
important to consider the consequences of dengue infections
occurring in IFN unresponsive mouse models. Since IFN
reportedly stimulates EC proliferation [76], IFN secretion by
dengue infected cells is also likely to contribute to vascular
repair following dengue infection, and the absence of the
IFN-signaling response may explain the enhanced pathogenesis of dengue infections in IFN receptor knockout AG129
mice [31, 67, 77]. This absence abrogates antiviral responses
that may naturally curb infection [72, 74]. Likewise, the
dengue NS5 protein, which interferes with downstream
IFN signaling to permit virus replication through STAT2
degradation, is unable to bind mouse STAT2 [78, 79]. These
differences cloud interpretation of results from dengueinfected mice and may in fact contribute significantly to
hemorrhagic responses that may or may not reflect normal
pathogenic mechanisms. Current work is ongoing to address
the lack of IFN responses within mice and create knock-in
mice, which harbor functional human STAT2 [79].
3.3. Nonhuman Primate Models. Limited studies have also
been conducted on nonhuman primates (NHPs) as an animal model. However, NHPs display almost no human symptoms of DF/DSS/DHF despite detectable virus replication
[80]. Gene profiling following infection in NHPs revealed a
potent antiviral response yet, in contrast to humans, almost
no production of type I or II interferons or inflammatory
cytokines [81]. Thus, murine models still appear to be the
most suitable animal model for studying dengue infection,
specifically in relation to cellular tropism and EC responses.
Further work continues to explore new mouse adaptations
that may one day produce animal models that fully mimic
human responses to dengue infection.
4. In Vitro Infection of Endothelial Cells
4.1. Use of Endothelial Cell Lines for Studying Dengue Virus
Infection. The difficulty of analyzing infections of the endothelium in vivo has driven the in vitro exploration of
dengue infection of ECs. In vitro, ECs from various sources
are permissive for dengue virus infection and have been
used to study pathophysiological changes occurring within
the endothelium following dengue virus infection. However,
not all EC lines are equivalent and this has led to confusing and often contradictory results [82, 83]. Cell lines
derived from endothelial and epithelial cell fusions are not
representative of primary ECs and the ECV304 endothelial
cell line has been shown to be bladder carcinoma and not
endothelial in nature [84]. However, early passage primary
human ECs permit investigation of dengue virus infection
that approximates the human endothelium for analysis of
dengue-altered EC responses.
Advances in Virology
4.2. Replication and Receptors for Dengue Virus on ECs. Dengue virus replicates within HUVECs (human umbilical vein
ECs), LSECs, HPMEC-ST1.6R cells (human pulmonary EC
line), ECV304 (endothelial cell line), and HMEC-1 cells
(human microvascular EC line) [26, 27, 70, 85–89]. Recent in
vitro studies demonstrated that efficient infection of primary
ECs by dengue virus occurs as a result of attachment to
heparan sulfate-containing cell surface proteins (HSPGs)
[88]. HSPGs, specifically heparan sulfate glycoproteins of
syndecan 2, also mediate attachment of dengue virus to
K562 monocytes [90]. Although more specific HSPGs on
ECs still need to be defined, an EC receptor blockade has
the potential to reduce viremia, immune targeting of dengue
virus infected ECs, and dengue virus-induced changes in ECs
that contribute to pathogenesis.
4.3. Responses Elicited by Dengue-Infected ECs In Vitro. Several studies have focused on changes in the levels of cellular
molecules or markers of EC activation, including VCAM
and ICAM [91, 92]. Dengue virus infection upregulates
cell surface markers of EC activation which can trigger the
expression and release of various cytokines, chemokines, and
complement factors that act on neighboring tissues, ECs,
and circulating immune cells. Analysis of dengue-induced
permeability responses suggested that EC permeability was
increased in vitro [93]. Although a productive infection was
not verified in this study, permeability occurred in conjunction with a decrease in VE-cadherin, which regulates the fluid
barrier function of adherens junctions [93, 94].
Additional studies examined the induction or secretion
of cytokines following dengue virus infection of primary
HUVECs and ECV304, LSEC-1, HMEC-1, or HPMECST1.6R cell lines [26, 27, 37, 85, 87, 89, 95]. Dengue infection
of the HPMEC-ST1.6R cell line increased IL-6 and IL-8
(6–8 days p.i.) as well as vascular endothelial cell growth
factor (VEGF) [27, 95]. Other studies have also singled
out RANTES, IL-6 and/or IL-8 as cytokines elicited by
dengue-infected ECs [26, 37, 46, 87], thus promoting the
endothelium as a source of potent chemotactic cytokines in
DSS/DHF patients. Both IL-8 and RANTES are chemotactic
agents that can increase vascular permeability by localized
attraction of neutrophils [96, 97]. Analyses of transcriptional
changes elicited in response to dengue infection also show
small increases in additional cytokines and antiviral IFNstimulated genes [27, 37, 46]. IFN is highly induced in
dengue patients and these findings suggest that ECs are likely
to contribute to circulating IFN responses.
4.4. Kinetics of Dengue Virus Infection In Vitro. Dengue infected endothelial cell lines reportedly induce a low level of
infection (<10%) and coordinately low-level transcriptional
responses [26, 85]. However, analysis of >90% uninfected
cells at late time points after infection (3–8 days) makes it
difficult to accurately assess the contribution from dengue
infected cells. This is compounded by innate antiviral
responses elicited by infecting a small number of ECs and
the stimulation of IFN responses by >90% uninfected ECs.
A recent kinetic analysis of primary EC responses to
dengue virus infection paints an important picture of the
Advances in Virology
endothelium’s role in dengue disease. Primary EC monolayers are ∼80% infected by dengue virus and rapidly produce
virus by 24 hours after infection [88]. Both the production
of progeny virus and the number of infected ECs within
monolayers decrease 2-3 days after infection [88]. Dengue
virus titers following EC infection were nearly identical to
viral titers observed in IFN-deficient VeroE6 cells [88], suggesting that virus regulates early cellular interferon responses
of ECs. In fact, the lack of viral spread reported in some
studies, where fewer than 10% of ECs were infected [37, 70],
is consistent with the paracrine effect of interferon produced
by a small number of initially infected cells. Thus, in vitro
studies point to the infected endothelium as a likely source of
viremia following dengue virus infection. These findings also
highlight the importance of assessing the impact of EC infections at early times following in vivo infection, something
more easily achieved within mouse models than patients.
Recovering DHF patients regain normal endothelial
function quickly, implying a transient effect and early recovery of EC functions following infection [3]. A rapid productive infection of the endothelium is difficult to assess in
humans, but in vitro studies show that infected ECs rapidly
release virus. This suggests that dengue infected ECs may
contribute to early viremia in dengue patients and present
dengue virus antigens on EC surfaces that may be targeted by
immune cells [88]. Infected ECs may also elicit cytokine and
chemokine responses that can act directly on the endothelium [26, 27, 37, 85, 87, 89, 95]. Endpoint sampling of ECs at
autopsy or analysis of endothelial function within recovering
patients does not take into consideration an early or transient
infection of ECs as is observed in vitro. The lack of apparent
spread within EC monolayers and the apparent decrease in
infected cells 2-3 days after infection [46] also suggest that
EC-elicited IFN responses may limit dengue virus spread
in vitro and contribute to high levels of circulating IFN
in dengue patients. Interestingly, IFN treatment directs EC
proliferation and may be a response that both limits viral
spread and activates the EC repair process [76]. In fact, EC
proliferation in response to IFN may explain the absence of
endothelial damage within DHF patients and the presence of
vascular leakage in IFN receptor deficient mouse models.
5. Conclusions
The endothelium is not a static channel that simply separates the vasculature from surrounding tissue [33–35]. The
endothelium dynamically elicits responses that may contribute to immune enhancement and vascular permeability
during dengue virus infection. Several hypotheses have been
offered to explain the development of severe dengue disease
and immune enhanced responses clearly impact barrier
functions of the endothelium, but pathogenic mechanisms
that result in DHF and DSS remain vague [1–3, 26]. Leakage
of the vascular endothelium is a central component of
dengue virus disease and studies discussed here suggest
that the dengue infected endothelium may contribute to
pathogenic immune responses and immune targeting of the
endothelium. However, the role of dengue virus infected
ECs in pathogenesis requires definitive in vivo kinetic studies
5
which are difficult to perform in patients. The ability of
the endothelium to respond to immune cells resulting in
capillary permeability further highlights the importance of
dengue infected ECs in pathogenesis. The central importance
of the endothelium in dengue disease suggests that stabilizing
fluid barrier functions of the endothelium may be a therapeutic approach for reducing vascular leakage in DHF and
DSS patients [64, 65].
Acknowledgments
The authors thank Irina Gavrilovskaya, Valery Matthys,
and Elena Gorbunova for critical paper review and helpful discussions. This work is supported by NIH, NIAID
Grants R01AI47873, PO1AI055621, R21AI1080984, and
U54AI57158 (NBC-Lipkin).
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