Role of p38 MAPK - Springer Link

C 2006) Journal of Clinical Immunology, Vol. 26, No. 4, July 2006 ( DOI: 10.1007/s10875-006-9024-4

Bluetongue Virus and Double-Stranded RNA Increase Human Vascular Permeability: Role of p38 MAPK EDDIE T. CHIANG,2 DIXIE-ANN PERSAUD-SAWIN,1 SANDHYA KULKARNI,1 JOE G. N. GARCIA,2 and FARHAD IMANI1,3

INTRODUCTION

Received January 23, 2006; accepted April 10, 2006 Published online: 20 June 2006

Viral-induced hemorrhagic fevers are associated with high morbidity and mortality and thus far, there are no approved treatments for these diseases other than a vaccine for yellow fever (1). Substantial evidence suggests that the sequaelae associated with viral hemorrhagic diseases are not only the consequence of direct viral damage to the cells but rather the result of a dysregulated inflammatory processes. Recent data from Hensley et al. showed that the TNF receptor superfamily is involved in Ebolaassociated pathologies observed in experimental monkeys (2). They also showed that infection of phagocytes with Ebola virus resulted in an increase in the expression of IFN-α, IFN-β, IL-6, IL-18, MIP-1α, and TNF-α (2). Furthermore, a study by Baize et al. showed that survivors of Ebola virus infections had an orderly and transient release of inflammatory cytokines early during the infections, which can control viral spread. In contrast, they reported that fatal infections were associated with low IL1β, low immunoglobulin titers, activation of monocytes and macrophages and with high levels of neopterin, IL-10 and circulating TNF-α(3). Bluetongue virus (BTV) is an endemic arthropod-borne infection of ruminants that causes considerable economic damage (4, 5). There are 25 different serotypes of BTV worldwide, which belongs to the orbivirus genus containing segmented dsRNA (10 segments) as the genome. BTV infection causes vasculitis, edema and eventually necrosis of epithelial and mucosal surfaces (5, 6, 7). As a model for virus-induced vascular permeability we have used BTV infection of human endothelial cells. A common viral structure for the induction of inflammatory responses is dsRNA (8). DsRNA can be present as the genomic structure as in the case for BTV and rotaviruses, or can form intracellularly as a replicative intermediate during the life cycle of most viruses (8). The

Endothelial cell (EC) involvement in viral hemorrhagic fevers has been clearly established. However, virally activated mechanisms leading to endothelial activation and dysfunction are not well understood. Several different potential mechanisms such as direct viral infection, alterations in procoagulant/anticoagulant balance, and increased cytokine production have been suggested. We utilized a model of EC barrier dysfunction and vascular endothelial leakage to explore the effect of bluetongue virus (BTV), a hemorrhagic fever virus of ruminants, on human lung endothelial cell barrier properties. Infection of human lung EC with BTV induced a significant and dose-dependent decrease in transendothelial electrical resistance (TER). Furthermore, decreases in TER occurred in conjunction with cytoskeletal rearrangement, suggesting a direct mechanism for viral infection-mediated endothelial barrier disruption. Interestingly, double-stranded RNA (dsRNA) mimicked the effects of BTV on endothelial barrier properties. Both BTV- and dsRNA-induced endothelial barrier dysfunction was blocked by treatment with a pharmacological inhibitor of p38 MAPK. The induction of vascular permeability by dsRNA treatment or BTV infection was concomitent with induction of inflammatory cytokines. Taken together, our data suggest that the presence of dsRNA during viral infections and subsequent activation of p38 MAPK is a potential molecular pathway for viral induction of hemorrhagic fevers. Collectively, our data suggest that inhibition of p38 MAPK may be a possible therapeutic approach to alter viral-induced acute hemorrhagic diseases. KEY WORDS: hemorrhagic fever; vascular permeability; doublestranded RNA (dsRNA); bluetongue virus; MAP kinase.

1 NIEHS/NIH,

Laboratory of Respiratory Biology, Durham, North Carolina 27709. 2 University of Chicago, Pritzker School of Medicine, Chicago, Illinois 60637. 3 To whom correspondence should be addressed to NIEHS/NIH MD 2-01, 111 Alexander Dr., RTP, North Carolina 27709; e-mail: [email protected].

406 C 2006 Springer Science+Business Media, Inc. 0271-9142/06/0700-0406/0

ROLE OF P38 MAPK

presence of intracellular dsRNA has been directly shown during infection with single-stranded RNA viruses such as dengue virus, influenza virus, rhinovirus and rubella virus (9–12). In the case of Ebola virus, there is indirect evidence for the presence of dsRNA in the infected cells. A report by Basler et al. showed that the Ebola virusencoded polypeptide VP35 was a dsRNA binding protein that is an antagonist of antiviral interferon (13), suggesting that dsRNA may form during Ebola infections. Central to vascular barrier regulation are signaling pathways, which alter the endothelial cytoskeleton, a dynamic cellular array of proteins responsive to cytosolic calcium, tyrosine kinases and MAP kinases. A variety of stimuli including lipopolysaccharide, osmotic shock, growth factors and viral infections can activate the MAPK pathways (14–17). These signaling pathways are key regulators of inflammatory responses (18–22). Moreover, we recently reported that viral and dsRNA induction of TNF-α and IL-1β follow a p38 MAPK-dependent pathway (23). Importantly, several reports, including our own, have clearly established a link between MAPKs and vascular barrier regulation (24–30). The inter-related MAPK pathways are exemplified by the p38 MAPK, ERK and JNK pathways and can be activated by viral infections and dsRNA treatment in different cells (19, 21, 22, 31, 32). Upon cell stimulation, p38 MAPK activates several transcription factors such as signal transducer and activator of transcription-1 (STAT-1) and ATF-2, which are both involved in induction of inflammation (33, 34, 35). In this study, we have examined the inflammatory effects of viral infection by focusing on bluetongue virus (BTV) effects on vascular integrity utilizing an in vitro model for vascular permeability. Our data, using primary human lung micro-vascular endothelial cells (HLMV), show that infection of these cells with BTV or treatment with dsRNA induces enhanced responses in vascular permeability, as assessed by a decrease in trans-endothelial electrical resistance (TER). Alteration in TER is delayed in onset and occurs concomitantly with the expression of TNF-α, IL-1β, IL-6 and IFN-β. We further show that pharmacologic inhibition of p38 MAPK reduces the viral induction of cytokines and vascular permeability.

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hibitor SB239063 was a generous gift from Dr. David C. Underwood (GlaxoSmithKline, King of Prussia, PA). DsRNA [high molecular weight 104 –106 base paired polyinosinic:polycytidylic acid (poly I:C)] was purchased from Sigma Chemical Company (St. Louis, MO) and was dissolved in tissue culture phosphate-buffered saline at 2 mg/ml stock concentration. All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Electrical Resistance Measurements Lung endothelial barrier properties were measured using an electrical cell-substrate impedance sensing (ECIS Model 1600R) system (Applied Biophysics, Troy, NY) as we have previously reported (24, 27, 36, 37, 38, 39). Cells are plated on gold-evaporated electrodes (10E8W-10 electrodes) at a density of 100,000 cells/well and grown to confluence. Trans-endothelial electrical resistance (TER) across the monolayer is measured over time to determine endothelial barrier function (40). Changes in electrical resistance represent alterations in cell-cell adhesion and thus changes in paracellular permeability. RT-PCR and ELISA

MATERIALS AND METHODS

For reverse transcription-polymerase chain reaction (RT-PCR), total cellular RNA was isolated by TRIzol RNA isolation system (Invitrogen, Bethesda, MD). One microgram of RNA was used for reverse transcription using SuperScript reverse transcriptase (Invitrogen, Bethesda, MD) for 1 h at 42◦ C. The specific primers were as follows; IL-1β-F-5 -aaa cag atg aag tgc tcc ttc agg-3 , IL-1β-R-5 -tgg aga aca cca ctt gtt gct cca-3 , TNF-F-5 cag agg gaa gag ttc ccc ag-3 , TNF-R-5 -cct tgg tct ggt agg aga cg-3 , IL-6-F- 5 -gcc aga gct gtg cag atg ag-3 , IL-6-R5 -agg aac tcc tta aag ctg cg-3 , IFN-β − F-5 -cac gac agc tct ttc cat ga-3 , IFN-β − R-5 -agc cag tgc tcg atg aat ct-3 , BTV-VP6-F-5 -gtt gag aga gga gga cgc aag, BTV-VP6R-5 -gtt ggc gct gtg aaa tat gcc g. Hybridization temperature, cycle number and magnesium concenteration were optimized for each primer set. ELISAs were performed using commercially available ELISA kits (eBioscience, and R&D) according to manufacturer’s instructions.

Endothelial Cell Culture and Reagents

Western Blot Analysis

Primary human micro-vascular endothelial cells were purchased from Cambrex Bioscience (Walkersville, MD). The cells were maintained in EGM-2 media with supplements (Cambrex Bioscience, Walkersville, MD) at 37◦ C in a 5% CO2 humidified chamber. The p38 MAPK in-

After each treatment, cells were washed 1X in phosphate -buffered saline and equal numbers of cells were lysed using 1X SDS-sample buffer containing 2.5% βmercaptoethanol. The proteins were then denatured and reduced by heating the samples at 95◦ C for 5 min. The

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chromosomal DNA was sheared by passing the samples through a 26-gauge needle. The proteins were resolved on a 12% SDS-PAGE and are electrotransferred onto nitrocellulose membranes. Polyclonal rabbit antip38 MAPK and anti-phospho-p38 MAPK (Cell Signaling, Worchester, MA) were used according to the manufacturer’s instructions. The immunoblotted proteins were visualized using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham, Arlington Heights, IL). For stripping, the blots were placed in a buffer containing 1% SDS, 62.5 mM Tris-HCl (pH 6.8), and 100 mM DTT. The blots were then heated to 65◦ C for 15 min. The striping buffer was then removed and the blots were washed extensively prior to re-probing with anti-p38 antibody. Actin and VE-Cadherin Visualization After treatment, staining for VE-cadherin was performed by fixing the cells with 3.7% formaldehyde and then permeabilizing with 0.25% Triton X-100. The permeabilized cells were then incubated for 1 h with antiVE-cadherin Ab, followed by three washes with PBSTween (0.1%) and incubation for 1 h at room temperature with an appropriate secondary fluorescent dye-conjugated Ab. After three washes with PBS/Tween, the coverslips were mounted and analyzed using Nikon video imaging system (Nikon Inc.). For actin staining, after permeabilization, cells were treated with Texas Red phalloidin or Rhodamine phalloidin (Molecular probes, Eugene, OR). BTV Propagation The serotype 10 of BTV was purchased from American type culture collection (Rockville, MD). For viral propagation, primary bovine pulmonary endothelial cells were infected with BTV at MOI of 0.5 pfu/cell. After evidence of cytopathic effects, cells were harvested in fresh medium and then lysed by repeated freeze/thaw procedure (3X). The cell debris was then removed by centrifugation 10,000 × g for 5 min. For titration, the BTV stock was serially diluted and a standard plaque assay was performed.

RESULTS

BTV Infection of Human Lung Microvascular Endothelium Decreases Trans-Endothelial Electrical Resistance (Ter) Endothelial cells play a key role in vascular permeability and as previously reported, during infections with hem-

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orrhagic fever viruses, including BTV, endothelial cells become infected (6, 7, 41, 42, 43). To establish a model for vascular permeability, we have used BTV infection of primary human lung micro-vascular endothelial monolayers. As a measure of vascular permeability, we examined the real-time changes in trans-endothelial electrical resistance across the endothelial monolayer. In this model, the confluent endothelial cell monolayers are either mock treated or are infected with BTV at a multiplicity of infection (MOI) of 5 pfu/cell and TER is continuously measured. Figure 1A demonstrates that after a lag time of 2 h during which the electrical resistance did not change, BTV infection induced significant decreases in TER. This decrease was sustained for several hours post viral infection and it is attributed to a reduction in intracellular adhesion. Since BTV is pathogenic to ruminant animals and infection of human endothelial cells has not yet been established, we next confirmed whether BTV could enter and replicate in human endothelial cells. Cells were infected with BTV at MOI of 5 pfu/cell and after indicated times, cells were harvested and total cellular RNA was extracted and subjected to RT-PCR. To detect BTV infection of human endothelial cells, we used specific primers to BTV VP6 gene which encodes a structural polypeptide. Figure 1B depicts accumulation of mRNA for BTV VP6 gene in the infected endothelium in a time-dependent fashion, suggesting that BTV enters and transcribes in these cells. DsRNA Mimics BTV-Induced Reduction in TER There are three interrelated facts that suggest a role for dsRNA in viral-induced vascular permeability. First, dsRNA is a common structure that is present in virus infected cells as a genomic fragment as the case for BTV, or as replicative intermediate as has been shown in cells infected with RNA viruses such as dengue virus, influenza virus, rhinovirus and rubella virus (9–12). Second, dsRNA is a potent inducer of inflammatory cytokines in many cell types (44–51). Lastly, inflammatory cytokines such as TNF-α, IL-1β and IL-6 are known inducers of vascular permeability (52–56). Therefore, we have determined whether dsRNA could mimic the BTV-induced reduction in TER. To first examine the effect of dsRNA on barrier disruption, confluent monolayers of HLMV endothelial cells were treated with increasing concentration of poly I:C. Figure 2 demonstrates that similar to BTV infection, dsRNA treatment induced vascular permeability as measured by a reduction in TER. It is important to note that at late times post dsRNA treatment, TER measurements returned to baseline levels. The return of TER to baseline


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Fig. 1 BTV infection causes a reduction in transendothelial resistance (TER). Panel A. Human lung microvascular (HLMV) endothelial cells were grown on tissue culture dishes containing gold electrodes as a means to measure the electrical cell substrate impedance across the cells. After the cells reached confluency and the basal electrical resistance was stabilized, the cells were incubated with BTV virus (serotype 10) at MOI of 5 pfu/cell. The infected cells were then allowed to incubate at 37◦ C and the electrical resistance was continuously measured (n = 3). Panel B. To determine the entry and replication of BTV in endothelial cells, HLMV endothelial cells were grown as a monolayer and were infected with BTV at MOI of 1 pfu/cell. The cells were then harvested at the indicated times post infection and total cellular RNA was then extracted. We then performed RT-PCR by using specific primers to BTV VP6 gene (n = 2).

Fig. 2 DsRNA treatment induces vascular permeability. To determine whether dsRNA was sufficient to induce vascular permeability, HLMV endothelial cells were grown on gold electrodecontaining plates. After the cells reached confluency, cells were treated (at time 0) with increasing concentrations of dsRNA (poly I:C) at 0, 10, 30 and 100 µg/ml, and TER was continuously measured for 20 h (n = 3).

suggests that dsRNA treatment is not activating an apoptotic pathway, rather, this represents a transient reduction in cellular contact. The dsRNA induced reduction in TER reached a maximum of approximately 40% at 7 h post treatment.


BTV and dsRNA Induction of Vascular Permeability is Mediated by Activation of p38 MAPK We have previously reported that activation of p38 MAPK is critical for induction of endothelial cell

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Fig. 3 p38 MAPK is activated in human endothelial cells by BTV infection or dsRNA treatment. To determine the effects of viral infection or dsRNA treatment on p38 MAPK activation, we infected primary endothelial cells with BTV at MOI of 5 pfu /cell (Panel A), treated the cells with dsRNA at increasing concentrations for 2 h (Panel B) or treated the cells with dsRNA at 30 µg/ ml (Panel C). The cells were then harvested at indicated times and total cellular proteins were extracted as described in Materials and Methods. To detect activation of p38 MAPK, the cellular proteins were separated by electrophoresis and immunoblotted by using a specific anti-phospho-p38 MAPK antibody. The level of total p38 MAPK protein in the cellular extracts was then determined by stripping the blot and reprobing with a specific anti-p38 MAPK antibody (n = 3).

barrier disruption (27). Since viral infections are known to activate p38 MAPK, we have tested the possibility that p38 MAPK may be involved in the BTV-induced reduction in TER. We first examined whether dsRNA treatment or BTV infection could activate p38 MAPK in human endothelial cells. HLMV endothelial cells were either mock treated, were infected with BTV (MOI 5 pfu/cell) (Fig. 3A) or were treated with dsRNA (Fig. 3B and C). Cells were harvested at indicated times and whole cells extracts were prepared. We then assessed the level of p38 MAPK phosphorylation by western blot analysis using a specific anti-phospho-p38 Ab. The level of total p38 protein was then determined by stripping the blot and re-probing with anti-p38 Ab. The data show that p38 is activated by BTV infection or dsRNA treatment of HLMV cells. To determine the role of p38 in vascular permeability, we pretreated HLMV endothelial cells with a pharmacologic inhibitor of p38 MAPK SB239063 at 20 µM followed by infection with BTV at MOI of 5 pfu/cell (Fig. 4A) or dsRNA treatment at 30 µg/ml (Fig. 4B). The trans-endothelial electrical resistance was then measured continuously. The data in Fig. 4 show

that SB239063 inhibits the dsRNA and BTV-induced reduction in TER, suggesting that viral-induced vascular permeability is following a p38 MAPK-dependent pathway. DsRNA Treatment Induces Cytoskeletal Rearrangement in Endothelial Cells We and others have demonstrated that endothelial cytoskeletal actin and VE-cadherin reorganization are critically involved in endothelial cell barrier disruption (57– 61). To test the effects of dsRNA on actin microfilament reorganization, we treated endothelial cells with 30 µg/ml of poly I:C (Fig. 5 A). After indicated times, the distribution pattern of actin was determined by fluorescence microscopy by using rhodamine phalloidin. Similar to the data in Fig. 2, data in panel A show that after 24 h of dsRNA treatment, the cellular distribution of actin returns to normal. Since the data in Fig. 5A show that there is substantial redistribution of actin microfilaments after dsRNA treatment, we next examined the effects of p38 MAPK inhibitor on the cytoskeletal rearrangement (Fig. 5B).


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Fig. 4 Effect of p38 MAPK inhibitor on BTV or dsRNA-induced endothelial barrier disruption. To determine whether inhibition of p38 MAPK attenuated BTV or dsRNA induced reduction in TER, HLMV endothelial cells were left untreated (control) or were treated with SB239063 at 20 µM. After 30 min, the cells were either infected with BTV at MOI of 5 pfu/cell (Panel A) or were treated with dsRNA at 30 µg/ml (Panel B). The trans-endothelial electrical resistance was then continously measured (n = 3).

HLMV cells were treated with SB239063 for 30 min prior to dsRNA treatment (30 µg/ml). The expression of actin and VE-cadherin was determined. The data show that dsRNA-induced cytoskeletal rearrangement is attenuated by pre-treatment of cells with p38 inhibitor. Therefore, dsRNA treatment induces a compromise in cellular contact and tethering forces, which is attenuated by SB239063. These data directly demonstrate that dsRNA induces cellular shape changes and intercellular gap formation.

DsRNA Treatment or Infection of Endothelial Cells with BTV Results in the Expression of Inflammatory Cytokines Recent reports show that the morbidity and mortality associated with VHFs is largely due to a dysregulated inflammatory response (2, 3). Therefore, we have determined the effects of BTV infection (MOI 5 pfu/ml) or dsRNA treatment (30 µg/ml) on cytokine expression in human endothelial cells (Fig. 6). At indicated times post BTV infection (Fig. 6A) or dsRNA treatment (Fig. 6B), cells were harvested and total cellular RNA was extracted. To assess the level of cytokine induction, we subjected the RNA to semi-quantitative RT-PCR by using specific primers to human TNF-α, IL-1β IL-6, IFN-β or GAPDH as a control. We then determined whether BTV infection resulted in expression of secreted cytokines in the culture supernatants. Data from ELISA experiments (Fig. 6C) demonstrate that both TNF-α and IL-1β are secreted after BTV infection of endothelial cells.


DISCUSSION

Severe viral hemorrhagic fever (VHF) diseases are characterized by generalized inflammation, vascular permeability and ultimately death. However, little is known regarding the molecular mechanisms by which viral infections induce vascular permeability. In this report, we have shown that dsRNA and BTV induce endothelial barrier disruption as detected by a decrease in TER. Consistent with our previous reports, BTV and dsRNA induction of endothelial permeability was mediated by activation of the p38 MAPK pathway. Furthermore, dsRNA and BTV proved to be potent inducers of inflammatory cytokines in endothelial cells as indicated by RT-PCR and ELISA detecting TNF-α and IL-1β expression. There is a causal relationship between dysregulated secretion of inflammatory cytokines and the clinical manifestations of VHF diseases. In animal and human studies, cytokines such as TNF-α, IL-1β, IL-6, and IFN-β have been reported to participate in the VHF-associated pathologies such as tissue damage, pro-coagulant activity, shock, vascular permeability and death (2, 3). Therefore, our data showing the involvement of dsRNA as a viral moiety and p38 as a key signaling molecule for viralinduced vascular permeability provide a novel insight in VHF diseases. Expression of inflammatory cytokines is beneficial to an antiviral immune response but it can also be deleterious to the host. A clear example of a cytokine that fits these paradoxical observations is TNF-α. It directly reduces viral replication and by recruiting and activating lymphocytes, initiates specific immune responses (62– 65). The deleterious effects of TNF-α are also well

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Fig. 5 Effect of dsRNA on cytoskeletal rearrangement in HLMV endothelial cells. Panel A. To determine the effect of dsRNA on actin filament reorganization confluent HLMV endothelial cells were grown as a monolayer. After reaching a confluent state, the cells were then left untreated or were treated with dsRNA (10 or 30 µg/ml) for 6, 8, or 24 h. The pattern of actin distribution in the cells was visualized (magnification of 400X) by permeabilizing and staining with Rhodamine Phalloidin stain (n = 2). Panel B. We next analyzed the effect of p38 MAP kinase inhibitor of the dsRNA induction of cytoskeletal reorganization. The cells were grown as in panel A. After reaching a confluent monolayer, cells were left untreated or were treated with dsRNA at 30 µg/ml for 6 hr in the absence or presence of p38 inhibitor (SB239063) (20 µM). The cells were then fixed and immuno-stained by using anti-VE-cadherin antibody (Ab) or were permeabilized and stained for actin by using Texas Red Phalloidin (n = 3).

studied, depending on the cell type and species, it can induce apoptosis, platelet dysfunction, tissue necrosis and vascular permeability (55, 66–69). Recently, we reported that the mechanism of TNF-α-induced vascular permeability involved key cytoskeletal rearrangement in endothelial cells, unrelated to increase in apoptosis. TNF-α

destabilization of the microtubules and actin microfilament cytoskeletal changes resulted in endothelial barrier disruption, which were attenuated by inhibition of p38 MAPK using SB203580 (55). Therefore, we first examined the effect of dsRNA on the integrity of human lung endothelial cells grown on


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Fig. 6 BTV infection and dsRNA treatment induce inflammatory cytokines expression in human endothelial cells. To establish whether virus infections or dsRNA treatment could induce cytokines in endothelial cells, human primary endothelial cells were infected with either bluetongue virus (BTV), Panel A, at MOI of 5 pfu/cell or were treated with dsRNA at 30 µg/ml, Panel B. The experiments were performed in the absence or presence of the specific p38 inhibitor SB239063 (20 µM). After indicated times, total cellular RNA was extracted and subjected to RT-PCRs by using specific primers to TNF-α, IL-1β IFN-β, IL-6 or GAPDH. The amplified products were resolved on a 1.5% agarose gel and the bands were visualized by ethidium bromide staining (n = 3). Panel C. To determine the presence of cytokine protein in culture supernatant, HLMV endothelial cells were infected with BTV at MOI of 5 pfu/cell. At indicated times cell-free culture supernatants were collected and were used in specific ELISA assays (eBioscience) (n = 2).

gold microelectrodes to measure trans-endothelial electrical resistance (TER). Since the activation of p38 MAPK is critically involved in specific models of cytoskeletal reorganization and vascular permeability, we next examined the effect of dsRNA treatment on p38 MAPK activation and endothelial cell barrier disruption. Our data show that dsRNA (10–100 µg/ml) treatment of isolated lung micro-vascular endothelial cells rapidly activates p38 MAPK (Fig. 4). Furthermore, dsRNA induced significant ( ∼ 40%) decline in TER after 7 h in association with cytoskeletal rearrangement, suggesting a direct role for dsRNA in endothelial barrier disruption. Pre-treatment with a pharmacological inhibitor of p38 MAPK (20 µ


SB239063) blocks the dsRNA-induced decrease in TER by blocking upregulation of TNF-α, IL-1β, IFN-β and IL-6. At this point, our data suggest but does not prove that BTV-induced vascular permeability is mediated through the intracellular or extracellulr expression of dsRNA. Studies by DeMaula et al. and Mortola et al. showed that BTV binding to ovine arterial endothelial cell induced apoptosis, which may provide an alternative interpretation for our observations (70, 71). However, based on our data presented in Fig. 5B, we do not detect BTV-induced cell lysis in primary human lung micro-vascularendothelial cells at 24 h post infection. We do not know the exact

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reason for this discrepency but it is possible that human cells are not sensitive to apoptotic effects of BTV. Additionaly, the observation of DeMaula et al. showed that BTV alone did not induce apoptosis in ovine or bovine lung microvascular cells. Therefore, there may be differences between arterial and microvascular endothelial cells. It is not yet clear whether the induction of vascular permeability by dsRNA is a direct effect on the endothelium or by the induction of inflammatory cytokines such as TNF-α. Nonetheless, our data suggest that the presence of dsRNA during viral infections and subsequent activation of p38 MAPK is a potential molecular pathway for viral induction of hemorrhagic fevers. Inhibition of p38 MAPK may be a possible therapeutic approach for viral-induced acute hemorrhagic diseases.

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