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Originally published online as doi:10.1189/jlb.0805441 on August 29, 2006

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(Journal of Leukocyte Biology. 2006;80:1242-1250.)
© 2006 by Society for Leukocyte Biology

A dominant role for Fc{gamma}RII in antibody-enhanced dengue virus infection of human mast cells and associated CCL5 release

Michael G. Brown*, Christine A. King*,1,, Christine Sherren*, Jean S. Marshall*,{dagger} and Robert Anderson*,2

Departments of
* Microbiology and Immunology and
{dagger} Pathology, Dalhousie University, Halifax, Nova Scotia, Canada

2 Correspondence: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. E-mail: robert.anderson{at}dal.ca

ABSTRACT

Dengue virus is a major mosquito-borne human pathogen with four known serotypes. The presence of antidengue virus antibodies in the serum of individuals prior to dengue virus infection is believed to be an important risk factor for severe dengue virus disease as a result of the phenomenon of antibody-dependent enhancement operating on Fc receptor (FcR)-bearing cells. In addition to blood monocytes, mast cells are susceptible to antibody-enhanced dengue virus infection, producing a number of inflammatory mediators including IL-1, IL-6, and CCL5. Using the human mast cell-like lines KU812 and HMC-1 as well as primary cultures of human cord blood-derived mast cells (CBMC), we aimed to identify the participating FcRs in antibody-enhanced mast cell dengue virus infection, as FcRs represent a potential site for therapeutic intervention. CBMC expressed significant levels of Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII, and mast cell-like HMC-1 and KU812 cells expressed predominantly Fc{gamma}RII. All four serotypes of dengue virus showed antibody-enhanced binding to KU812 cells. Specific Fc{gamma}RII blockade with mAb IV.3 was found to significantly abrogate dengue virus binding to KU812 cells and CBMC in the presence of dengue-specific antibody. Dengue virus infection and the production of CCL5 by KU812 cells were also inhibited by Fc{gamma}RII blockade.

Key Words: dengue hemorrhagic fever • FcR binding • dengue pathogenesis • virus-antibody complexes

INTRODUCTION

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe complications of dengue virus infections, believed, in part, to result from antibody-dependent enhancement (ADE) of virus infection via Fc receptors (FcRs). The presence of heterotypic subneutralizing antibodies to dengue virus as a result of primary infection has been shown to potentiate secondary infection of monocytes and macrophages via ADE [1 ]. In addition to blood monocytes, considered to be major targets for dengue virus infection, dengue virus can infect other cells, such as mast cells, resulting in the release of potent vasoactive cytokines IL-1β and IL-6 [2 ] and chemokines, such as CCL3, CCL4, and CCL5 [3 ]. We aimed to define the FcRs involved in antibody-enhanced dengue virus infection of human mast cells, which express a number of FcRs including those for IgG (Fc{gamma}R) and IgE (Fc{epsilon}R) [4 5 6 ]. Three main classes of receptors for IgG, namely Fc{gamma}RI (CD64), Fc{gamma}RII (CD32), and Fc{gamma}RIII (CD16), have been identified in humans [7 ].

We used a number of complementary human mast cell/basophil cell models including KU812 and HMC-1 cells and cord blood-derived mast cells (CBMC). The KU812 cell line possesses properties of basophils and mast cells and has been shown to express mast cell tryptase as well as mRNA for carboxypeptidase and low levels of major basic protein [8 , 9 ]. KU812 cells express Fc{epsilon}R1, the high-affinity IgE receptor, Fc{gamma}RII, and the stem cell factor receptor (c-kit) but lack chymase and the basophil-specific marker 2D7 [10 11 12 13 ]. The HMC-1 mast cell line has been found to express chymase mRNA and higher levels of tryptase mRNA than KU812 cells [12 13 14 15 ]. In contrast to KU812 cells, HMC-1 cells express Fc{gamma}RII but do not express Fc{epsilon}RI consistently [16 , 17 ]. Although work has been done on CD34+ cultured mast cells [5 ], there are little data on CBMC. In the context of dengue infection, we therefore sought to examine various types of mast cells in a comparative manner with respect to the role and expression of Fc{gamma}R.

MATERIALS AND METHODS

Mast cell culture
Human KU812 cells [9 ] were maintained in RPMI 1640 (Sigma Chemical Co., St Louis, MO), supplemented with 10% FCS, 10 mM HEPES. HMC-1 cells [14 ] were maintained in IMDM, supplemented with 10% FCS. All cell lines were passaged two to three times per week.

Primary human-cultured CBMC
Primary mast cells were generated by culture of human CBMC using a modification of the method of Saito et al. [18 ] and characterized as described previously [19 ]. These cells were predominantly positive by flow cytometry for CD117 (c-kit) and CD13 but not CD14. Cells were cultured at an initial concentration of 0.6 x 106/ml in RPMI supplemented with 20% FCS, 100 U penicillin per ml, 100 µg streptomycin per ml, 20% CCL-204 cell supernatant as a source of IL-6, 10–7 M PGE2 (Sigma Chemical Co.), and 50–100 ng stem cell factor (Peprotech, Rocky Hill, NJ)/ml. The medium was changed once weekly for 5–10 weeks. The purity of each CBMC preparation was assessed by toluidine blue (pH 1.0) staining of cytocentrifuge preparations and examination of cells for the presence of multiple metachromatic granules and appropriate nuclear morphology. Only mast cell preparations, which were ≥95% pure, were used for this study. The mean purity of cord blood mast cells was 97%.

Dengue virus propagation
Dengue viruses, type 2 (strain 16681) [1 ] as well as type 1 (strain Hawaii), type 3 (strain K-68/88, 0477/88), and type 4 (strain 814669) were propagated in African green monkey kidney Vero cell monolayers cultured in endotoxin-free RPMI 1640 (Sigma, Oakville, Ontario, Canada), supplemented with 1% FCS (Life Technologies, Grand Island, NY).

Sera
For ADE assays of dengue virus infection, a dengue-immune serum pool was prepared by using nine convalescent-phase sera from patients recovering from a dengue virus serotype 2 infection. The individual dengue-convalescent patient sera and dengue virus-specific mAb 1B7 (subclass IgG2a) and 3H5 (subclass IgG1) were obtained from a collection provided by Dr. Bruce Innis (Armed Forces Research Institute of Medical Science, Bangkok, Thailand) and described briefly in ref. [20 ]. The dengue virus-specific mAb 7E11 (subclass IgG2a) was kindly obtained from Drs. Robert Putnak and Tim Endy [Walter Reed Army Institute of Research (WRAIR), Washington, DC]. Normal human AB sera were obtained from volunteer donors.

Antibodies to FcRs and controls
The mAb used were mouse anti-human Fc{gamma}RI (clone 10.1, subclass IgG1, BD PharMingen, San Diego, CA), mouse anti-human Fc{gamma}RII (clone C1KM5, subclass IgG1, Caltag, Burlingame, CA), and mouse anti-human Fc{gamma}RIII (clone 3G8, subclass IgG1, Caltag) as well as the isotype-matched mouse IgG1 control (BD PharMingen) and were used at a final concentration of 5 µg/ml. Detection of Fc{gamma}RII and Fc{gamma}RIII used a rat anti-mouse FITC-conjugated secondary antibody; the Fc{gamma}RI antibody was FITC-conjugated. Anti-Fc{gamma}RII mAb IV.3 (subclass IgG2b) is an Fc{gamma}RII-blocking antibody, originally from American Type Culture Collection (Manassas, VA), kindly provided by Dr. Andrew Issekutz (Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada). Human IgE (10 µg/ml, Chemicon, Temecula, CA) was used for blocking Fc{epsilon}RI.

Plaque assay
Supernatants from dengue virus-infected Vero or KU812 cells were harvested, serially diluted, and inoculated onto Vero cell monolayers for plaque assay. At 30 h postinfection, cells were washed once with PBS, fixed with methanol for 10 min, and then placed at 4°C in PBS until immunostaining, which was performed using dengue-specific antibody and goat secondary AP conjugate followed by development with the one-step NBT/5-bromo-4-chloro-3-indolyl-phosphate AP developer (Pierce, Rockford, IL).

FACS analysis
Cells were harvested at indicated times postinfection, fixed in 1% paraformaldehyde, permeabilized using 0.1% saponin, and stained for intracellular expression of dengue E protein using mAb 1B7 [21 ], NS1 protein using mAb 7E11, or an IgG2a isotype control antibody. A goat anti-mouse Alexa 488 conjugate (Molecular Probes, San Diego, CA) was used as a secondary antibody. In some cases, cells were then blocked with normal mouse sera prior to the addition of a mouse antihuman CD117 (c-kit)-allophycocyanin conjugate (BD PharMingen). Washed cells were resuspended in 1% paraformaldehyde and subjected to FACS analysis. Data were obtained using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using FCS Express 2 and CellQuest.

Radiolabeling of dengue virus with [35S]methionine-[35S]cysteine
At 24 h postinfection, dengue virus-infected Vero cells were incubated in methionine- and cysteine-free medium supplemented with [35S]methionine-[35S]cysteine (Perkin Elmer, Woodbridge, Ontario, Canada), as described previously [22 ]. Cell-free supernatants were harvested at 36 h postinfection, and dengue virus was purified using a 15/55% sucrose gradient by ultracentrifugation at 45 K rpm for 2 h at 4°C.

Virus-binding assay
Purified, radiolabeled dengue-2 virus (prepared as above) was mixed with various concentrations of dengue-immune serum for 1 h on ice. KU812, HMC-1, and CBMC (5.0x105 cells) were then inoculated with the virus-antibody preparation and incubated for 90 min on ice, mixed every 15 min. For blocking experiments, cells were pretreated with mAb IV.3 or IgE for 60 min prior to the addition of virus-antibody mixtures. Following adsorption, cells were washed extensively with 0.1% BSA in PBS. Cell pellets were resuspended in SDS-PAGE loading buffer and boiled immediately for 5 min to inactivate endogenous mast cell proteases. Samples were then placed at –20°C until use. A total of 50,000 cells was loaded per condition and resolved using 15% polyacrylamide gel, and gels were impregnated with 1 M sodium salicylate and fluorographed by exposure to Kodak X-ray film at –70°C.

CCL5 immunoprecipitation assay
KU812 cells, inoculated with dengue-2 virus and dengue immune sera, were radiolabeled with [35S]methionine-[35S]cysteine at 48 h postinfection. At 72 h postinfection, cell-free supernatants were harvested, and radiolabeled CCL5 protein was immunoprecipitated with anti-CCL5 antibody (Endogen, Rockford, IL) and protein A-bearing, formalin-fixed Staphylococcus aureus. Immunoprecipitates were resolved by SDS-PAGE using 15% polyacrylamide gels. Gels were impregnated with 1 M sodium salicylate and fluorographed by exposure to Kodak X-ray film at –70°C. Data shown are representative of at least two separate experiments.

RESULTS

FcR expression on mast cells
FcR surface expression on human mast cells was evaluated with a panel of mAb using FACS. To prevent nonspecific binding, 1 µl human AB serum per 100 µl final volume was added during the primary and secondary antibody incubation steps. Cell analysis was performed using CellQuest and FCS Express software. The percent positive cells for each Fc{gamma}R were determined by Overton histogram subtraction [23 ]. The majority of KU812 cells (84±10%; Fig. 1B ) and HMC-1 cells (64±15%; Fig. 1E ) showed Fc{gamma}RII expression. In contrast, low levels of Fc{gamma}RI (Fig. 1A and 1D) and Fc{gamma}RIII (Fig. 1C and 1F) were expressed on KU812 and HMC-1 cells. Median fluorescent intensity (MFI) of surface Fc{gamma}RII on KU812 cells (21±11%) versus HMC-1 cells (7±0.6%) indicated that KU812 cells also have a greater number of Fc{gamma}RII receptors on the surface than do HMC-1 cells. Conversely, CBMC from five individuals demonstrated widespread surface expression of all three classes of Fc{gamma}Rs with Fc{gamma}RI (76±8%; Fig. 1G ), Fc{gamma}RII (75±16%, Fig. 1H ), and Fc{gamma}RIII (44±12%, Fig. 1I ). To our knowledge, this is the first report of human mast cell surface Fc{gamma}RIII expression, although Fc{gamma}RIII mRNA has been detected in human CD34+ cultured mast cells [5 ], and surface expression has been observed on murine mast cells [24 ]. MFI values of surface Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII on CBMC were 7 ± 6%, 8 ± 6%, and 5 ± 3.5%, respectively.


Figure 1
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  		Figure 1. Human mast cells express high levels of Fc{gamma}RII. KU812, HMC-1, and CBMC were analyzed by FACS for surface expression of Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII. Shaded lines indicate isotype control; black lines indicate Fc{gamma}R-specific antibody. Data are representative of at least two separate experiments.

Antibody-enhanced dengue virus infection of mast cells
Although we have shown previously that cells of a mast cell/basophil lineage are susceptible to antibody-enhanced dengue virus infection [2 , 3 ], a comparative analysis of various mast cell lines has not yet been performed. Moreover, as c-kit (CD117) is one of many reliable markers for mast cells [12 , 25 ], we wished to verify that dengue virus infection localized to c-kit (+) cells. HMC-1 cells express a constitutively activated mutant c-kit [26 , 27 ], and KU812 cells have been shown to respond to stem cell factor, suggesting a more functional c-kit receptor [28 ]. KU812 and HMC-1 cells as well as CBMC were inoculated with dengue-2 virus (multiplicity of infection of one) and a subneutralizing concentration (1:10,000 dilution) of dengue immune serum. The results (Fig. 2 ) show that antibody-enhanced infection of mast cells was most pronounced in KU812 cells, followed by HMC-1 and CBMC. In view of the similarities in Fc{gamma}RII expression by HMC-1 cells and CBMC (Fig. 1) , the low level of infection in CBMC may suggest a postattachment restriction for productive dengue virus infection within CBMC. It is most important that the bulk of dengue infection was associated with c-kit (+) cells in the CBMC, thus confirming the mast cell nature of the cells involved in dengue infection.


Figure 2
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  		Figure 2. Antibody-enhanced infection of KU812, HMC-1, and CBMC. Cells were incubated for 2 h at 4°C with medium alone (RPMI 1640 containing 1% FCS) or a mixture of dengue virus and a subneutralizing dilution (1:10,000) of dengue-immune sera. After washing, cells were resuspended in fresh media and incubated at 37°C for 24 h before processing for FACS analysis as described in Materials and Methods. The high granule content of CBMC results in undesirable autofluorescence, which was excluded to eliminate interference with the fluorochromes.

The productive nature of the dengue virus infection in CBMC was confirmed by FACS analysis (Fig. 3A ) and virus plaque assay (Fig. 3B) . Detection of intracellular E protein by FACS verified an increase in the presence of E protein-expressing c-kit (+) CBMC over the course of infection (mean of 0.66% at 0 h compared with 2.47% at 24 h postinfection from two independent experiments; Fig. 3A ). Isotype control values were no higher than those at the 0-h time-points (data not shown). Furthermore, plaque assay using supernatants from infected CBMC at 0 and 24 h postinfection clearly confirmed the production of infectious dengue virus (Fig. 3B) .


Figure 3
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  		Figure 3. CBMC are productively infected by dengue virus. Cells were incubated for 2 h at 4°C with a mixture of dengue virus and a subneutralizing dilution (1:1000) of pooled dengue-immune sera. After washing, cells were harvested at 0 h or resuspended in fresh media and incubated at 37°C for 24 h before being harvested and processed for FACS analysis as described in Materials and Methods (A). Supernatants from dengue virus-infected CBMC were harvested at 0 and 24 h postinfection, and the presence of infectious dengue virus particles was determined by plaque assay (B). The results are the average from two independent experiments.

As the KU812 cell line was shown to be most permissive to antibody-enhanced dengue virus infection, these cells were used to observe the kinetics of the infection. Cells were harvested at 0, 12, 24, 36, and 48 h postinfection and then fluorescently stained for intracellular dengue virus envelope (E) and nonstructural-1 (NS1) proteins and analyzed by flow cytometry. In addition, infected KU812 cell supernatants were inoculated onto Vero cell monolayers to determine the presence of infectious virus particles at the various time-points postinfection, and titers were determined by plaque assay. It was found that the peak of infection was reached by 24 h, as determined by the highest detection of dengue virus E and NS1 proteins (Fig. 4B and 4C , respectively), as well as the supernatant virus titers (Fig. 4A) . The relative differences in E and NS1 protein levels may be a result of different sensitivities of the respective antibodies and/or to the fact that intracellular protein is partly lost by the export to released progeny virions.


Figure 4
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  		Figure 4. Time course of antibody-enhanced infection of KU812 cells, which were inoculated with dengue virus in the presence of a 1:10,000 dilution of pooled dengue-immune serum, incubated at 37°C, and harvested at 0, 12, 24, 36, and 48 h postinfection. Production of infectious virus titers from dengue virus-infected KU812 cells was determined by plaque assay (A). Cells were also stained for intracellular dengue E (B) or NS1 (C) proteins using mAb 1B7 or mAb 7E11, respectively, and analyzed by FACS. As controls, negligible virus titers and dengue E and NS1 staining were observed for mock-inoculated KU812 cells or KU812 cells inoculated with dengue virus alone or dengue virus with dengue nonimmune serum (data not shown). Data from two experiments with different multiplicities of infection of four (black diamonds) and 0.4 (gray circles) are shown.

Antibody-enhanced dengue virus-binding to mast cells
We assessed binding of dengue-2 virus to KU812, HMC-1 cells, and CBMC using 35S-radiolabeled dengue virus. Cell-binding assays were conducted for 90 min on ice in the absence or presence of subneutralizing concentrations of dengue-immune serum. Cells were washed, and cell-bound radiolabeled dengue virus was detected by SDS-PAGE and fluorography [29 ]. As shown in Figure 5A , KU812 and HMC-1 cells and CBMC demonstrate antibody-enhanced binding of dengue virus. Virus binding was stronger for KU812 cells than for HMC-1 cells (Fig. 5A) , possibly reflecting higher expression of Fc{gamma}RII on KU812 cells (Fig. 1B) . In comparison with KU812 and HMC-1 cells, CBMC exhibit the weakest binding of radiolabeled dengue virus. Densitometric analysis of data from three individual experiments revealed significantly enhanced (P≤0.05) binding of virus to CBMC with 1:1000 and 1:10,000 dilutions of dengue-immune serum (5.1±4.6- and 5.0±3.0-fold increases, respectively, compared with virus-CBMC binding with nonimmune serum). Similar results to those shown in Figure 5 were found using other pools of dengue patient sera (data not shown). Binding assays using individual, dengue-immune sera all showed enhanced binding, although the optimum serum dilution varied from patient to patient. Enhanced dengue virus-cell binding was never observed with dengue nonimmune sera (data not shown). Antibody-enhanced virus-cell binding, as well as a requirement for the viral E protein, was demonstrated with KU812 cells using a variety of E protein-specific mouse mAb, and mAb 3H5 showed the best binding (Fig. 5B) . Furthermore, binding experiments undertaken using radiolabeled dengue-1, dengue-2, dengue-3, and dengue-4 viruses mixed with subneutralizing concentrations of dengue-immune sera clearly demonstrated that all four serotypes of dengue virus exhibit antibody-enhanced binding to KU812 cells (Fig. 5C) . In the absence of dengue immune serum, there was little to no virus-cell binding (data not shown).


Figure 5
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  		Figure 5. Antibody-enhanced binding of dengue virus to KU812, HMC-1 cells, and CBMC. (A) Cell binding of radiolabeled dengue virus. Radiolabeled dengue-2 virus was incubated with KU812, HMC-1 cells, or CBMC for 90 min on ice in the presence or absence of various dilutions of dengue-immune sera. Washed cells were then assayed for radiolabeled virus by fluorographic SDS-PAGE. (B) Dependence of antibody-enhanced virus binding on viral E protein. Radiolabeled dengue-2 virus was incubated with KU812 cells for 90 min on ice in the presence of various dilutions of pooled human dengue-2 convalescent serum (Poly) or with mAb 3H5 specific for the E protein [21 ]. Washed cells were then assayed for radiolabeled virus by fluorographic SDS-PAGE. (C) Antibody-enhanced binding to KU812 cells for all four serotypes of dengue virus. Radiolabeled dengue-1, dengue-2, dengue-3, or dengue-4 viruses were mixed with pooled dengue-immune serum (1:10,000 final dilution) and with KU812 cells for 90 min on ice and washed as above. In the absence of dengue-immune serum, virtually no virus binding to KU812 cells was observed (data not shown). Indicated in A are the positions of the viral proteins, E, prM, and M (little C protein is found in dengue virus particles produced in Vero cells; ref. [30 ]). (B and C) Regions of the gel fluorogram encompassing the dengue viral E protein.

Antibody-enhanced binding of dengue virus to mast cells is mediated primarily by Fc{gamma}RII
Dengue virus has been shown to use Fc{gamma}RI [31 ] and Fc{gamma}RII [32 ] during antibody-enhanced infection of monocyte-like U937 cells and erythroleukemic K562 cells, respectively. To determine the FcR responsible for antibody-enhanced binding of dengue virus to mast cells, KU812 cells and CBMC were pretreated with normal mouse serum or anti-Fc{gamma}RII mAb IV.3, followed by binding of radiolabeled dengue-2 virus in the presence of normal or subneutralizing dengue immune serum. As shown in Figure 6A , pretreatment of mast cells with mAb IV.3 but not normal mouse serum dramatically reduced antibody-enhanced dengue virus binding to KU812 cells (compare lanes 5 and 6 with lanes 2 and 3) and CBMC (compare corresponding lanes). Given the importance of the high-affinity IgE receptor Fc{epsilon}RI in mast cell biology, we also investigated the potential contribution of Fc{epsilon}RI in antibody-enhanced binding of dengue virus to the surface of KU812 cells. Blocking studies using KU812 cells preincubated with human IgE (10 µg/ml) [33 ], 1:4 dilution of ascites containing mAb IV.3, or irrelevant IgG control antibody (10 µg/ml) showed that antibody-enhanced binding of dengue virus was mediated predominately by Fc{gamma}RII (Fig. 6B) . Experiments using heat-inactivated (56°C, 2 h), dengue-immune sera, which results in the destruction of IgE biological function [33 , 34 ], showed no decrease in the level antibody-enhanced dengue virus binding to KU812 cells (data not shown).


Figure 6
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  		Figure 6. Fc{gamma}RII is the major receptor for antibody-bound dengue virus on KU812 cells and CBMC. (A) Radiolabeled dengue-2 virus was mixed with normal or dengue-immune serum and then added to KU812 cells or CBMC, which had been pretreated for 1 h on ice with normal mouse serum or anti-Fc{gamma}RII mAb IV.3. Following adsorption for 90 min on ice, cells were washed to remove unbound virus and then subjected to SDS-PAGE followed by fluorography to detect cell-bound, radiolabeled virus. The region of the gel containing the dengue viral E protein is shown. (B) Similar assay as A, except duplicate KU812 cell cultures were pretreated for 1 h on ice with IgE (10 µg/ml), mAb IV.3, or irrelevant control IgG2b followed by 90 min on ice for adsorption of the cells with radiolabeled virus.

Fc{gamma}RII is required for productive dengue virus infection and for CCL5 response
We have shown previously that dengue virus infection of human mast cells triggers dramatic cytokine and chemokine responses, of which CCL5 was one of the strongest induced [2 , 3 ]. In addition to mediating cell binding of antibody-complexed dengue virus, we investigated whether Fc{gamma}RII was also required for productive antibody-enhanced dengue virus infection and CCL5 production. KU812 cells were inoculated with dengue virus in the presence of a subneutralizing concentration of human dengue immune serum (1:1000 final dilution), and the synthesis of viral proteins and CCL5 was monitored by radiolabeling with [35S]methionine-[35S]cysteine and immunoprecipitation as described previously [2 , 35 ]. This was an independent method of assaying CCL5 production, which we have previously measured using ELISA [3 ]. Blocking of Fc{gamma}RII clearly impeded dengue virus infection (Fig. 7A ; compare lanes 5 and 6 with lanes 7 and 8). Concomitant with blocking infection, pretreatment of KU812 cells with mAb IV.3 also abrogated the CCL5 response (compare lanes 3 and 4 in Fig. 7B ).


Figure 7
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  		Figure 7. Blockade of Fc{gamma}RII abrogates antibody-enhanced dengue virus infection and CCL5 production by human mast cells. (A) Duplicate cultures of KU812 cells, pretreated with Fc{gamma}RII-blocking mAb IV.3 or control IgG, were inoculated with dengue virus in the presence of human dengue-immune serum (final dilution 1:1000) and radiolabeled, and dengue viral proteins were immunoprecipitated (mAb 3H5) from supernatant culture media at 24 h postinfection. Shown is fluorographic SDS-PAGE of the immunoprecipitated proteins. (B) Similarly treated KU812 cell cultures were harvested at 72 h postinfection, and radiolabeled CCL5 protein was immunoprecipitated from supernatant culture media with anti-CCL5 antibody. *, Position of CCL5 protein. The data are representative of two separate experiments.

DISCUSSION

The role of mast cells as key effector cells in allergic disease has been characterized extensively; however, the role of mast cells has recently been expanded to include sentinel functions in innate immune defense [36 ]. Being constantly exposed to the external environment, with an intimate spatial relationship with skin, blood vessels, airways, and the gastrointestinal tract, mast cells function as first-line defenders reacting to the constant onslaught of pathogenic invaders. Hence, mast cells have been categorized as an important cellular element of the innate immune system, which also includes epithelial cells, dendritic cells, phagocytic cells (macrophages and granulocytes), NK cells, and {gamma}{delta} T cells [37 ]. Mast cells have been shown to respond to a number of viral pathogens including respiratory syncytial virus, influenza virus, sendai virus, adenovirus, HIV, newcastle disease virus, and dengue virus [38 39 40 ]. However, to our knowledge, only dengue virus, adenovirus, and HIV-1 have been shown to infect human mast cells [41 , 42 ]. Moreover, mast cell responses to pathogens vary, implying that mast cells can sense and respond specifically to exposure with different viruses. Through the signature release of mediators, including cytokines and chemokines, mast cells can influence the type of immune reaction mounted against a particular pathogen.

Mast cells express a number of Ig receptors or FcRs, which allow them to bind soluble antibodies or antibody-antigen complexes to their surface. Our previous studies have shown that mast cells can become a target for dengue virus infection in vitro through binding of virus-antibody complexes, which leads to the specific release of a number of cytokines (IL-1β and IL-6) and chemokines (CCL3, CCL4, and CCL5) [2 , 3 ]. Although dengue virus infection of mast cells has not been detected in vivo, a number of clinical observations implicate mast cell involvement in severe dengue disease. Increased levels of blood and urinary histamine have been detected in patients suffering from DHF/DSS [43 , 44 ]. A recent study also observed mast cell recruitment occurring in the lungs of mice infected with dengue virus [45 ].

The antibody-dependent nature of mast cell infection by dengue virus led us to examine which, if any FcR expressed by mast cells, predominated in the binding of virus-antibody complexes. As a result of the classical role of Fc{epsilon}RI-mediated activation of mast cells driving inflammatory reactions to various antigens, it was important to determine whether Fc{epsilon}RI played a role in antibody-dependent dengue virus binding and infection of mast cells or mast cell responses to dengue virus infection. Although elevated levels of total and dengue-specific IgE serum levels in the acute stage of disease have been documented [46 ], it is unknown whether virus-specific IgE can potentiate antibody-dependent dengue virus binding of mast cells. Following blockade of Fc{epsilon}RI, the level of antibody-enhanced binding of dengue virus to KU812 cells was unaffected. Blocking of Fc{gamma}RII using the mAb IV.3 impeded antibody-enhanced binding of dengue virus to KU812 cells and CBMC. In addition, it was found that the CCL5 release from KU812 cells is dependent on antibody-enhanced dengue virus infection mediated by Fc{gamma}RII. The existence of the different isoforms of Fc{gamma}RII, Fc{gamma}RIIA (activating) and Fc{gamma}RIIB (inhibitory) [47 ], may play an additional role in antibody-enhanced binding and infection of FcR-bearing cells (including mast cells) by dengue virus. Although both isoforms share extensive homology in their extracellular motifs, their cytoplasmic sequences differ significantly. Fc{gamma}RIIA contains a cytoplasmic ITAM and has been shown to be involved in the endocytosis of bound complexes, and Fc{gamma}RIIB contains an ITIM and provides inhibitory signaling [48 ]. Unfortunately, as a result of the extensive homology in the extracellular domains of these isoforms, no antibodies are available to absolutely discriminate between Fc{gamma}RIIA or Fc{gamma}RIIB expression. Nevertheless, mAb IV.3 demonstrates preferential binding to and blocking of Fc{gamma}RIIA compared with Fc{gamma}RIIB [48 49 50 ]. Hence, blockade of Fc{gamma}RIIA using mAb IV.3 may interfere with attachment and/or endocytosis of virus-immune complexes, providing an explanation for the observed, diminished binding and subsequent infection of KU812 cells and CBMC.

In summary, our present findings highlight a critical role for Fc{gamma}RII in mediating antibody-enhanced dengue virus infection of mast cells as well as cytokine/chemokine responses such as CCL5. This is the first report of FcR involvement in dengue virus infection of mast cells, although Fc{gamma}RI and Fc{gamma}RII have been shown to mediate ADE of dengue virus infection in U937 and K562 cell lines, respectively [31 , 32 ]. Taken together, the results indicate a crucial requirement for specific FcRs in antibody-dependent dengue infection, which may prove useful for therapeutic targeting strategies.

ACKNOWLEDGEMENTS

The Canadian Institutes of Health Research (CIHR) supported this work. We are grateful to Drs. Bruce Innis, Alan King, and David Vaughn, formerly of the Armed Forces Research Institute of Medical Science, Bangkok, Thailand, for providing the dengue virus stocks, human dengue immune sera, and dengue-specific mAb used in this study. We also thank Drs. Tim Endy and Robert Putnak (WRAIR) for providing us with mAb 7E11. Special thanks goes to Ula Kadela-Stolarz and Yi-Song Wei for their excellent technical assistance in the generation of human CBMC. M. G. B. and C. A. K. contributed equally to this work.

FOOTNOTES

1 Current address: Cambridge University, Department of Medicine, Addenbrooke’s Hospital, Level 5, Box 157, Hills Rd., Cambridge CB2 2QQ, UK. Back

Received August 6, 2005; revised June 23, 2006; accepted July 3, 2006.

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