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Journal of Virology, August 2002, p. 8408-8419, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8408-8419.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Dengue Virus Selectively Induces Human Mast Cell Chemokine Production

Christine A. King,1 Robert Anderson,1 and Jean S. Marshall1,2*

Departments of Microbiology and Immunology,1 Pathology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada2

Received 25 January 2002/ Accepted 20 May 2002


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ABSTRACT
 
Severe dengue virus infections usually occur in individuals who have preexisting anti-dengue virus antibodies. Mast cells are known to play an important role in host defense against several pathogens, but their role in viral infection has not yet been elucidated. The effects of dengue virus infection on the production of chemokines by human mast cells were examined. Elevated levels of secreted RANTES, MIP-1{alpha}, and MIP-1ß, but not IL-8 or ENA-78, were observed following infection of KU812 or HMC-1 human mast cell-basophil lines. In some cases a >200-fold increase in RANTES production was observed. Cord blood-derived cultured human mast cells treated with dengue virus in the presence of subneutralizing concentrations of dengue virus-specific antibody also demonstrated significantly (P < 0.05) increased RANTES production, under conditions which did not induce significant degranulation. Chemokine responses were not observed when mast cells were treated with UV-inactivated dengue virus in the presence or absence of human dengue virus-specific antibody. Neither antibody-enhanced dengue virus infection of the highly permissive U937 monocytic cell line nor adenovirus infection of mast cells induced a RANTES, MIP-1{alpha}, or MIP-1ß response, demonstrating a selective mast cell response to dengue virus. These results suggest a role for mast cells in the initiation of chemokine-dependent host responses to dengue virus infection.


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INTRODUCTION
 
Dengue viruses are lipid-enveloped RNA viruses that belong to the family Flaviviridae. Four antigenically distinct serotypes, dengue virus types 1 to 4, are transmitted to humans via the mosquito vector Aedes aegypti (24). It is estimated that up to100 million individuals are infected with dengue virus annually (24). Increased vascular permeability, shock, and severe thrombocytopenia (12, 22, 23, 24, 37) are associated with severe forms of dengue virus infection, such as dengue hemorrhagic fever (DHF) and/or dengue shock syndrome (DSS). Substantial T cell activation is also observed in severe dengue disease (reviewed in reference 38). The pathogenesis of severe forms of dengue virus infection is not completely understood. Symptoms of severe disease most often occur in individuals experiencing secondary dengue virus infections (56). A number of cytokines and chemokines are found to be elevated in DHF and DSS patients (4, 17, 20, 31, 40, 54). The presence of heterotypic subneutralizing antibodies to dengue virus as a result of primary infection has been shown to potentiate secondary infection via antibody-dependent enhancement (23). Due to the lack of an animal model for DHF or DSS, most of the work investigating primary target cells and potential mechanisms of pathogenesis has been done in vitro. During antibody-enhanced dengue virus infection of monocytes (12), increased uptake of antibody-virus complexes occurs via Fc receptor-mediated binding to cells (53). Such infection of monocytes stimulates cytokine production (particularly tumor necrosis factor alpha), which perturbs endothelial cell function (23).

For many years there has been speculation as to the involvement of mast cells in dengue pathogenesis. Dengue patients exhibit increased levels of urinary histamine, a major granule product of mast cells, which correlates with disease severity (69). However, the potential role of mast cells has not yet been explored with regard to dengue pathogenesis. Mast cells play an important role in a wide variety of inflammatory reactions and in host defense against bacterial pathogens (19, 44). These cells selectively produce and secrete a variety of mediators including chemokines, cytokines, lipid mediators, and granule-associated products. Production of a wide variety of cytokines and chemokines, including tumor necrosis factor alpha (5), interleukin 6 (IL-6) (9), IL-1ß (46), IL-16 (58), IL-8 (47), ENA-78 (43), MIP-1{alpha} (75), MIP-1ß (63), and RANTES (55), has been demonstrated by human mast cells. Mast cells reside mainly in the tissues and have been shown to associate closely with blood vessels (64) and nerves (73). Human mast cells can express both Fc{varepsilon} receptor I (Fc{varepsilon}RI) (21, 67) and some Fc{gamma} receptors, including Fc{gamma}RI (50, 51) and Fc{gamma}RII (52, 72), and they contain Fc{gamma}RIII mRNA (52).

The mast cell is a potential target cell for dengue infection in view of its Fc receptor expression. This laboratory previously reported that the human KU812 mast cell-basophil line is permissive to dengue virus infection (33). In view of the critical role of chemokines in mobilizing effective immunity, the present study sought to investigate the production of the key chemokines RANTES, MIP-1{alpha}, and MIP-1ß by mast cells in response to viral infection. Human cord blood-derived mast cells (CBMC) as well as human mast cell lines were examined. The data suggest that mast cells may act as an early and important source of such chemokines during dengue virus-induced disease.


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MATERIALS AND METHODS
 
Dengue virus propagation. Dengue virus type 2 strain 16681 (26) was propagated in African green monkey kidney Vero cell monolayers cultured in endotoxin-free RPMI 1640 (Sigma, Oakville, Ontario, Canada) supplemented with 1% fetal calf serum (FCS; Life Technologies, Grand Island, N.Y.). For some experiments, virus was inactivated by UV irradiation (254 nm; 1,000 J/m2) (2).

Adenovirus and respiratory syncytial virus (RSV) propagation. Adenovirus type 37 (65, 70) was propagated in human lung epithelial A549 cell monolayers cultured in endotoxin-free RPMI 1640 medium (Sigma) supplemented with 1% FCS (Life Technologies). RSV (Long strain) was propagated in HEp-2 cells with the same medium.

Sera. For antibody-dependent enhancement assays of dengue virus infection, a serum pool was prepared by using nine convalescent-phase sera from patients recovering from a dengue virus serotype 2 infection. Anti-dengue virus immunoglobulin G (IgG) titers among the nine sera ranged from 207 to 465 enzyme immunoassay units per ml of serum. The individual patient sera and the dengue virus-specific monoclonal antibody 1B7 were obtained from a collection provided by Bruce Innis and described briefly in reference 29. Normal human AB sera were obtained from volunteer donors.

Mast cell culture. Human prebasophilic KU812 cells were maintained in RPMI 1640 (Life Technologies) supplemented with 10% FCS, 10 mM HEPES, 100 U of penicillin per ml, and 100 µg of streptomycin (Life Technologies) per ml. HMC-1 cells were maintained in Iscove's modified Dulbecco's medium (Life Technologies) supplemented with 10% FCS, 100 U of penicillin per ml, and 100 µg of streptomycin (Life Technologies) per ml. The human monocytic U937 cell line was maintained in RPMI 1640 (Life Technologies) supplemented with 10% FCS, 100 U of penicillin per ml, and 100 µg of streptomycin (Life Technologies) per ml. 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. (59) and characterized as previously described (42). These cells were consistently positive by flow cytometry for 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 of penicillin per ml, 100 µg of streptomycin (Life Technologies) per ml, 20% CCL-204 supernatant as a source of IL-6, 10-7 M prostaglandin E2 (Sigma), and 50 to 100 ng of stem cell factor (Peprotech, Rocky Hill, N.J.)/ml. The medium was changed once weekly for 5 to 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 that were >95% pure were used for this study. The mean purity of cord blood mast cells was 97%.

Dengue virus infection. Infection experiments with primary cultured human mast cell lines HMC-1 and U937 were carried out as previously described (33). Briefly, human KU812, HMC-1, and U937 were washed and resuspended at 106 cells/ml in appropriate media. Cells were adsorbed (90 min at 4°C) with aliquots of dengue virus (multiplicity of infection [MOI], 0.1 to 0.3), UV-inactivated dengue virus, combinations of UV-inactivated virus and human dengue virus immune serum (1:1,000 or 1:10,000 final dilution), combinations of virus and human dengue virus immune serum (1:1,000 or 1:10,000 final dilution), or virus and normal human sera (1:1,000 final dilution) (premixed 4°C for 90 min). Two dilutions of human dengue virus immune serum were used for every experiment to control for variation in dengue virus titer from experiment to experiment, since subneutralizing concentrations of antibody are necessary for antibody-dependent enhancement infection. In some experiments, KU812 cells were used to monitor effective dengue infection (33). Cells were then washed twice with RPMI culture medium (KU812, U937, and primary cultured human mast cells) or Iscove's culture medium (HMC-1), resuspended at a concentration of 106 cells/ml in the appropriate medium, and incubated at 37°C and 5% CO2. CBMC were cultured in RPMI 1640 (Life Technologies) supplemented with 20% FCS, 100 U of penicillin per ml, 100 µg of streptomycin (Life Technologies) per ml, 20 ng of SCF (Peprotech) per ml, and 100 µg of soybean trypsin inhibitor (Sigma) per ml.

Adenovirus and RSV infection. Human KU812, HMC-1, and U937 were washed once and resuspended at 106 cells/ml in the appropriate media supplemented as described above. Cells were adsorbed (3 h at 37°C and 5% CO2) with aliquots of adenovirus (MOI, 1), UV-inactivated adenovirus, or RSV (MOI, 1) in the absence or presence of human RSV-positive serum (1:1,000 and 1:10,000 dilutions). Human lung epithelial A549 cell monolayers were used to monitor adenovirus infection by immunoprecipitation. Cells were washed twice with the appropriate media supplemented as described above and incubated at 37°C and 5% CO2. An aliquot was removed to assess infection by immunoprecipitation of radiolabeled viral proteins.

Radiolabeling of KU812, HMC-1, and U937 cells with [35S]methionine-[35S]cysteine. At 24 h postinfection, culture supernatants were removed, and cells were resuspended in [35S]methionine-[35S]cysteine (NEN) in methionine- and cysteine-free medium (Gibco, BRL, Burlington, Ontario, Canada) for 3 to 4 h, with a 12- to 14-h chase at 37°C and 5%CO2. Culture supernatants were harvested and immunoprecipitated with dengue virus immune sera as previously described (33) or adenovirus rabbit immune sera and fixed Staphylococcus aureus as previously described (29).

Chemokine and cytokine analysis. RANTES, MIP-1{alpha}, MIP-1ß, IL-8, and ENA-78 were examined in 72-h-postinfection supernatants harvested from primary cultured human mast cells, i.e., KU812, HMC-1, and U937 cells inoculated with dengue virus (MOI, 0.1 to 0.3) alone, UV-inactivated dengue virus, combinations of UV-inactivated virus and human dengue virus immune serum (1:1,000 or 1:10,000 final dilution), combinations of virus and human dengue virus immune serum (1:1,000 or 1:10,000 final dilution), or virus and normal human sera (1:1,000 final dilution). Activation with 25 ng of phorbol myristate acetate (PMA) per ml and 5 x 10-7 M A23187 was used as a positive control, and medium alone was a negative control. RANTES and IL-8 were analyzed via an in-house enzyme-linked immunosorbent assay (ELISA). ELISA involved capture antibodies (RANTES antibody P-230-E; Endogen, Woburn, Mass.; IL-8 monoclonal antibody 208, R&D Systems, Minneapolis, Minn.). Nonspecific binding was blocked by using 1% bovine serum albumin (BSA) in phosphate-buffered saline for IL-8 and 1% BSA in Na2HPO4 (pH 8.3 to 8.5) for RANTES for 1 h at 37°C. Matched biotinylated chemokine detection antibodies were BAF 278 for RANTES and BAF 208 (R&D Systems) for IL-8. A commercial ELISA amplification system (Life Technologies) was used for detection. The sensitivities of both the RANTES and IL-8 assays were 7.8 pg/ml. MIP-1{alpha} and MIP-1ß in culture supernatants were analyzed with commercial ELISA kits (Endogen).

Short-term mediator release and {alpha}-hexosaminidase assay. CBMC (5 x 104/ml) were incubated for 30 min at 37°C in the presence or absence 50 µl of dengue virus containing medium alone, combinations of UV-inactivated virus and human dengue virus immune serum (1:1,000 or 1:10,000 final dilution), combinations of virus and human dengue virus immune serum (1:1,000 or 1:10,000 final dilution), or combinations of medium and human dengue virus immune serum (1:1,000 or 1:10,000 final dilution). Activation with 5 x 10-7 M A23187 was used as a positive control, and medium alone was a negative control. The reaction was stopped with the addition of 450 µl of cold HEPES-Tyrode's buffer. Cells were centrifuged at 300 x g for 10 min at 4°C. After collection of supernatants, the pellets were resuspended in the 500 µl of the buffer and disrupted by sonication. The modified HEPES-Tyrode's buffer was prepared with (concentrations in millimolar units) Na (137), glucose (5.6), KCl (2.7), NaH2PO4 (0.5), CaCl2 (1), and HEPES (10), plus 0.1% BSA, pH 7.3.

A ß-hexosaminidase assay was carried out by using a previously reported method (62). Briefly, 50 µl of supernatant and pellet samples, in duplicate, were incubated with 50 µl of 1 mM p-nitrophenyl-N-acetyl-ß-D-glucosaminide (Sigma) dissolved in 0.1 M citrate buffer, pH 4.5, in a 96-well microtiter plate at 37°C for 1 h. The reaction was stopped with 200 µl of 0.1 M carbonate buffer, pH 10.5, per well. The plate was read at 405 nm in an ELISA reader. The net percent ß-hexosaminidase release was calculated as follows: [(ß-hexosaminidase in supernatant)/(ß-hexosaminidase in supernatant + ß-hexosaminidase in pellet)] x 100.

Inflammatory cytokine stimulation of mast cells. KU812 cells (106/ml) were treated for 24 h with various concentrations of recombinant human IL-6 (R&D Systems) or IL-1ß (R&D Systems) in RPMI 1640 (Life Technologies) supplemented with 10% FCS, 10 mM HEPES, 100 U of penicillin per ml, and 100 µg of streptomycin (Life Technologies) per ml. Cell supernatants were harvested and analyzed for RANTES concentration by ELISA.

Fluorescence microscopy. KU812, HMC-1, and U937 cells inoculated with dengue virus (MOI, 0.1 to 0.3) or dengue virus-antiserum combinations were harvested 24 h postinfection. Cells were fixed with 4% paraformaldehyde, washed, resuspended in 10% dimethyl sulfoxide in phosphate-buffered saline, and frozen at -80°C. Following permeabilization with 0.1% saponin for 1 h at room temperature, samples were washed and resuspended in 1% BSA-0.2% sodium azide solution. Mouse anti-dengue virus monoclonal antibody 1B7 (30) and isotype-matched mouse IgG2a antibody (negative control) were employed as primary antibodies and incubated on ice for 1 h. Subsequently, samples were washed and incubated with Texas red-labeled anti-mouse IgG antibody (Molecular Probes, Eugene, Oreg.) for 1 h on ice. Cytospins were made of each sample and viewed via fluorescence microscopy to assess the number of infected cells. A total of 1,000 cells were counted under bright field, with the number of positive red fluorescent cells (Texas red) recorded to give the percentage of cells infected with dengue virus.

Statistical analysis. Statistical significance of changes in RANTES production by KU812 cells was assessed by using a parametric approach. Repeated-measures analysis of variance was performed followed by examination of specific groups using the Bonferroni multiple-comparison test. The statistical significance of RANTES, MIP-1{alpha}, and MIP-1ß production by HMC-1 and U937 cells, as well as KU812 MIP-1{alpha} and MIP-1ß, in response to dengue virus and adenovirus infection was assessed by a nonparametric approach. CBMC RANTES production in response to dengue virus was assessed in the same manner. These data were initially analyzed with Friedman's test for all data obtained, followed by examination of specific groups using the Wilcoxon signed ranks test.


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RESULTS
 
Antibody-enhanced infection of dengue virus in mast cells and monocytes. The human mast cell lines KU812 and HMC-1 and the monocytic cell line U937 were mock inoculated or inoculated with dengue virus in the presence or absence of human dengue virus immune serum (1:1,000 and 1:10,000 final dilution). Immunoprecipitation analysis (Fig. 1A) demonstrated that U937 cells as well as the mast cell-basophil line KU812 and the mast cell line HMC-1 were permissive to dengue virus infection in the presence of human dengue virus immune sera. The degree of infection was lower in HMC-1 cells than in KU812 and U937 cells. KU812, HMC-1, and U937 cells inoculated with dengue virus in the presence of normal human serum (NHS) (1:1,000 dilution), UV-inactivated dengue virus alone, or human dengue virus immune serum at either the 1:1,000 or 1:10,000 dilution showed no evidence of infection (data not shown).




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  		FIG. 1. Antibody-enhanced dengue virus infection of KU812, HMC-1, and U937 cells. (A) Cultures of KU812, HMC-1, and U937 cells were inoculated with UV-inactivated dengue virus (MOI, 0.1 to 0.3) or with combinations of virus with NHS (final dilution, 1:1,000) or human dengue virus immune serum (final dilution, 1:1,000 or 1:10,000) to ensure subneutralizing concentrations), and dengue proteins were immunoprecipitated. Exposure times were 2 days for KU812 and U937 cells and 8 days for HMC-1 cells. Data are representative of four separate experiments. (B to E) Immunofluorescence of dengue virus-inoculated KU812 and U937 cells. Dengue virus-infected cells were visualized by fluorescent microscopy using Texas red-labeled secondary antibody. (B) KU812 cells inoculated with dengue virus and NHS (1:1,000 final dilution); (C) antibody-enhanced dengue virus-infected KU812 cells with 1:1,000 final dilution of human dengue virus immune serum; (D) U937 cells inoculated with dengue virus and NHS (1:1,000 final dilution); (E) antibody-enhanced dengue virus-infected U937 cells with 1:1,000 final dilution of human dengue virus immune serum. Data are representative of three separate experiments.

Relative permissiveness of mast cells versus monocytes infected with dengue virus in the presence of antibody. In order to assess the proportion of mast cells-basophils or monocytes infected, fluorescence microscopy was employed. KU812, HMC-1, and U937 cells inoculated with dengue virus-NHS and dengue virus-human dengue virus immune serum combinations were harvested 24 h postinfection and examined following staining with an antibody specific for the envelope protein of the virus (Fig. 1). None of the cell lines showed virus-positive cells when inoculated with dengue virus in the presence of NHS. The percentages of KU812, HMC-1, and U937 dengue virus-positive cells in three experiments are shown in Table 1. Ranges of 3.10 to 14.19% and 1.2 to 3.0% of the populations of KU812 and HMC-1 cells, respectively, were infected with dengue virus, while the proportion of infected U937 cells ranged from 3.18 to 6.38%. IgG2a isotype control antibody-stained slides were less than 0.2% positive.


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  		TABLE 1. Percentage of dengue virus-infected KU812, HMC-1, and U937 cells

Chemokine production by antibody-enhanced dengue virus-infected cells. To analyze chemokine production in response to antibody-enhanced dengue virus infection of KU812, HMC-1, and U937 cells, 72-h cell supernatants were harvested from cultures and screened by ELISA. RANTES production was 260-fold greater in supernatants obtained from dengue virus-infected KU812 cultures than in cells incubated with medium alone. KU812 cells treated with UV-inactivated dengue virus or dengue virus in the presence of NHS (Fig. 2A) did not demonstrate enhanced RANTES production. MIP-1{alpha} and MIP-1ß production was also significantly enhanced in antibody-enhanced dengue virus-infected KU812 cells (Fig. 2B and C). In contrast, neither IL-8 nor ENA-78 levels were elevated (data not shown). Analysis of HMC-1 chemokine production indicated that HMC-1 cells produce significantly increased amounts of RANTES (Fig. 2D) and MIP-1{alpha} (Fig. 2E) but not MIP-1ß (Fig. 2F) in response to antibody-enhanced dengue virus infection. However, U937 cells failed to produce RANTES (Fig. 2G), MIP-1{alpha} (Fig. 2H), or MIP-1ß (Fig. 2I) in response to any of the conditions employed.



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  		FIG. 2. Chemokine production by KU812, HMC-1, and U937 cells following inoculation with dengue virus. (A to C) KU812 chemokine production at 72 h postinfection. RANTES data (A) are from nine separate experiments with duplicate samples; MIP-1{alpha} (B) and MIP-1ß (C) data are from five separate experiments with duplicate samples. (D to F) HMC-1 chemokine production at 72 h postinfection. The data are from five separate experiments with duplicate samples. (G to I) U937 chemokine production at 72 h postinfection. The data are from four separate experiments with duplicate samples. Experiments used either pooled sera from convalescent dengue patients or one patient serum, 7873. Cells incubated with medium alone exhibit constitutive production; PMA- and A23187-treated cells were used as positive controls. Culture supernatants were analyzed by ELISA. Significant differences from the values for medium-alone samples are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Please note y-axis differences. Data are means ± SEM. a, >5,000 pg/ml; b, >7,000 pg/ml

Time course of chemokine production by antibody-enhanced dengue virus-infected KU812 cells. KU812 cells were inoculated with dengue virus-NHS and dengue virus-human dengue virus immune serum combinations. Time course analyses of antibody-enhanced dengue virus-infected KU812 cells were carried out in 4-, 24-, 48-, and 72-h cell supernatants harvested from KU812 cultures. RANTES, MIP-1{alpha}, MIP-1ß, and IL-8 levels were assessed by ELISA. The majority of RANTES production occurred by 24 h postinfection; levels persisted in supernatants for up to 72 h (Fig. 3A). A continuous increase in MIP-1{alpha} (Fig. 3B) and MIP-1ß (Fig. 3C) production by dengue virus-infected cells was noted from 4 to 72 h postinfection, with the highest levels being observed by 72 h. Examination of IL-8 production revealed no modulation by any of the infection conditions at any time point (Fig. 3D).



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  		FIG. 3. Time course of RANTES, MIP-1{alpha}, and MIP-1ß production by antibody-enhanced dengue virus-infected KU812 cells. RANTES (A), MIP-1{alpha} (B), MIP-1ß (C), and IL-8 (D) were measured in cell supernatants at 4, 24, 48, and 72 h postinfection by ELISA. KU812 cells incubated with medium alone exhibit constitutive production. The data (means ± SEM) are from two separate experiments with duplicate samples and pooled sera from convalescent dengue patients.

Concurrent production of virus and RANTES by antibody-enhanced dengue virus-infected KU812 cells. A more detailed time course examining the RANTES response in KU812 cells was undertaken to determine the temporal relationship between dengue virus-antibody treatment, RANTES production, and infectious-virion production as assessed by 50% tissue culture infective doses (33). KU812 cells were inoculated with dengue virus-NHS (1:1,000 final dilution), dengue virus-human dengue virus immune serum (1:1,000 and 1:10,000 final dilutions), or dengue virus alone. RANTES and infectious virus production were assessed at 4, 8, 16, 24, 48, and 72 h postinfection. The analysis indicated that there was a large increase in RANTES production by antibody-enhanced dengue virus-infected KU812 cells between 8 and 16 h postinfection (Fig. 4), with coincident virus production. RANTES levels continued to increase up to 72 h postinfection, while infectious virion production persisted until 24 h and began to decline by 48 h postinfection. The amount of preformed RANTES associated with unactivated KU812 cells was <0.0026 fg/cell (n = 2).



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  		FIG. 4. Concurrent infectious virus and RANTES production by antibody-enhanced dengue virus-infected KU812 cells. KU812 cells were inoculated with dengue virus alone (Den), dengue virus-NHS, or dengue virus-immune serum (1:1,000 and 1:10,000 final dilutions). Both RANTES and virus production were assessed in cell supernatants at 4, 8, 16, 24, 48, and 72 h postinfection by ELISA and 50% tissue culture infective doses, respectively. The data (means ± SEM) are from two separate experiments with duplicate samples and pooled sera from convalescent dengue patients.

Cord blood-derived human mast cell induction of chemokines by dengue virus. Under conditions which were effective in inducing dengue virus infection in KU812 cells, five of five CBMC preparations examined demonstrated increased RANTES production (Fig. 5A) for one or both of the dengue virus-human dengue virus immune sera combinations. Statistical analysis demonstrated a significant (P < 0.05) increase in RANTES production (10- to 70-fold) compared to treatment with UV-inactivated dengue virus at both concentrations of specific antibody. In addition, four of four CBMC preparations examined had increased levels (1.5- to 3.5-fold) of MIP-1ß when treated with dengue virus in the presence of dengue virus-specific antibody (Fig. 5B). This increased chemokine production was restricted to conditions in which live dengue virus-human dengue virus immune sera combinations were employed. UV-inactivated virus alone (Fig. 5A and B) or in combination with human dengue virus immune sera at both 1:1,000 and 1:10,000 final dilutions (Table 2) as well as dengue virus-NHS combinations failed to modulate either mediator in CBMC.



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  		FIG. 5. Cord blood-derived mast cell production of RANTES and MIP-1ß in response to dengue virus and antibody. RANTES (A) and MIP-1ß (B) were measured in cell supernatants at 72 h postinfection by ELISA. Cord blood-derived mast cells treated with medium alone exhibit constitutive production; PMA- and A23187-treated cord blood-derived mast cells were used as positive controls. These data are from five (RANTES) or four (MIP-1ß) subjects with duplicate samples. Experiments used pooled sera from convalescent dengue patients. a, >400 pg/ml; ND, not done. Statistical analysis of RANTES production indicated that dengue virus-dengue virus immune serum versus UV-inactivated dengue virus at both serum dilutions induced significant increases (P < 0.05).


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  		TABLE 2. Fc receptor cross-linking by dengue virus-antibody complexes fails to induce appreciable levels of RANTES in comparison to levels produced in response to active infection

Cross-linking of surface Fc receptors is not responsible for the chemokine response. To further investigate the mechanism of the mast cell response to dengue virus, specifically the importance of Fc receptor cross-linking, KU812 cells were inoculated with UV-inactivated dengue virus or UV-inactivated virus-human dengue virus immune serum combinations (1:1,000 and 1:10,000 final dilutions). These conditions would be expected to produce Fc receptor cross-linking but not active infection. As further controls, dengue virus-NHS (1:1,000 final dilution) or dengue virus-human dengue virus immune serum combinations (1:1,000 or 1:10,000 final dilution) were employed. The RANTES response was examined at 72 h postinfection. Low levels of RANTES (Table 2) were produced by KU812 cells treated with virus and antibody combinations that did not yield infection. The RANTES production in response to Fc receptor cross-linking was a mean of 0.62% of the response to live virus plus antibody for KU812 cells (n = 3) and a mean of 4.9% for CBMC (n = 3).

Dengue virus alone or in combination with human dengue virus-specific antibody does not induce mast cell degranulation. ß-Hexosaminidase release by CBMC incubated with live or inactivated virus in the presence or absence of human dengue virus-specific antibody or with medium containing antibody alone was assessed to determine if surface cross-linking of Fc receptors upon binding of the virus-antibody complexes or antibody alone could activate mast cells. There was no significant ß-hexosaminidase release in response to any of the conditions tested, as indicated by data from two separate experiments (means ± standard errors of the means [SEM]): inactivated virus with 1:1,000 antibody, 8.79% ± 6.22%; live virus with 1:1,000 antibody, 10.12% ± 7.16%; and medium with 1:1,000 antibody, 7.53% ± 5.32% (spontaneous levels were 7.94% ± 5.62%). The value for positive control calcium ionophore was 16.06% ± 11.36%.

Inflammatory cytokines do not induce significant levels of RANTES in KU812 cells. KU812 cells were stimulated with 10, 50, 100, or 200 pg of recombinant human IL-1ß (rhIL-1ß)/ml and 5, 10, or 20 ng of rhIL-6 for 24 h/ml. Cell supernatants were harvested and analyzed by ELISA for RANTES content. Data from four separate experiments indicated no significant increase in production of RANTES by KU812 cells for any of the conditions tested (rhIL-1ß at 10 pg/ml, 209.1 ± 61.4; rhIL-1ß at 50 pg/ml, 258.7 ± 69.3; rhIL-1ß at 100 pg/ml, 366.5 ± 92.5; rhIL-1ß at 200 pg/ml, 317.8 ± 74.2; rhIL-6 at 5 ng/ml, 242.2 ± 62.7; rhIL-6 at 10 ng/ml, 200.6 ± 82.6; rhIL-6 at 20 ng/ml, 250.9 ± 97.1) compared to medium alone (204.2 ± 60.7). PMA and A23187 were used as a positive control and gave significant levels of RANTES (526.5 ± 71.9, P < 0.01). Data are expressed as means ± SEM.

Dengue virus, but not two unrelated viruses, activate the chemokine response in mast cells-basophils. To assess the specificity of the chemokine mast cell-basophil response to dengue virus infection, two unrelated viruses were employed, adenovirus type 37 and RSV. To assess mast cell permissiveness to adenovirus, the cell lines KU812 and HMC-1, the monocytic cell line U937, and A549 human lung epithelial cells were mock inoculated with UV-inactivated adenovirus or with adenovirus alone. Supernatants were harvested and immunoprecipitated. KU812, HMC-1, and U937 cells were permissive to adenovirus infection (Fig. 6) with A549 cells as a positive control. At 72 h postinfection, supernatants were analyzed for chemokines. Neither mast cells-basophils nor U937 cells demonstrated enhanced production of RANTES, MIP-1{alpha}, or MIP-1ß in response to adenovirus infection (Table 3). Furthermore, similar experiments using RSV, in both the presence and absence of RSV immune serum, indicated that KU812 cells were not permissive to RSV infection, and treatment did not result in RANTES production at 24, 48, or 72 h posttreatment (data not shown).



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  		FIG. 6. Adenovirus infection of A549, KU812, HMC-1, and U937 cells. Cultures of KU812, HMC-1, and U937 cells were inoculated with adenovirus or UV-inactivated adenovirus virus (MOI, 1). Cultures were incubated at 37°C and radiolabeled with [35S]methionine-[35S]cysteine from 24 h postinfection for 3 to 4 h followed by a 12- to 14-h chase. Cell supernatants were harvested, immunoprecipitated, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography. The positions of radiolabeled viral hexon, penton, and fiber proteins are indicated. Exposure times were 16 h for the A549 cells and 2 days for KU812, HMC-1, and U937 cells. Data are representative of three separate experiments.


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  		TABLE 3. Adenovirus infection does not modulate chemokine production in KU812, HMC-1, and U937 cells


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DISCUSSION
 
These findings demonstrate that dengue virus plus dengue virus-specific antibody treatment results in selective production of the T-cell chemoattractants RANTES, MIP-1{alpha}, and MIP-1ß by human mast cells. To our knowledge, this is the first report of a mast cell chemokine response to a viral pathogen.

Dengue virus infection of peripheral blood monocytes is increased dramatically in the presence of subneutralizing concentrations of virus-specific antibody (10, 11, 18, 25, 48), due to enhanced virus-cell attachment via surface Fc{gamma} receptors (53). The present study investigated the mast cell production of essential chemokines involved in T-cell mobilization in response to treatment with dengue virus and specific antibody. A number of complementary human mast cell-basophil models were employed in this study. The KU812 cell line has been documented to possess properties of both basophils (34) and mast cells (7). KU812 cells have been demonstrated to express mRNA for mast cell tryptase and mast cell carboxypeptidase with low-level expression of major basic protein (7). KU812 cells express Fc{varepsilon}R1, the high-affinity IgE receptor, (1), and Fc{gamma}RII (32). The HMC-1 mast cell line (16) has been found to express chymase mRNA and higher levels of tryptase mRNA than KU812 cells (74). In contrast to KU812 cells, HMC-1 cells have Fc{gamma}RII (72) but do not consistently express Fc{varepsilon}RI (74).

We have previously demonstrated antibody-enhanced dengue virus infection of the mast cell-basophil cell line KU812 (33). This infection resulted in the selective production of the vasoactive cytokines IL-1ß and IL-6. In the present study, antibody-enhanced dengue virus infection of a further mast cell line, HMC-1, was demonstrated. The level of infection was lower in HMC-1 cells than in either KU812 or U937 cells (Fig. 1A; Table 1). Assessment of the number of dengue virus-infected cells by using immunofluorescence to detect the dengue virus envelope protein indicated that a greater proportion of KU812 cells were infected than monocytic U937 cells under the same infection conditions. However, immunoprecipitation indicated greater amounts of viral protein by 24 h postinfection in U937 cells (Fig. 1A) than in either KU812 cells or HMC-1 cells.

Antibody-enhanced dengue virus infection of KU812 and HMC-1 cells resulted in the induction of significant levels of RANTES, MIP-1{alpha}, and MIP-1ß in both KU812 cells and HMC-1 cells. The levels of RANTES production observed in dengue virus-infected KU812 cells were very substantial, with a 260-fold increase over controls. In some cases, levels of RANTES detected exceeded 50 fg/cell and could not be accounted for by preformed RANTES pools of <0.003 fg/cell. Interestingly, lymphocytes stimulated with PMA and monocytes stimulated with lipopolysaccharide have been reported to produce only 1 to 5 fg of RANTES per cell (27). In contrast, production of the neutrophil chemoattractants IL-8 (Fig. 3D) and ENA-78 (data not shown) was not enhanced in antibody-enhanced dengue virus-infected KU812 cells compared to controls. KU812 cells stimulated with the vasoactive cytokines IL-6 and IL-1ß, previously shown to be produced by dengue virus-infected KU812 cells (33), at concentrations observed by 72 h postinfection or greater did not induce the production of significant levels of RANTES, suggesting that the RANTES observed in our system is not a result of feedback from cytokines already present in our system. This suggests a highly selective mast cell response to infection. Both KU812 cells and HMC-1 cells have been demonstrated to produce IL-8 in response to treatment with stromal cell-derived factor 1{alpha} (42) or PMA and ionophore (47). Antibody-independent dengue virus infection of the human umbilical cord vein endothelial cell line ECV304 has been shown to result in significant IL-8 production (10 fg/cell) by 72 h postinfection (4). Furthermore, influenza A virus and Sendai virus infections of human macrophages result in significant IL-8 production, indicating that IL-8 can be induced by other viral infections (45). While U937 cells are fully permissive to antibody-enhanced dengue virus infection, they do not produce any of the chemokines examined, suggesting differential responses to dengue virus infection in mast cells and monocytes.

Significantly increased RANTES production was not detected following UV-inactivated virus-human dengue virus immune serum treatment of either KU812 cells or CBMC (Table 2) derived from three individuals. These data suggest that Fc receptor cross-linking is not responsible for the majority of the observed increase in chemokine production. In addition, CBMC fail to degranulate in response to virus-antibody complexes or antibody alone, as measured by percent ß-hexosaminidase, further supporting the concept that a virus-specific rather than degranulation-related signal is responsible for inducing chemokine production. We have not directly addressed the role of IgE, which may be involved in our system; however, heat inactivation (56°C, 30 min) of serum does not influence infection (R. Anderson, personal communication) while it is known to prevent human IgE binding to Fc{varepsilon}RI (57), suggesting that the role of IgE in dengue virus infection is minimal. Furthermore, no increase in chemokine production was observed when mast cells (KU812 cells, HMC-1 cells, and CBMC) were treated with dengue virus-NHS combinations, further demonstrating the critical importance of specific antibodies in enhancing dengue virus infection in mast cells.

Confirmation of the chemokine response observed with the cell lines was demonstrated using human CBMC (59). Exposure of CBMC to dengue virus in the presence of dengue virus-specific antibodies resulted in a significant RANTES response by mast cells from five subjects (Fig. 5A) and an increased MIP-1ß (Fig. 5B) response by mast cells from four subjects. These data support the hypothesis that mast cells may contribute to the initiation of chemokine-dependent host responses during dengue virus infections. The amount of RANTES produced by CBMC was significantly lower than that produced by KU812 cells but was similar to data obtained with the HMC-1 cell line. Low levels of dengue virus infection may relate to the lower levels of RANTES produced. Perhaps due to the high levels of protease activity in CBMC, we have been unable to demonstrate antibody-enhanced infection of these cells by immunoprecipitation.

To determine whether the observed mast cell chemokine response occurred in response to infection with an alternate virus, we used adenovirus, a nonenveloped DNA virus. KU812 and HMC-1 mast cells were permissive to adenovirus infection. However, analysis of the mast cell chemokine response to infection did not demonstrate enhanced RANTES, MIP-1{alpha}, and MIP-1ß production. In vitro adenovirus 21 infection of human respiratory epithelial HEp-2 cells and human embryonic WI-38 lung fibroblasts has been shown to result in significant (2 to 4 fg/cell) RANTES production (8). These observations provide further evidence for a selective response of mast cells to dengue virus, though they do not rule out the possibility of other virus activators of RANTES in these cells. Furthermore, RSV treatment of KU812 mast cells in the presence or absence of subneutralizing concentrations of human RSV immune serum also failed to induce a significant RANTES response (data not shown). Studies are planned to investigate mast cell responses to flaviviruses other than dengue virus.

Mast cells have been implicated in several viral diseases, including dengue virus infection. Dengue virus-infected patients exhibit increased levels of urinary histamine, a major granule product of mast cells. Increased histamine was also found to correlate with disease severity (69). A large study from Thailand that examined 100 dengue virus-infected patients demonstrated that mast cells in the connective tissue around the thymus showed swelling, vacuolation of the cytoplasm, and loss of granule integrity (6), suggestive of mast cell activation. However, if selective chemokine production by mast cells occurred in vivo in the absence of mast cell degranulation, as observed in our in vitro studies, mast cells could be an important source of such mediators in the absence of classical degranulation.

Insights into the mechanism of the RANTES response in antibody-enhanced dengue virus-infected KU812 cells were obtained from examination of the early time course of infection. Levels of both RANTES and infectious virus production were low at 4 h postinfection and rose rapidly between 8 and 16 h postinfection (Fig. 4). Dengue virus RNA replication in vertebrate cells has been demonstrated by 6 h postinfection followed by increasing viral protein synthesis (13). Taken together, these observations are consistent with expression of a viral gene product(s) in infected mast cells contributing to the observed RANTES response. ß-Chemokine induction, as a result of replicating virus, has been demonstrated in other viral systems, specifically epithelial cells infected with RSV (28). The lack of a very early RANTES response following virus inoculation suggests that the RANTES response in mast cells was not the result of preformed RANTES release as a result of dengue virus-antibody complexes but rather de novo synthesis. The finding that KU812 cells contain little preformed RANTES, <0.0026 fg/cell, supports this concept. The lack of a mast cell RANTES, MIP-1{alpha}, or MIP-1ß response to UV-inactivated virus-human dengue virus immune serum combinations further suggests that viral attachment and entry were not sufficient signals for the mast cell RANTES production. Elucidation of the RANTES mechanism is under way in our laboratory.

While this is the first report of chemokine production in response to viral infection in mast cells, enhanced ß-chemokine production following virus infection has been demonstrated in other cells undergoing viral infection including dengue virus. Antibody-independent dengue virus infection of ECV304 cells has been reported to result in significant RANTES elevation (4). In addition, in vitro infection of CBMC by a clinical isolate of dengue virus type 3 resulted in higher levels of MIP-1{alpha} (49). Furthermore, MIP-1{alpha} and MIP-1ß were recently found to be induced by dengue virus type 2 infection in the human K562 myelomonocytic cell line and peripheral blood mononuclear cells (PBMC) (66). Human immunodeficiency virus infection of both activated and naive Th1 cells results in significant levels of RANTES, MIP-1{alpha}, and MIP-1ß by 4 to 6 days postinfection (3). In addition, PBMC obtained from individuals infected with human T-lymphotropic virus type II spontaneously secreted significant levels of MIP-1{alpha} and MIP-1ß and to a lesser extent RANTES compared to uninfected controls (41). Both influenza A virus and Sendai virus infection of macrophages results in upregulation of, among others, RANTES, MIP-1{alpha}, and MIP-1ß (45). RSV infection of HEp-2, MRC-5, and WI-38 cells results in significant levels of MIP-1{alpha} and RANTES production (8). However, ß-chemokine production is not enhanced in all models of viral infection, as cytomegalovirus and herpes simplex virus type 1 infection in those cell lines failed to modulate MIP-1{alpha} or RANTES (8).

Selective production of RANTES, MIP-1{alpha}, and MIP-1ß by antibody-enhanced dengue virus infection of mast cells may provide additional insights into the pathogenic mechanisms of severe dengue disease. Our finding of increased RANTES, MIP-1{alpha}, and MIP-1ß production in antibody-enhanced dengue virus-infected mast cells raises the possibility that mast cells-basophils may represent either a systemic or local source of chemokines involved in mobilization of immune effector cells, including T lymphocytes, in dengue virus infection. Extravasation of T cells through the endothelium to the site of inflammation is dependent on both adhesion molecule expression and ß-chemokine-receptor interaction on recruited cells (15). Dengue virus-specific CD4+ and CD8+ T lymphocytes have been shown to be generated following primary dengue virus infection (35, 36, 39). Dengue virus-specific memory CD4+ T cells have been detected in PBMC isolated from patients following primary exposure. Furthermore, these memory T cells are serotype cross-reactive (36). Cross-reactive CD8+ dengue virus-specific T cells have also been isolated from PBMC obtained from a dengue virus-infected individual (14). The ß-chemokine RANTES has been demonstrated to attract CD4+ memory T cells (60), while MIP-1ß has been shown to preferentially attract naive CD4+ T cells. MIP-1{alpha} is a more potent lymphocyte chemoattractant, mainly attracting B cells and CD8+ T cells (61). Interestingly, mast cells have been implicated in regulating T-lymphocyte traffic into peripheral lymph nodes during development of an immune response. Mast cells accumulated in the lymph nodes of contact-sensitized mice were found to be the predominant source of MIP-1ß (68, 71). The finding that dengue virus induced significant RANTES, MIP-1{alpha}, and MIP-1ß production by human mast cells suggests a potential role for mast cells in both recruiting and activating T lymphocytes at the site of infection during secondary dengue virus infection.

The observation that dengue virus-antibody complexes are much more potent than dengue virus alone in inducing mast cells to produce these chemokines shows an interesting parallel with the known epidemiological evidence that preexisting immunity is a risk factor for DHF/DSS in human infections (24). The underlying mechanisms of hemorrhagic disease induced by a number of viruses are poorly understood, including the more severe forms of dengue virus infection, DHF and DSS. DHF and DSS patients are characterized by increased capillary permeability and abnormal homeostasis (24). Our results strongly indicate that antibody-enhanced dengue virus infection of mast cells and basophils in vitro leads to the production of mediators which may contribute to cell recruitment during severe dengue virus infection in vivo. The location of mast cells in skin and in close association with blood vessels in most tissues (64) may enhance the effectiveness of this process. Mast cell responses could play a role in host defense or in enhancing the process of vascular damage.

In conclusion, we have demonstrated a selective and potent induction of chemokines by dengue virus in the presence of dengue virus-specific antibody in both mast cell-basophil lines and CBMC. The consequences of this novel aspect of the mast cell response to viral infection require further study.


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ACKNOWLEDGMENTS
 
This work was supported by the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council (NSERC).

We are grateful to B. Innis and A. King of the Walter Reed Army Institute of Research for providing the human dengue virus immune serum and monoclonal antibodies used in this study. Thanks go to A. Stadnyk for assistance with the ENA-78 ELISA. Special thanks go to Cheng-Hsien Chang for help with the adenovirus experiments and to Yi-Song Wei and Ula Kadela-Stolarz for excellent technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. Phone: (902) 494-5118. Fax: (902) 494-5125. E-mail: Jean.Marshall{at}Da.ca. Back


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Journal of Virology, August 2002, p. 8408-8419, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8408-8419.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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