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Dengue virus selectively induces human mast cell chemokine production - PubMed

Dengue virus selectively induces human mast cell chemokine production

Christine A King et al. J Virol. 2002 Aug.

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-1alpha, and MIP-1beta, 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-1alpha, or MIP-1beta 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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Figures

FIG. 1.
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.

FIG. 1.
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.

FIG. 2.
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-1α (B) and MIP-1β (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

FIG. 3.
FIG. 3.

Time course of RANTES, MIP-1α, and MIP-1β production by antibody-enhanced dengue virus-infected KU812 cells. RANTES (A), MIP-1α (B), MIP-1β (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.

FIG. 4.
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.

FIG. 5.
FIG. 5.

Cord blood-derived mast cell production of RANTES and MIP-1β in response to dengue virus and antibody. RANTES (A) and MIP-1β (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-1β) 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).

FIG. 6.
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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