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Cytokine production by dengue virus antigen‐responsive human T lymphocytes in vitro examined using a double immunocytochemical technique
Author(s) -
Mori Masuko,
Kurane Ichiro,
Janus Jurand,
Ennis Francis A.
Publication year - 1997
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.61.3.338
Subject(s) - biology , cytokine , antigen , immune system , immunology , dengue virus , cd8 , tumor necrosis factor alpha , peripheral blood mononuclear cell , elispot , microbiology and biotechnology , virology , dengue fever , in vitro , biochemistry
A number of studies suggest that cytokines may contribute to the pathogenesis of viral infections, including dengue. In this study, we developed a double immunocytochemical method and characterized cytokine‐producing cells in the peripheral blood mononuclear cells (PBMC) of dengue virus‐immune donors after in vitro stimulation with specific dengue antigens. We found that double immunostaining using immunoalkaline phosphatase (Vector blue) for cytokines [interferon‐γ (IFN‐γ), interleukin (IL) ‐2, ‐4, ‐1α, ‐1β, and ‐6, tumor necrosis factor β (TNF‐β), and TNF‐α] and immunoperoxidase [diaminobenzidine (DAB)] for cell surface markers (CD3, CD4, CD8, CD20, and CD68) provided the best distinction of double‐positive cells from single‐positive or ‐negative cells. The number of IFN‐γ, IL‐2, IL‐4, and TNF‐β‐positive cells increased 2 or 3 days after stimulation with specific dengue antigens. No or very few cytokine‐producing cells were detected in the PBMC of non‐immune donors stimulated with dengue antigens and the PBMC of immune donors stimulated with a control antigen. The analysis of cell surface markers showed that mainly CD4 + and CD8 + T cells produced these cytokines. The results obtained by immunocytochemistry were consistent with cytokine levels detected in the culture medium assayed by enzyme‐linked immunosorbent assay. In conclusion, this double immunocytochemistry technique is suitable for the detection and characterization of cytokine‐producing cells in PBMC. Furthermore, the results support the hypothesis that antigen‐stimulated CD4 + and CD8 + T cells produce cytokines that may play a role in the pathogenesis of dengue virus infection. J. Leukoc. Biol . 61: 338–345; 1997.