title: Cytokine production by dengue virus antigen-responsive human T lymphocytes in vitro examined using a double immunocytochemical technique.
abstract: 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-gamma (IFN-gamma), interleukin (IL) -2, -4, -1alpha, -1beta, and -6, tumor necrosis factor beta (TNF-beta), and TNF-alpha] 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-gamma, IL-2, IL-4, and TNF-beta-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.
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, Worcester 01655, USA.
authors.forename: M
authors.initials: M
authors.lastname: Mori
authors.forename: I
authors.initials: I
authors.lastname: Kurane
authors.forename: J
authors.initials: J
authors.lastname: Janus
authors.forename: F A
authors.initials: FA
authors.lastname: Ennis
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism*; D008233:Lymphotoxin-alpha / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D000956:Antigens, Viral; D007376:Interleukin-2; D007378:Interleukins; D008233:Lymphotoxin-alpha; D015847:Interleukin-4; D007371:Interferon-gamma