CYTOKINE PRODUCTION BY DENGUE VIRUS ANTIGEN-RESPONSIVE HUMAN T-LYMPHOCYTES IN-VITRO EXAMINED USING A DOUBLE IMMUNOCYTOCHEMICAL TECHNIQUE

A number of studies suggest that cytokines may contribute to the patho genesis of viral infections, including dengue, In this study, we devel oped a double immunocytochemical method and characterized cytokine-pro ducing cells in the peripheral blood mononuclear cells (PBMC) of dengu e virus-immune donors after in vitro stimulation with specific dengue antigens, We found that double immunostaining using immuno-alkaline ph osphatase (Vector blue) for cytokines [interferon-gamma (IFN-gamma), i nterleukin (IL) -2, -4, -1 alpha, -1 beta, and -6, tumor necrosis fact or beta (TNF-beta), and TNF-alpha] and immunoperoxidase [diaminohenzid ine (DAB)] for cell surface markers (CD3, CD4, CD8, CD20, and CD68) pr ovided the best distinction of double-positive cells from single-posit ive or -negative cells, The number of IFN-gamma, IL-2, IL-4, and TNF-b eta-positive cells increased 2 or 3 days after stimulation with specif ic dengue antigens, No or very few cytokine-producing cells were detec ted in the PBMC of non-immune donors stimulated with dengue antigens a nd the PBMC of immune donors stimulated with a control antigen, The an alysis of cell surface markers showed that mainly CD4(+) and CD8(+) T cells produced these cytokines, The results obtained by immunocytochem istry were consistent with cytokine levels detected in the culture med ium assay-ed by enzyme-linked immunosorbent assay, In conclusion, this double immunocytochemistry technique is suitable for the detection an d characterization of cytokine-producing cells in PBMC, Furthermore, t he results support the hypothesis that antigen-stimulated CD4(+) and C D8(+) T cells produce cytokines that may play a role in the pathogenes is of dengue virus infection.