Measurement of antigen-dependent interleukin-4 production by human peripheral blood mononuclear cells. Introduction of an amplification step using ionomycin and phorbol myristate acetate.
The study of T cell responses in parasitic disease and allergy in humans has been limited by difficulties in the measurement of interleukin-4 (IL-4) in supernatants from antigen-stimulated peripheral blood mononuclear cells (PBMC). To obtain measurable amounts of IL-4 in vitro, we have added an amplification step to the antigen-specific response. Human PBMC were stimulated by tetanus toxoid (TT) or tuberculin (PPD) for 6 days and then pulsed with ionomycin and phorbol myristate acetate (PMA) for 24 h. TT-stimulated cells from nine revaccinated donors but not from seven unvaccinated donors and four that had only received childhood vaccinations against tetanus produced high levels of IL-4 (median (range) 1500 (300-3800), 316 (0-1600), and 270 (100-410) pg/ml, respectively, as measured by an enzyme linked immunosorbent assay, P = 0.005). PPD did not increase IL-4 production above the background level, although the majority of PPD-stimulated PBMC proliferated and produced interferon-gamma (IFN-gamma) in cultures without ionomycin and PMA. TT-induced IL-4 production correlated positively with proliferation. Culture supernatants did not interfere with IL-4 immunoreactivity and failed to affect ionomycin and PMA induced IL-4 production. The findings suggest that proliferating antigen-specific T cells were the source of IL-4 in these experiments. The method should prove useful for comparing the IL-4 producing ability of antigen-specific T cells from different individuals.
Kurtzhals
J A
JA
Department of Infectious Diseases, University Hospital, Copenhagen, Denmark.
Hansen
M B
MB
Hey
A S
AS
Poulsen
L K
LK
D000941:Antigens / Q000276:immunology; D002478:Cells, Cultured; D006801:Humans; D066298:In Vitro Techniques; D015847:Interleukin-4 / Q000096:biosynthesis*; D015759:Ionomycin / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008213:Lymphocyte Activation; D013745:Tetanus Toxoid / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014373:Tuberculin / Q000276:immunology
D000941:Antigens; D013745:Tetanus Toxoid; D014373:Tuberculin; D015847:Interleukin-4; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate