Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen: Cytokine detection by immunoassay and intracellular immunostraining

Abstract

Production of IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-γ, GM-CSF (granulocyte-macrophage colony stimulating factor) and macrophage colony stimulating factor by murine peripheral blood and spleen cells was analyzed following primary and secondary mitogen stimulation in vitro. Individual cytokine producing cells were detected by an intracytoplasmic staining technique. Cytokine production in cells from peripheral blood and spleen was comparable and more rapidly induced by calcium ionophore and phorbol 12-myristate 13-acetate than by concanavaling A. Restimulation in vitro induced both a swift production of cytokines and, for some cytokines, higher frequencies of producing cells. This was especially evident for IL-10 secreting cells, which increased 30–80 times in secondary responses. These analyses using the dual approaches of immunoenzymetric and fluorescent immunohistochemical techniques provide important evidence that cytokine induction kinetics can differ following primary or secondary stimulation.