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Upon primary activation, T helper (Th) cell populations express different cytokines transiently and with different kinetics. Stimulation of naive murine splenic Th cells with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in vitro results in expression of IL-2, IFN-gamma and IL-10 with fast, intermediate and slow kinetics, respectively. This first report of a functional analysis of cells separated alive according to cytokine expression shows that these cytokines are not produced by different Th cell subpopulations, but can be expressed sequentially by individual Th cells. Th cells, activated with SEB for 1 day and isolated according to expression of IL-2, using the cellular affinity matrix technology, upon continued stimulation with SEB later secrete most of the IFN-gamma and IL-10. Likewise, after 2 days of SEB culture, cells expressing IFN-gamma, separated according to specific surface-associated IFN-gamma as detected by magnetofluorescent liposomes, 1 day later secrete IL-10. Thus, individual Th1 cells can contribute to the control of their own IFN-gamma expression by sequential expression of first IL-2, supporting their proliferation, and later IL-10, down-regulating the production of IFN-gamma-inducing monokines and limiting the pro-inflammatory effects of IFN-gamma.