CELLULAR AND MOLECULAR MECHANISMS OF IFN-GAMMA PRODUCTION INDUCED BY IL-2 AND IL-12 IN A HUMAN NK CELL-LINE

Interferon-gamma (IFN-gamma) is an important immunoregulatory protein produced predominantly by T cells and large granular lymphocytes (LGL) in response to different extracellular signals. In particular, two interleukins (ILs), IL-2 and IL-12, have been shown to be potent inducers of IFN-gamma gene expression in both T cells and LGL, Although it has been reported that there are some T cell lines that produce IFN-gamma in response to IL-2 and IL-12 stimulation, there has as yet been no report of a natural killer (NK) cell line that responds in a similar manner, In this report we present evidence that the cell line NK3.3 derived from human NK cells, responds to both IL-2 and IL-12, as measured by increases in IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) cytoplasmic mRNA and protein expression, In addition, when used together IL-2 and IL-12 synergized in the induction of IFN-gamma and GM-CSF and this synergy was attributed to an increased accumulation and stability of the IFN-gamma and GM CSF mRNAs, To investigate the signaling pathways involved in the gene induction, five inhibitors, cyclosporin A (CsA), transforming growth factor-p, cycloheximide, genistein, and staurosporine A, were used in analyzing the effects of IL-2 and IL-12 on NK3.3 cells, The results suggest that activation of protein kinase C, but not new protein synthesis, is required for IL-2 induction of IFN-gamma and GM CSF cytoplasmic mRNA, In contrast, IL-12 induction of IFN-gamma cytoplasmic mRNA appears to only partially depend on activation of protein kinase C, Furthermore, both transforming growth factor-beta and genistein, a tyrosine kinase inhibitor, could suppress IL-2 and IL-12 signaling but CsA was generally inactive, It also was observed that suppression of cytokine gene expression by these agents was independent of the inhibition of proliferation, In addition, IL-2 but not IL-12 induced nuclear factors NF-KB and AP1, and regulation of the nuclear levels of these two DNA binding protein complexes is correlated with IFN-gamma and GM-CSF gene expression, These data indicate that IL-2 and IL-12 may have distinct signaling pathways leading to the induction of IFN-gamma and GM-CSF gene expression, and that the NK3.3 cell line may serve as a novel model for dissecting the biochemical and molecular events involved in these pathways.