Cellular and molecular mechanisms of IFN-γ production induced by IL-2 and IL-12 in a human NK cell line

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Abstract
Interferon-γ (IFN-γ) 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 (ELs), IL-2 and IL-12, have been shown to be potent inducers of IFN-γ gene expression in both T cells and LGL. Although it has been reported that there are some T cell lines that produce IFN-γ 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-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) cytoplasmic mRNA and protein expression. In addition, when used together EL-2 and IL-12 synergized in the induction of IFN-γ and GM-CSF and this synergy was attributed to an increased accumulation and stability of the IFN-γ and GM-CSF mRNAs. To investigate the signaling pathways involved in the gene induction, five inhibitors, cyclosporin A (CsA), transforming growth factor-β, cydoheximide, 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-γ and GM-CSF cytoplasmic mRNA. In contrast, IL-12 induction of IFN-γ cytoplasmic mRNA appears to only partially depend on activation of protein kinase C. Furthermore, both transforming growth factor-β 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 nudear factors NF-κB and API, and regulation of the nudear levels of these two DNA binding protein complexes is correlated with IFN-γ 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-γ 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. J. Leukoc. Biol. 58: 225–233; 1995.

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