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-beta, 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-kappa B 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.
Ye
J
J
Laboratory of Experimental Immunology, NCI-FCRDC, Frederick, Maryland 21702-1201, USA.
Ortaldo
J R
JR
Conlon
K
K
Winkler-Pickett
R
R
Young
H A
HA
D000470:Alkaloids / Q000494:pharmacology; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis*; D018664:Interleukin-12 / Q000494:pharmacology*; D007376:Interleukin-2 / Q000494:pharmacology*; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology*; D007700:Kinetics; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D019311:Staurosporine; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism
D000470:Alkaloids; D007376:Interleukin-2; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D014157:Transcription Factors; D018664:Interleukin-12; D007371:Interferon-gamma; D016572:Cyclosporine; D011493:Protein Kinase C; D019311:Staurosporine