REVERSAL OF INTERFERON-GAMMA-RESISTANT PHENOTYPE BY POLY(I-C) - POSSIBLE INVOLVEMENT OF ISGF2 (IRF1) IN INTERFERON-GAMMA-MEDIATED INDUCTION OF THE IDO GENE

Indoleamine 2,3-dioxygenase (IDO) is induced in many cell lines by interferon-gamma (IFN-gamma) treatment. IDO mRNA increases rapidly from 4 h after IFN-gamma treatment to at least 24 h after treatment in ME180 cells. The IFN-gamma-resistant mutant of ME180, IR3B6B, expresses only one-sixth the amount of IDO message after IFN-gamma treatment and very low levels of IDO. However, pretreatment of these mutants with poly(I:C) restores normal levels of IDO mRNAs and IDO activity. Since IRF1 mRNA induction is also low in IR3B6B cells after IFN-gamma treatment, we examined whether there was any relationship between IRF1 induction and IDO induction by IFN-gamma. The steady-state level of IRF1 mRNA was elevated by treating IR3B6B cells with poly(I:C) and IFN-gamma. Poly(I:C)-mediated reversal of IFN-gamma-resistant phenotype and induction of IDO and IRF1 messages are inhibited by 2-aminopurine. Transient transfection of IRF1 cDNA in ME180 cells resulted in activation of IDO transcription. Nuclear extracts prepared from IFN-gamma-treated ME180 and IR3B6B cells affected differently the mobility of a 80-bp DNA fragment of the 5' regulatory region of IDO gene. Pretreatment of IR3B6B cells with poly(I:C) and addition of IFN-gamma resulted in increased DNA binding of nuclear proteins to the DNA. Pre- and post-treatment of nuclear extract of IFN-gamma-treated ME180 cells with anti-IRF1 antibody resulted in a super shift in mobility of the probe with the abolishment of normal gel-shift pattern. In vitro dephosphorylation of nuclear extract prepared from IFN-gamma-treated ME180 cells almost completely abolished DNA-binding activity of nuclear proteins. Thus, we conclude that accumulation of IRF1 and its phosphorylation appear to be necessary for its binding to IDO-5' regulatory region and induction of the IDO gene.