2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.     

Growth arrest of epithelial cells during measles virus infection is caused by upregulation of interferon regulatory factor 1

Natural infection with measles virus (MeV) is initiated when the virus reaches epithelial cells in the respiratory tract, oropharynx, or conjunctivae. Human epithelial cells infected with MeV frequently show growth suppression. In this study, we investigated the possible mechanisms for this suppression. The bronchiolar epithelial cell A549 showed growth arrest in G(0)/G(1) following MeV infection or treatment with gamma interferon (IFN-gamma). IFN regulatory factor-1 (IRF-1) was upregulated during MeV infection, although A549 did not produce IFN-gamma. Cells of the cervical squamous cell line SiHa persistently infected with various strains of MeV displayed slower growth than uninfected SiHa cells, although the growth rates varied depending on the MeV strain. Transfection of antisense-oriented IRF-1 cDNA released the MeV-infected SiHa cells from growth suppression. Although these infected cells did not produce IFN-gamma and suppressed IFN-alpha/beta-induced Jak1 phosphorylation, Jak1 was constitutively phosphorylated. The growth rates negatively correlated with levels of both IRF-1 expression and constitutively phosphorylated Jak1. These results indicate that MeV upregulates IRF-1 in a manner that is independent of IFN but dependent on the JAK/STAT pathway. This induction of IRF-1 appears to suppress cell growth, although the extent seems to vary among MeV strains.     

Deletional analysis of the murine IL-12 p35 promoter comparing IFN-gamma and lipopolysaccharide stimulation.

IL-12, pivotal to the development of Th1 cells and formed by association of p35 and p40 subunits, is made by macrophages and the macrophage cell line RAW264.7. In this study, the promoter for p35 was cloned and analyzed. The murine IL-12 p35 gene has promoters upstream from each of the first two exons. The exon 1 and exon 2 promoters, cloned into a reporter vector, were responsive to LPS or IFN-gamma/CD40 ligation in transfected RAW264.7 cells. The exon 2 promoter containing bp -809 to +1 has significant homology to the human p35 promoter. Thus, deletion analysis was performed to determine the regions required for responsiveness to LPS, CD40, and/or IFN-gamma. Base pairs -809 to -740 influenced responsiveness to LPS. In contrast, bp -740to -444 and bp -122 to -100 were required for responses to IFN-gamma, IFN-gamma/LPS, or IFN-gamma/CD40 ligation. Removal of bp -444 to -392 increased the response of the exon 2 promoter to each stimulant. IFN regulatory factor (IRF)-1 is involved in the activity of this promoter at bp -108 to -103 because levels of nuclear IRF-1 correlated with exon 2 promoter activity in response to IFN-gamma and IRF-1 overexpression stimulated and enhanced exon 2 promoter activity. Also, site or deletion mutation of the IRF-1 element at bp -108 to -103 reduced the responsiveness of the promoter and IRF-1 bound to an oligonucleotide containing bp -108 to -103. The data suggest that the response of the p35 promoter to IFN-gamma requires a distinct IRF-1 positive regulatory element at bp -108 to -103.     

Characterization of an interferon-stimulated response element (ISRE) in the Il23a promoter

We have demonstrated previously that IFN-γ plays a protective role in the initiation of chronic intestinal inflammation through attenuation of Toll-like receptor-mediated IL-23 induction in macrophages. Here, an interferon-stimulated response element (ISRE) is identified in a region of conserved nucleotide sequences in the Il23a promoter. This ISRE mediated, in part, Il23a promoter induction by LPS and inhibition of LPS-induced activity by IFN-γ. LPS and IFN-γ recruit interferon regulatory factors (IRFs) to the Il23a ISRE in murine bone marrow-derived macrophages (BMMs). Functionally, IRF-1 is a negative regulator of Il23a in LPS-stimulated BMMs. IRF-1(-/-) BMMs demonstrated enhanced LPS-induced Il23a expression compared with WT BMMs. Moreover, IRF-1 deficiency resulted in prolonged occupancy of RelA on the Il23a promoter. Consequently, IRF-1(-/-) mice were more susceptible to colonic injury by trinitrobenzenesulfonic acid, and IL-10/IRF-1 double-deficient (IL-10/IRF-1(-/-)) mice demonstrated more severe colonic inflammation compared with IL-10(-/-) mice. The severity of colitis in both models correlated with increased colonic IL-23. CD11b(+) lamina propria mononuclear cells, comprising predominantly macrophages, were identified as the major source of IL-23 in colitis-prone mice. Basal and heat-killed Escherichia coli-stimulated levels of Il23a were increased in IL-10/IRF-1(-/-) compared with WT and IL-10(-/-) colonic CD11b(+) lamina propria mononuclear cells. In conclusion, these experiments characterize IRF-ISRE interactions on the Il23a promoter, which have in vivo relevance as a homeostatic checkpoint in chronic intestinal inflammation.     

Varying functions of specific major histocompatibility class II transactivator promoter III and IV elements in melanoma cell lines.

Melanoma cells commonly express MHC class II molecules constitutively. This is a rare, or possibly unique, phenotype for a nonprofessional antigen-presenting cell, where MHC class II expression ordinarily occurs only after IFN-gamma treatment. Despite the fact that constitutive expression of MHC class II on melanoma cells has been observed for decades and that the regulation of the MHC class II genes is well understood for many different cell types, there is no data regarding the basis for constitutive MHC class II expression in melanoma cells. Here we report that MHC class II expression in melanoma cells can be traced to constitutive expression of the class II transactivator protein (CIITA), which mediates both IFN-gamma-inducible and -constitutive MHC class II expression in all other cell types. In addition, we determined that constitutive CIITA expression is the result of the activation of both the B cell-specific CIITA promoter III and the IFN-gamma-inducible CIITA promoter IV, the latter of which previously has never been known to function as a constitutive promoter in any cell type. The recently described B cell-related ARE-1 activity is important for promoter III activation in the melanoma cells. Constitutive promoter IV activation involves the IFN regulatory factor element (IRF-E), which binds members of the IRF family of proteins, although the major, IFN-gamma inducible member of this family, IRF-1, is not constitutively expressed in these cells. In cells with constitutively active promoter IV, the promoter IV IRF-E is most likely activated by IRF-2. The relevance of these results to the pathway of melanoma development is discussed.