Delayed Translational Silencing of Ceruloplasmin Transcript in Gamma Interferon-Activated U937 Monocytic Cells: Role of the 3′ Untranslated Region

ABSTRACT Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-γ) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-γ for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-γ treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-γ for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3′ untranslated region (3′-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3′-UTR added as a “decoy” and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3′-UTR indicated an internal 100-nucleotide region of the Cp 3′-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.

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Transcript-Selective Translational Silencing by Gamma Interferon Is Directed by a Novel Structural Element in the Ceruloplasmin mRNA 3′ Untranslated Region

Molecular and Cellular Biology ◽

10.1128/mcb.23.5.1509-1519.2003 ◽

2003 ◽

Vol 23 (5) ◽

pp. 1509-1519 ◽

Cited By ~ 83

Author(s):

Prabha Sampath ◽

Barsanjit Mazumder ◽

Vasudevan Seshadri ◽

Paul L. Fox

Keyword(s):

Translational Control ◽

Untranslated Region ◽

Gamma Interferon ◽

In Vitro Translation ◽

Initiation Factor ◽

Luciferase Reporter ◽

Structural Element ◽

Local Inflammatory Response ◽

Ifn Γ

ABSTRACT Transcript-selective translational control of eukaryotic gene expression is often directed by a structural element in the 3′ untranslated region (3′-UTR) of the mRNA. In the case of ceruloplasmin (Cp), induced synthesis of the protein by gamma interferon (IFN-γ) in U937 monocytic cells is halted by a delayed translational silencing mechanism requiring the binding of a cytosolic inhibitor to the Cp 3′-UTR. Silencing requires the essential elements of mRNA circularization, i.e., eukaryotic initiation factor 4G, poly(A)-binding protein, and poly(A) tail. We here determined the minimal silencing element in the Cp 3′-UTR by progressive deletions from both termini. A minimal, 29-nucleotide (nt) element was determined by gel shift assay to be sufficient for maximal binding of the IFN-γ-activated inhibitor of translation (GAIT), an as-yet-unidentified protein or complex. The interaction was shown to be functional by an in vitro translation assay in which the GAIT element was used as a decoy to overcome translational silencing. Mutation analysis showed that the GAIT element contained a 5-nt terminal loop, a weak 3-bp helix, an asymmetric internal bulge, and a proximal 6-bp helical stem. Two invariant loop residues essential for binding activity were identified. Ligation of the GAIT element immediately downstream of a luciferase reporter conferred the translational silencing response to the heterologous transcript in vitro and in vivo; a construct containing a nonbinding, mutated GAIT element was ineffective. Translational silencing of Cp, and possibly other transcripts, mediated by the GAIT element may contribute to the resolution of the local inflammatory response following cytokine activation of macrophages.

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Toll-Like Receptor 2 Controls the Gamma Interferon Response to Francisella tularensis by Mouse Liver Lymphocytes

Infection and Immunity ◽

10.1128/iai.00561-07 ◽

2007 ◽

Vol 75 (11) ◽

pp. 5338-5345 ◽

Cited By ~ 27

Author(s):

Kee-Jong Hong ◽

Jason R. Wickstrum ◽

Hung-Wen Yeh ◽

Michael J. Parmely

Keyword(s):

Francisella Tularensis ◽

Cell Types ◽

Gamma Interferon ◽

Interferon Response ◽

Interleukin 12 ◽

Toll Like Receptor ◽

Wild Type ◽

Ifn Γ ◽

Deficient Mice

ABSTRACT The production of gamma interferon (IFN-γ) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-γ expression. The present study was undertaken to determine whether or not macrophages (Mφ) or dendritic cells (DC) provide coactivating signals for the liver IFN-γ response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mφ and DC significantly augmented the IFN-γ response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-γ production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-γ at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-γ responses to F. tularensis by regulating DC IL-12 production. While Mφ also coinduced hepatic IFN-γ production in response to F. tularensis, they did so in a fashion less dependent on TLR2.

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Gamma Interferon Mediates Experimental Cerebral Malaria by Signaling within Both the Hematopoietic and Nonhematopoietic Compartments

Infection and Immunity ◽

10.1128/iai.01035-16 ◽

2017 ◽

Vol 85 (11) ◽

Cited By ~ 5

Author(s):

Ana Villegas-Mendez ◽

Patrick Strangward ◽

Tovah N. Shaw ◽

Ivana Rajkovic ◽

Vinko Tosevski ◽

...

Keyword(s):

Endothelial Cells ◽

Cerebral Malaria ◽

Myeloid Cell ◽

Gamma Interferon ◽

Cell Populations ◽

Experimental Cerebral Malaria ◽

Cerebral Pathology ◽

Multiple Cell ◽

Ifn Γ

ABSTRACT Experimental cerebral malaria (ECM) is a gamma interferon (IFN-γ)-dependent syndrome. However, whether IFN-γ promotes ECM through direct and synergistic targeting of multiple cell populations or by acting primarily on a specific responsive cell type is currently unknown. Here, using a panel of cell- and compartment-specific IFN-γ receptor 2 (IFN-γR2)-deficient mice, we show that IFN-γ causes ECM by signaling within both the hematopoietic and nonhematopoietic compartments. Mechanistically, hematopoietic and nonhematopoietic compartment-specific IFN-γR signaling exerts additive effects in orchestrating intracerebral inflammation, leading to the development of ECM. Surprisingly, mice with specific deletion of IFN-γR2 expression on myeloid cells, T cells, or neurons were completely susceptible to terminal ECM. Utilizing a reductionist in vitro system, we show that synergistic IFN-γ and tumor necrosis factor (TNF) stimulation promotes strong activation of brain blood vessel endothelial cells. Combined, our data show that within the hematopoietic compartment, IFN-γ causes ECM by acting redundantly or by targeting non-T cell or non-myeloid cell populations. Within the nonhematopoietic compartment, brain endothelial cells, but not neurons, may be the major target of IFN-γ leading to ECM development. Collectively, our data provide information on how IFN-γ mediates the development of cerebral pathology during malaria infection.

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Gamma Interferon Production, but Not Perforin-Mediated Cytolytic Activity, of T Cells Is Required for Prevention of Toxoplasmic Encephalitis in BALB/c Mice Genetically Resistant to the Disease

Infection and Immunity ◽

10.1128/iai.72.8.4432-4438.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4432-4438 ◽

Cited By ~ 52

Author(s):

Xisheng Wang ◽

Hoil Kang ◽

Takane Kikuchi ◽

Yasuhiro Suzuki

Keyword(s):

T Cells ◽

T Cell ◽

Nude Mice ◽

Genetic Resistance ◽

Cytolytic Activity ◽

Gamma Interferon ◽

Toxoplasmic Encephalitis ◽

Wild Type ◽

Ifn Γ

ABSTRACT We previously showed the requirement of both T cells and gamma interferon (IFN-γ)-producing non-T cells for the genetic resistance of BALB/c mice to the development of toxoplasmic encephalitis (TE). In order to define the role of IFN-γ production and the perforin-mediated cytotoxicity of T cells in this resistance, we obtained immune T cells from spleens of infected IFN-γ knockout (IFN-γ−/−), perforin knockout (PO), and wild-type BALB/c mice and transferred them into infected and sulfadiazine-treated athymic nude mice, which lack T cells but have IFN-γ-producing non-T cells. Control nude mice that had not received any T cells developed severe TE and died after discontinuation of sulfadiazine treatment due to the reactivation of infection. Animals that had received immune T cells from either wild-type or PO mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-γ−/− mice developed severe TE and died as early as control nude mice. T cells obtained from the spleens of animals that had received either PO or wild-type T cells produced large amounts of IFN-γ after stimulation with Toxoplasma gondii antigens in vitro. In addition, the amounts of IFN-γ mRNA expressed in the brains of PO T-cell recipients did not differ from those in wild-type T-cell recipients. Furthermore, PO mice did not develop TE after infection, and their IFN-γ production was equivalent to or higher than that of wild-type animals. These results indicate that IFN-γ production, but not perforin-mediated cytotoxic activity, by T cells is required for the prevention of TE in genetically resistant BALB/c mice.

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Development of a Human Gamma Interferon Enzyme Immunoassay and Comparison with Tuberculin Skin Testing for Detection ofMycobacterium tuberculosis Infection

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.5.4.531-536.1998 ◽

1998 ◽

Vol 5 (4) ◽

pp. 531-536 ◽

Cited By ~ 45

Author(s):

Nuket Desem ◽

Stephen L. Jones

Keyword(s):

Enzyme Immunoassay ◽

Skin Test ◽

Skin Testing ◽

Gamma Interferon ◽

Tuberculin Skin Testing ◽

In Vitro Test ◽

Detection Range ◽

Ifn Γ

ABSTRACT A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-γ) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-γ per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-γ. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-γs. Cross-reactions with other human cytokines or with IFN-γs derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-γ in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-γ were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-γ responsiveness in vitro. These results support the further study of the blood culture–IFN-γ EIA system as an alternative to skin testing for the detection of human TB infection.

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Effects of Gamma Interferon, Interleukin-10, and Transforming Growth Factor β on the Survival of Mycobacterium avium subsp. paratuberculosis in Monocyte-Derived Macrophages from Naturally Infected Cattle

Infection and Immunity ◽

10.1128/iai.72.4.1974-1982.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 1974-1982 ◽

Cited By ~ 52

Author(s):

M. S. Khalifeh ◽

J. R. Stabel

Keyword(s):

Growth Factor ◽

Cell Cultures ◽

Mycobacterium Avium ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Interleukin 10 ◽

Gamma Interferon ◽

Ifn Γ ◽

Culture Supernatants

ABSTRACT Gamma interferon (IFN-γ) plays a significant role in the control of mycobacterial infections, including Mycobacterium avium subsp. paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection with M. avium subsp. paratuberculosis on the production of IFN-γ, IL-10, and TGF-β by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-γ, IL-10, and TGF-β on M. avium subsp. paratuberculosis survival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-β in culture supernatants in response to stimulation with live M. avium subsp. paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-β, but after stimulation with live M. avium subsp. paratuberculosis, TGF-β levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viable M. avium subsp. paratuberculosis recovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection with M. avium subsp. paratuberculosis. The addition of exogenous IL-10 and TGF-β to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. avium subsp. paratuberculosis recovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-β during infection with M. avium subsp. paratuberculosis that may be directly related to their effects on macrophage activation and killing of M. avium subsp. paratuberculosis.

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Virulent Mycobacterium fortuitum Restricts NO Production by a Gamma Interferon-Activated J774 Cell Line and Phagosome-Lysosome Fusion

Infection and Immunity ◽

10.1128/iai.70.10.5628-5634.2002 ◽

2002 ◽

Vol 70 (10) ◽

pp. 5628-5634 ◽

Cited By ~ 24

Author(s):

Tânia Regina Marques da Silva ◽

Juliana Ribeiro de Freitas ◽

Queilan Chagas Silva ◽

Cláudio Pereira Figueira ◽

Eliana Roxo ◽

...

Keyword(s):

Gamma Interferon ◽

No Production ◽

Mycobacterium Fortuitum ◽

Vitro Assay ◽

J774 Cells ◽

Nitrogen Radicals ◽

Ifn Γ ◽

Phagosome Fusion ◽

J774 Cell Line

ABSTRACT The virulence of different isolates of Mycobacterium has been associated with two morphologically distinguishable colonial variants: opaque (SmOp) and transparent (SmTr). In this report we used an in vitro assay to compare macrophage (Mφ) responses to SmOp and SmTr Mycobacterium fortuitum variants, taking advantage of the fact that these variants were derived from the same isolate. Cells preactivated or not with gamma interferon (IFN-γ) were infected with SmOp or SmTr M. fortuitum. We showed that SmOp and SmTr induced different levels of nitric oxide (NO) production by IFN-γ-stimulated Mφ. Indeed, the amount of IFN-γ-induced NO production by J774 cells was 4.8 to 9.0 times higher by SmOp (23.1 to 37.7 μM) compared to SmTr infection (3.9 to 4.8 μM) (P = 0.0332), indicating that virulent SmTr bacilli restricted NO production. In addition, IFN-γ-induced NO production by Mφ was higher when correlated with reduction of only avirulent SmOp bacillus viability. SNAP (S-nitroso-N-acetyl-dl-penicillamine)-induced NO production did not modify SmTr viability, indicating its resistance to nitrogen radicals. Electron microscopy studies were performed to evaluate the capacity of phagosomes to fuse with lysosomes labeled with bovine serum albumin-colloidal gold particles. By 24 h postinfection, 69% more phagosome-containing SmOp variant had fused with lysosomes compared to the SmTr-induced phagosomes. In conclusion, these data indicate that virulent SmTr bacilli may escape host defense by restricting IFN-γ-induced NO production, resisting nitrogen toxic radicals, and limiting phagosome fusion with lysosomes.

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Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2315-2326.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2315-2326

Author(s):

J H Teruya ◽

S Y Kutsunai ◽

D H Spear ◽

P A Edwards ◽

C F Clarke

Keyword(s):

Transcription Initiation ◽

Untranslated Region ◽

In Vitro Translation ◽

Cdna Clones ◽

Rat Tissues ◽

Farnesyl Pyrophosphate ◽

S1 Nuclease ◽

Testis Cdna ◽

Low Efficiency

A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.

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