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. 2011 Mar 10;117(10):2855-63.
doi: 10.1182/blood-2010-07-294702. Epub 2011 Jan 11.

IκBζ augments IL-12- and IL-18-mediated IFN-γ production in human NK cells

Yashaswini Kannan  1 , Jianhua Yu, Raquel M Raices, Sudarshan Seshadri, Min Wei, Michael A Caligiuri, Mark D Wewers
Affiliations

IκBζ augments IL-12- and IL-18-mediated IFN-γ production in human NK cells

Yashaswini Kannan et al. Blood. .

Abstract

Interferon-γ (IFN-γ) production by natural killer (NK) cells and cytotoxic lymphocytes is a key component of innate and adaptive immune responses. Because inhibitor of κB-ζ (IκBζ), a Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) inducible transcription factor, regulates IFN-γ production in KG-1 cells, we tested IκBζ's role in the classic lymphocyte pathway of IL-12/IL-18-induced IFN-γ. Upon stimulation with IL-12/IL-18, monocyte-depleted human peripheral blood lymphocytes expressed the 79-kDa form of IκBζ and released IFN-γ. CD56(+) NK cells were shown to be the IκBζ-producing lymphocyte subpopulation, which also released abundant IFN-γ in response to IL-12/IL-18. Importantly, IκBζ was undetectable in CD56(-) lymphocytes where IFN-γ release was 10-fold lower. In addition, small interfering RNA knockdown of IκBζ suppressed IFN-γ expression in CD56(+) cells. The association of IκBζ with the IFN-γ promoter was documented by chromatin immunoprecipitation. IFN-γ promoter activity from IκBζ overexpression was confirmed by luciferase reporter assay. Finally, IκBζ coprecipitated with p65 and p50 NF-κB in NK cells in response to IL-12/IL-18, suggesting that IκBζ's effects on IFN-γ promoter activity are coregulated by NF-κB. These results suggest that IκBζ functions as an important regulator of IFN-γ in human NK cells, further expanding the class of IκBζ-modulated genes.

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Figures

Figure 1
Figure 1
IκBζ and IFN-γ expression in human lymphocytes. Human lymphocytes were assessed for the expression of IκBζ and IFN-γ in response to recombinant IL-12 and IL-18 (10 ng/mL each) at 6 and 24 hours. (A) Time course of IκBζ protein expression in human lymphocytes by immunoblot compared with lipopolysaccharide-stimulated monocyte lysate (Control). (B) IκBζ mRNA expression in human lymphocytes assessed by quantitative PCR. (C) IFN-γ release from human lymphocytes in response to IL-12 and IL-18 was determined by ELISA. (D) IFN-γ mRNA expression in human lymphocytes. The immunoblot is representative of 3 independent experiments. Bar values represent mean ± SEM for 3 to 5 experiments. **P < .05 vs controls. RCN indicates relative copy number.
Figure 2
Figure 2
IκBζ expression in CD56+ and CD56− cells. Human lymphocytes were isolated from PBMCs after CD14 depletion followed by positive selection for CD56+ cells. Cells were stimulated (5 × 106 cells) with recombinant IL-12 and IL-18 (10 ng/mL each) for 6 hours and 24 hours. Protein normalized cell lysates were immunoblotted for IκBζ and actin. Results are representative of 2 independent experiments. *Nonspecific band (IκBζ blot), which confirms equal protein loading as the actin expression is very high.
Figure 3
Figure 3
IκBζ and IFN-γ expression in human NK cells. IκBζ and IFN-γ expression was evaluated in CD56+ selected human NK cells over time in response to recombinant IL-12 and IL-18 (10 ng/mL each). (A) Time course of IκBζ, IFN-γ, and IL-8 protein expression for IL-12 and IL-18 stimuli in human NK cells (2 × 106 cells/mL) as assessed by immunoblot of cell lysates for IκBζ and ELISA of supernatants for IFN-γ and IL-8. (B) Time course of IκBζ, IFN-γ, and IL-8 mRNA expression in human NK cell by quantitative PCR. Immunoblot is representative of 3 independent experiments and bar graphs represent the mean ± SEM for 3 experiments. Values in the undetectable range have been assigned a random insignificant number and plotted in the graph. Vertical lines have been inserted to indicate a repositioned gel lane. RCN indicates relative copy number.
Figure 4
Figure 4
Cellular localization of IκBζ in human NK cells. The cellular localization of IκBζ expression was evaluated in CD56+ human NK cells stimulated with recombinant IL-12 and IL-18 (10 ng/mL each) over time. The cellular lysates were fractionated into the nucleus and the cytosol separately. The cell lysates were immunoblotted for IκBζ, lamin B1 (nuclear marker), and LDH (cytosolic marker–lactate dehydrogenase). The immunoblot is representative of 3 independent experiments. *Nonspecific band. **Nonspecific band.
Figure 5
Figure 5
Effect of siRNA-mediated knockdown of IκBζ in human NK cells. To assess the role of IκBζ in IFN-γ production, human NK cells were nucleofected with siRNA targeting IκBζ (30nM) or siControl (30nM). After nucleofection, the cells were stimulated with recombinant IL-12 and IL-18 (10 ng/mL each). (A) Protein expression for IκBζ and IFN-γ. IκBα, actin, and IL-8 are also shown as controls. (B) Gene expression profiles for IFN-γ, IκBα, and IL-8 in IκBζ knockdown cells. The immunoblot is representative of 7 independent experiments. Bar values represent the mean ± SEM for 5 to 7 experiments. *Values in the nondetectable range. **P < .05 vs controls. ***P < .005 vs controls. RCN indicates relative copy number.
Figure 6
Figure 6
IκBζ associates with the IFN-γ promoter and activates luciferase expression in human NK cells. The binding of IκBζ to the IFN-γ promoter was studied by ChIP in human NK cells. IκBζ's function in promoting IFNG gene expression was studied in a luciferase reporter assay in HEK 293 cells stably transfected with TLR4/IL-1R/MD-2. (A) Top: Diagrammatic representation of 4 NF-κB binding sites (κB, C3-1P, C3-3P, and C3 1st intron as reported) present on the IFN-γ promoter. Bottom: The effect of IL-12/IL-18 stimulation on the relative enrichment of IκBζ association with the 4 NF-κB sites as determined by ChIP. (B) Top: Diagrammatic representation of the IFN-γ luciferase reporter construct used in the luciferase reporter assay. Bottom: The ability of IκBζ-L to induce expression of the IFN-γ luciferase reporter. Values are mean ± SEM for 3 independent experiments. **P < .05 vs controls. RLU indicates relative luciferase units.
Figure 7
Figure 7
IκBζ coimmunoprecipitates with NF-κB specifically in the nucleus of human NK cells. (A) To assess the ability of IκBζ to bind to NF-κB, human NK cells were stimulated with recombinant IL-12 and IL-18 (10 ng/mL each) for 6 hours. The cell lysates were immunoprecipitated with the indicated antibodies overnight at 4°C. The immunoprecipitated lysates were immunoblotted with respective antibodies as indicated in the figure. (A) IκBζ binding to the p65 NF-κB subunit. (B) IκBζ binding to the p50 NF-κB subunit. (C) Binding of IκBζ to NF-κB specifically in the nucleus. Arrowhead represents p50 NF-κB subunit; and arrow, immunoglobulin heavy chain. *(Black) IκBζ-L band. *(White) Nonspecific smudge mark on the Western blot. Results are representative of 3 independent experiments. Lamin B1, nuclear marker. LDH indicates lactate dehydrogenase (cytosolic marker).
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References

    1. Steeg PS, Moore RN, Johnson HM, Oppenheim JJ. Regulation of murine macrophage Ia antigen expression by a lymphokine with immune interferon activity. J Exp Med. 1982;156(6):1780–1793. - PMC - PubMed
    1. Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004;75(2):163–189. - PubMed
    1. Schoenborn JR, Wilson CB. Regulation of interferon-gamma during innate and adaptive immune responses. Adv Immunol. 2007;96:41–101. - PubMed
    1. Boehm U, Klamp T, Groot M, Howard JC. Cellular responses to interferon-gamma. Annu Rev Immunol. 1997;15:749–795. - PubMed
    1. Collart MA, Belin D, Vassalli JD, de Kossodo S, Vassalli P. Gamma interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin 1, and urokinase genes, which are controlled by short-lived repressors. J Exp Med. 1986;164(6):2113–2118. - PMC - PubMed

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