leupeptins and 3-3--4-5--tetrahydroxystilbene

Type I interferons (IFN) exert multiple effects on both the innate and adaptive immune system in addition to their antiviral and antiproliferative activities. Little is known, however about the direct effects of type I IFNs on germinal center (GC) B cells, the central components of adaptive B cell responses. We used Burkitt's lymphoma (BL) lines, as a model system of normal human GC B cells, to examine the effect of type I IFNs on the expression of BCL-6, the major regulator of the GC reaction. We show that type I IFNs, but not IFNγ, IL-2 and TNFα rapidly down-regulate BCL-6 protein and mRNA expression, in cell lines derived from endemic, but not from sporadic BL. IFNα-induced down-regulation is specific for BCL-6, independent of Epstein-Barr virus and is not accompanied by IRF-4 up-regulation. IFNα-induced BCL-6 mRNA down-regulation does not require de novo protein synthesis and is specifically inhibited by piceatannol. The proteasome inhibitor MG132 non-specifically prevents, while inhibitors of alternate type I IFN signaling pathways do not inhibit IFNα-induced BCL-6 protein downregulation. We validate our results with showing that IFNα rapidly down-regulates BCL-6 mRNA in purified mouse normal GC B cells. Our results identify type I IFNs as the first group of cytokines that can down-regulate BCL-6 expression directly in GC B cells.

Topics: Adaptor Proteins, Signal Transducing; Animals; B-Lymphocytes; Burkitt Lymphoma; Cell Line, Transformed; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation, Neoplastic; Germinal Center; Herpesvirus 4, Human; Humans; Interferon Regulatory Factors; Interferon-alpha; Interferon-gamma; Interleukin-2; Kinetics; Leupeptins; Mice; Protein Biosynthesis; Proto-Oncogene Proteins c-bcl-6; RNA-Binding Proteins; RNA, Messenger; Signal Transduction; Stilbenes; Transcription Factors; Tumor Necrosis Factor-alpha

Studies of patients with congenital immunodeficiency due to mutation of the NF-kappaB essential modulator (NEMO) gene have demonstrated that NEMO integrity is required for NK cell cytotoxicity. Thus, we have studied the physiology of NF-kappaB activation in NK cells during the cytolytic program. In resting ex vivo human NK cells or cell lines, IkappaB was degraded after 10 min exposure to PMA and ionomycin, or TNF and was maximally degraded by 30 min. Ligation of several NK cell activation receptors including NKp30 induced a similar response and was blocked by pretreatment with the proteosome inhibitor MG132. There was no short-term effect on p100 processing, the signature of noncanonical NF-kappaB activation. NK cell IkappaB degradation corresponded to increases in nuclear NF-kappaB as detected by EMSA. Supershift of stimulated NK cells and fluorescence microscopy of individual NK cells in cytolytic conjugates demonstrated that the p65/p50 heterodimer was the primary NF-kappaB used. NF-kappaB function was evaluated in NK92 cells transduced with a kappaB GFP reporter, and their conjugation with K562 cells or ligation of NKp30 ligation resulted in rapid GFP accumulation. The latter was prevented by the Syk inhibitor piceatannol. Thus, NK cell activation signaling specifically induces transcriptional activation and synthesis of new NF-kappaB dependent proteins during the initiation of cytotoxicity.

Topics: Cell Line; Green Fluorescent Proteins; Humans; Ionomycin; K562 Cells; Killer Cells, Natural; Leupeptins; Natural Cytotoxicity Triggering Receptor 3; NF-kappa B; Receptors, Immunologic; Signal Transduction; Stilbenes; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha