Full-length IL-33 is a member of the IL-1 family of cytokines, which can act in an autocrine or paracrine manner by binding to the IL-33R on several different target cell types. In addition, IL-33 can act in an intracrine fashion by translocating to the nucleus, where it binds to the chromatin and modulates gene expression. In this article, we report that full-length IL-33, but not mature IL-33, interacts with the transcription factor NF-κB. This interaction occurs between the N-terminal part of IL-33 from aa 66–109 and the N-terminal Rel homology domain of NF-κB p65. Coimmunoprecipitation experiments in cells overexpressing IL-33 or endogenously expressing IL-33 revealed rhIL-1β–stimulated association between IL-33 and p65, whereas binding to the p50 subunit was constitutive. The biological consequence of IL-33/NF-κB complex formation was reduction in NF-κB p65 binding to its cognate DNA and impairment of p65-triggered transactivation. Overexpression of IL-33 resulted in a reduction and delay in the rhIL-1β–stimulated expression of endogenous NF-κB target genes such as IκBα, TNF-α, and C-REL. We suggest that nuclear IL-33 sequesters nuclear NF-κB and reduces NF-κB–triggered gene expression to dampen proinflammatory signaling.
Dendritic cells (DCs)* fulfill an important regulatory function at the interface of the innate and adaptive immune system. The thymus and activation-regulated chemokine (TARC/CCL17) is produced by DCs and facilitates the attraction of activated T cells. Using a fluorescence-based in vivo reporter system, we show that CCL17 expression in mice is found in activated Langerhans cells and mature DCs located in various lymphoid and nonlymphoid organs, and is up-regulated after stimulation with Toll-like receptor ligands. DCs expressing CCL17 belong to the CD11b+CD8−Dec205+ DC subset, including the myeloid-related DCs located in the subepithelial dome of Peyer's patches. CCL17-deficient mice mount diminished T cell–dependent contact hypersensitivity responses and display a deficiency in rejection of allogeneic organ transplants. In contrast to lymphoid organs located at external barriers of the skin and mucosa, CCL17 is not expressed in the spleen, even after systemic microbial challenge or after in vitro stimulation. These findings indicate that CCL17 production is a hallmark of local DC stimulation in peripheral organs but is absent from the spleen as a filter of blood-borne antigens.
Dendritic cells are key players of the immune system as they efficiently induce primary immune responses by activating naive T cells. We generated human dendritic cells from CD14+ blood precursors and investigated expression of the actin-bundling protein fascin during maturation by western blotting, immunofluorescence, and cytofluorometry. Cells obtained by culture of CD14+ blood precursors in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, which were only weakly positive for the maturation marker CD83, expressed low amounts of fascin. Addition of a cytokine cocktail including tumor necrosis factor alpha, interleukin-1beta, interleukin-6, and prostaglandin E2 induced maturation of the cells and enhanced fascin expression in parallel with CD83 expression. Isolated mature CD83+ cells displayed especially high fascin levels on western blots, as did gated CD83+ dendritic cells in cytofluorometry. Dendritic cells generated from CD34+ blood precursors expressed high levels of fascin as well. Confocal microscopy revealed that location of fascin within the cell was restricted to the area of the submembranous actin cytoskeleton and to the dendritic processes. Suppression experiments using antisense constructs of fascin hint at a retarded morphologic maturation of dendritic cells, supporting the view that fascin expression is pivotal for dendrite formation. Our data suggest that fascin could serve as a marker molecule to monitor the maturation state of in vitro generated dendritic cells for use in clinical trials.
Strong cell-type-specific promoters are basic tools in gene therapy allowing for novel applications and focused strategies by transcriptionally targeting gene expression to selected cells. In immunotherapy, dendritic cells (DC) are of central importance, since they represent the principal inducers of immune responses. Here we describe isolation and use of the promoter of the murine actin-bundling protein fascin to target transcriptionally gene expression to cutaneous DC. Using the reporter gene enhanced green fluorescent protein (EGFP), we demonstrate that the fascin promoter mediates a strong antigen expression that is restricted to mature DC. DNA vaccination with antigenencoding expression vectors under control of the fascin promoter using a gene gun resulted, consistently, in limited antigen expression by few directly transfected DC. Nevertheless, nearly as many antigen-specific CD8 + T cells directed against the encoded antigens EGFP and bgalactosidase, respectively, were induced as with expression constructs under control of the ubiquitously expressed CMV promoter. This result impressively underlines the pivotal role of directly transfected DC in DNA vaccination. Immunization using the fascin promoter induced markedly lower levels of antigen-specific antibodies following single or repeated immunization. Thus, our DC-targeted DNA vaccination approach induces qualitatively distinct, predominantly cellular immune responses and provides new opportunities for immunotherapy.
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