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Sin-Hyeog Im

Publications and source records attributed to Sin-Hyeog Im.

13 recordsLinked to original sources
PubMed2005

Differential regulation of the IL-10 gene in Th1 and Th2 T cells.

Interleukin-10 (IL-10), an immunoregulatory cytokine, modulates the function of various immune and nonimmune cells, yet little information is available on the molecular mechanism of transcriptional regulation at the chromatin level. During T cell differentiation from naive T cells into Th1 and Th2 cells, the expression of IL-10 in Th1 cells slowly disappears, whereas Th2 cells produce more IL-10. We examined the chromatin structural changes associated with IL-10 gene transcription by naive and differentiated murine Th1 and Th2 cells. Naive T cells lack DNase I hypersensitivity (HS) sites in the vicinity of the IL-10 gene, whereas differentiated T cells display a strong 3' constitutive HS site as well as several inducible sites. In committed Th1 cells, the mechanism of IL-10 gene silencing is associated with a closed chromatin structure, the lack of an HS site at the promoter region, and the development of repressive histone modification near the IL-10 promoter and introns 3 and 4. We confirm that the majority of HS sites coincide with conserved noncoding sequences (CNSs) identified by comparative genomic sequence alignment between human and mouse genomes. Potential transcription factor binding sites were located by comparing CNSs with the TRANSFAC database. Predicted in vivo binding of specific factors on the CNS locus were confirmed by chromatin immunoprecipitation assays. Our results suggest that the combination of HS site and comparative genomic approaches allows identification of regulatory elements involved in differential IL-10 gene expression between Th1 and Th2 cells during T cell differentiation.

Animals↗
PubMed2005

A molecular dissection of lymphocyte unresponsiveness induced by sustained calcium signalling.

In lymphocytes, integration of Ca2+ and other signalling pathways results in productive activation, while unopposed Ca2+ signalling leads to decreased responsiveness to subsequent stimulation (anergy). The Ca(2+)-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. NFAT cooperates with the transcription factor AP-1 (Fos/Jun) to up-regulate genes involved in productive activation of lymphocytes. However, in the absence of AP-1, NFAT imposes an opposing genetic programme that leads to lymphocyte anergy. Anergy is implemented at least partly through proteolytic degradation of the key signalling proteins PKCtheta and PLCgamma1. Sustained Ca(2+)-calcineurin signalling increases mRNA and protein levels of the E3 ubiquitin ligases Itch, CblB and Grail and induces expression of Tsg1O1, the ubiquitin-binding component of the ESCRT1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promotes membrane translocation of Itch and the related protein Nedd4, resulting in PKCtheta and PLCgamma1 degradation. T cells from Itch- and CblB-deficient mice are resistant to anergy induction. Anergic T cells show impaired calcium mobilization after TCR triggering and are unable to maintain a mature immunological synapse. Thus Ca(2+)-calcineurin-NFAT signalling links gene transcription to a multi-step programme that leads to impaired signal transduction in anergic T cells.

Calcium Signaling↗
PubMed2004

Activation and deactivation of gene expression by Ca2+/calcineurin-NFAT-mediated signaling.

Ca2+/calcineurin-NFAT-mediated signaling pathways are involved in diverse cellular reactions by regulating gene expression either positively or negatively. The transcriptional activity of NFAT proteins can be either activating or deactivating depending on which binding partners are involved. Interaction of NFAT with AP-1 turns on the genes involved in active immune responses, while NFAT without cooperative binding of AP-1 turns on a T cell anergy program and blocks T cell activation and proliferation. In addition, interaction of NFAT with histone deacetylase (HDAC) proteins induces gene silencing. In this review we focus on the dual function, activator or deactivator, of NFAT and the binding partners that determine the role of NFAT in gene expression.

Animals↗
PubMed2004

Chromatin-level regulation of the IL10 gene in T cells.

The immunoregulatory cytokine interleukin 10 (IL-10) modulates the function of diverse immune and non-immune cells. Here, we examine the chromatin structural changes associated with IL10 gene transcription by naive and differentiated murine T cells. Naive T cells lack DNase I hypersensitive (HS) sites in the vicinity of the IL10 gene, whereas differentiated T cells display a strong 3' constitutive HS site as well as several inducible sites. The majority of HS sites map to regions that are strongly conserved in sequence between mouse and human genomes. In committed Th1 cells, the mechanism of IL10 gene silencing is associated with the development of repressive histone modifications near the IL10 promoter and also near intronic hypersensitive regions of the IL10 gene. Our results constitute the first report of chromatin structural differences within the IL10 gene in differentiated Th1 and Th2 cells and emphasize the surprising diversity of mechanisms used to regulate cytokine gene expression at the chromatin level.

Animals↗
PubMed2004

Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins.

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

Animals↗
PubMed2004

T-cell anergy.

Self-reactive T cells that escape negative selection in the thymus must be inactivated in the periphery. Anergy constitutes one means of imposing peripheral tolerance. Anergic T cells are functionally inactivated and unable to initiate a productive response even when antigen is encountered in the presence of full co-stimulation. Recent studies have provided new insights into the mechanisms responsible for the induction and maintenance of T-cell anergy. These studies have helped clarify the nature of the signals that induce tolerance, the cells able to deliver them and the molecular processes that underlie the unresponsive state.

Animals↗
PubMed2004

Immunosuppression of rat myasthenia gravis by oral administration of a syngeneic acetylcholine receptor fragment.

A syngeneic rat recombinant fragment of the extracellular domain of the acetylcholine receptor (AChR) alpha-subunit (Ralpha1-205), administered orally, suppresses ongoing experimental autoimmune myasthenia gravis (EAMG) in rats. The underlying mechanism is a shift from Th1 to Th2 regulation as evidenced by downregulated mRNA expression levels of IFN-gamma and TNF-alpha, upregulated IL-10, changes in anti-AChR IgG isotypes and diminished Th1 signaling via CD28/CTLA-4:B7. Unlike the xenogeneic fragment, the syngeneic Ralpha1-205 does not induce elevation in TGF-beta and elicitation of autoregulatory cells. The ability to suppress EAMG by a non-immunogenic syngeneic fragment of AChR is encouraging and may in the future be applied for the treatment of myasthenia gravis in humans.

Animals↗
PubMed2003

Experimental autoimmune myasthenia gravis in mice expressing human immunoglobulin loci.

Antibodies (Abs) specifically directed against the muscular acetylcholine receptor (AChR) mediate the pathogenesis of myasthenia gravis (MG). The animal model experimental autoimmune MG (EAMG) can be induced by passive transfer or by active immunization of anti-AChR Abs. We report a new EAMG mouse model that generates human anti-AChR Abs upon immunization with Torpedo AChR (tAChR). Mice transgenic for human mu, gamma1, and kappa germ line genes (HuMAb-Mice) were immunized with tAChR. Serum titers of anti-tAChR Abs were in the nanomolar range, and anti-rodent AChR Abs were in picomolar range. Some HuMAb-Mice had signs of muscle weakness, clearly indicating their susceptibility to EAMG. Human Ab-mouse AChR complexes were found at the neuromuscular junction, while AChR loss was up to 65%. Spleen and lymph nodes were used for producing hybridomas. Of the anti-tAChR monoclonal Ab-producing hybridomas, 2% had cross-reactivity with rodent AChR and none with human AChR. Immunization with a fusion protein, Trx-Halpha1-210, displaying the human main immunogenic region did not result in EAMG or the generation of human anti-human AChR monoclonal Abs. These experiments show that the HuMAb-Mouse represents a suitable model to generate and study the effects of human anti-AChR Abs in vivo.

Animals↗
PubMed2003

Breakage of tolerance to hidden cytoplasmic epitopes of the acetylcholine receptor in experimental autoimmune myasthenia gravis.

The acetylcholine receptor (AChR) is the major autoantigen in the antibody-mediated disease myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). This study demonstrates that rats immunized with a recombinant fragment corresponding to the normally exposed extracellular region of the rat AChR alpha-subunit first develop antibodies to the injected extracellular portion only, but later develop antibodies to intracellular cytoplasmic epitopes of AChR. The presence of autoantibodies to intracellular epitopes seems to be correlated with development of clinical signs of disease. We propose that a similar process of epitope spreading may take place in the natural course of myasthenia.

Animals↗
PubMed2003

Suppression of myasthenia gravis by antigen-specific mucosal tolerance and modulation of cytokines and costimulatory factors.

We have shown that mucosal administration of recombinant fragments corresponding to the human acetylcholine receptor (AChR) alpha subunit suppresses chronic ongoing experimental autoimmune myasthenia gravis (EAMG) in rats. Treated animals exhibit a Th1 to Th2/Th3 shift in their cytokine profile and downregulation of costimulatory factors. However, application of a xenogeneic recombinant fragment may have limitations when considered as a possible approach for the treatment of MG in humans. We therefore tested the potential of a syngeneic fragment and of long synthetic peptides to suppress EAMG. We found that a syngeneic fragment corresponding to the extracellular region of the rat AChR alpha subunit was as effective as the formerly described human xenogeneic fragment in suppressing ongoing EAMG. This is encouraging in view of the potential use of mucosally administered recombinant AChR fragments for the treatment of MG in humans. However, in severely affected individuals, this antigen-specific approach may need to be supported by direct modulation of cytokines and costimulatory factors known to be involved in the pathogenesis of EAMG. To test the potential of this approach, myasthenic rats were injected by antibodies either to the proinflammatory cytokine IL-18 or to the costimulatory factor CD40L. These treatments act via different mechanisms, but both lead to the alleviation of clinical symptoms even when given at the chronic phase of EAMG. We suggest that antagonists to key cytokines and/or costimulatory factors be used to augment antigen-specific treatments of myasthenia such as mucosal administration of AChR recombinant fragments.

Animals↗
PubMed2002

Transcriptional mechanisms underlying lymphocyte tolerance.

In lymphocytes, integration of Ca2+ and other signaling pathways results in productive activation, while unopposed Ca2+ signaling leads to tolerance or anergy. We show that the Ca2+-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. Ca2+/calcineurin signaling induces a limited set of anergy-associated genes, distinct from genes induced in the productive immune response; these genes are upregulated in vivo in tolerant T cells and are largely NFAT dependent. T cells lacking NFAT1 are resistant to anergy induction; conversely, NFAT1 induces T cell anergy if prevented from interacting with its transcriptional partner AP-1 (Fos/Jun). Thus, in the absence of AP-1, NFAT imposes a genetic program of lymphocyte anergy that counters the program of productive activation mediated by the cooperative NFAT:AP-1 complex.

Animals↗
PubMed2002

Rat interleukin-18 binding protein: cloning, expression, and characterization.

Interleukin-18 binding protein (IL-18BP) is a constitutively expressed and secreted protein that lacks a transmembrane domain. IL-18BP binds specifically to mature IL-18 and inhibits its activity. To study the immunomodulating role of IL-18BP in models of autoimmune diseases in rats, we cloned and characterized rat IL-18BP. Rat IL-18BP has 193 amino acid residues and is highly homologous to human and mouse IL-18BP. Recombinant rat IL-18BP binds to rat IL-18, reacts with antibodies to human or mouse IL-18BP, and inhibits IL-18-dependent interferon-gamma (IFN-gamma) production in vitro. Thus, rat IL-18BP can be employed to antagonize the proinflammatory responses induced by endogenous IL-18 in rat models of autoimmune diseases.

Amino Acid Sequence↗