1Diabetes-prone (DP) BB rats spontaneously develop insulin-dependent diabetes resembling human type 1 diabetes. They also exhibit lifelong T-cell lymphopenia. Functional and genetic data support the hypothesis that the gene responsible for the lymphopenia, Lyp, is also a diabetes susceptibility gene, named Iddm1. We constructed a 550-kb P1-derived artificial chromosome contig of the region. Here, we present a corrected genetic map reducing the genetic interval to 0.2 cM and the physical interval to 150 -290 kb. A total of 13 genes and six GenomeScan models are assigned to the homologous human DNA segment on HSA7q36.1, 8 of which belong to the family of immune-associated nucleotides (Ian genes). Two of these are orthologous to mouse Ian1 and -4, both excellent candidates for Iddm1. In normal rats, they are expressed in the thymus and T-cell regions of the spleen. In the thymus of lymphopenic rats, Ian1 exhibits wild-type expression patterns, whereas Ian4 expression is reduced. Mutational screening of their coding sequences revealed a frameshift mutation in Ian4 among lymphopenic rats. The mutation results in a truncated protein in which the COOH-terminal 215 amino acids-including the anchor localizing the protein to the outer mitochondrial membrane-are replaced by 19 other amino acids. We propose that Ian4 is identical to Iddm1.
Apoptosis is a regulated cell death program controlled by extrinsic and intrinsic signaling pathways. The intrinsic pathway involves stress signals that activate pro-apoptotic members of the Bcl-2 family, inducing permeabilization of mitochondria and release of apoptogenic factors. These proteins localize to the outer mitochondrial membrane. Ian4, a mitochondrial outer membrane protein with GTP-binding activity, is normally present in thymocytes, T cells, and B cells. We and others have recently discovered that a mutation in the rat Ian4 gene results in severe T cell lymphopenia that is associated with the expression of autoimmune diabetes. The mechanism by which Ian4 controls T cell homeostasis is unknown. Here we show that the absence of Ian4 in T cells causes mitochondrial dysfunction, increased mitochondrial levels of stress-inducible chaperonins and a leucine-rich protein, and T cell-specific spontaneous apoptosis. T cell activation and caspase 8 inhibition both prevented apoptosis, whereas transfection of T cells with Ian4-specific small interfering RNA recapitulated the apoptotic phenotype. The findings establish Ian4 as a tissuespecific regulator of mitochondrial integrity. Apoptosis is a regulated cell death program that can be initiated by two different signaling pathways. The extrinsic pathway involves ligation of cell surface death receptors and recruitment of proteins to a death-inducing signaling complex (1). The intrinsic pathway is death receptor-independent and involves stress signals that activate pro-apoptotic members of the Bcl-2 family; this action in turn induces permeabilization of mitochondria and the release of apoptogenic factors (2). These proteins are localized to the outer mitochondrial membrane, but the mechanisms responsible for regulating mitochondrial homeostasis localize to the inner membrane (3). How the two systems interact is unknown.Immune-associated nucleotide-binding protein 4 (Ian4) was originally identified as a highly expressed protein in Bcr͞Abl-transformed 32D cells (4). It localized to the mitochondrial outer membrane and displayed GTP-binding activity in vitro (4). Cells transfected with mutated Bcr͞Abl constructs lacked oncogenic potential and displayed lower Ian4 expression. Homologues of Ian4 are present in mouse, rat, and human (5). More recently, Ian4 was found to be disrupted in diabetes-prone BB (BBDP) rats (5, 6), which develop severe T lymphopenia due to apoptosis of recent thymic emigrants (7). We hypothesized that mitochondrial Ian4 (also known in the rat as Ian4l1 and Ian5) plays an important role in regulating T cell survival through control of T cell apoptosis. MethodsAnimals. Diabetes-resistant BB (BBDR) rats and BBDP rats were obtained from Biomedical Research Models (Worcester, MA). Approximately 90% of BBDP rats develop spontaneous autoimmune diabetes; they are Ian4 Ϫ/Ϫ and congenitally lymphopenic (8). BBDR rats are Ian4 ϩ/ϩ and never become spontaneously diabetic (8). Wistar Furth (WF) rats were obtained from Harlan-Sprague-Dawley. A congenic, no...
IntroductionRheumatoid arthritis (RA) is a chronic progressive, inflammatory and destructive autoimmune disease, characterised by synovial joint inflammation and bone erosion. To better understand the pathophysiology and underlying immune mechanisms of RA various models of arthritis have been developed in different inbred strains of mice. Establishment of arthritis models with components of adaptive immunity in the C57BL/6J strain of mice has been difficult, and since most genetically modified mice are commonly bred on this background, there is a need to explore new ways of obtaining robust models of arthritis in this strain. This study was undertaken to establish and characterise a novel murine model of arthritis, the delayed-type hypersensitivity (DTH)-arthritis model, and evaluate whether disease can be treated with compounds currently used in the treatment of RA.MethodsDTH-arthritis was induced by eliciting a classical DTH reaction in one paw with methylated bovine serum albumin (mBSA), with the modification that a cocktail of type II collagen monoclonal antibodies was administered between the immunisation and challenge steps. Involved cell subsets and inflammatory mediators were analysed, and tissue sections evaluated histopathologically. Disease was treated prophylactically and therapeutically with compounds used in the treatment of RA.ResultsWe demonstrate that DTH-arthritis could be induced in C57BL/6 mice with paw swelling lasting for at least 28 days and that disease induction was dependent on CD4+ cells. We show that macrophages and neutrophils were heavily involved in the observed pathology and that a clear profile of inflammatory mediators associated with these cell subsets was induced locally. In addition, inflammatory markers were observed systemically. Furthermore, we demonstrate that disease could be both prevented and treated.ConclusionsOur findings indicate that DTH-arthritis shares features with both collagen-induced arthritis (CIA) and human RA. DTH-arthritis is dependent on CD4+ cells for induction and can be successfully treated with TNFα-blocking biologics and dexamethasone. On the basis of our findings we believe that the DTH-arthritis model could hold potential in the preclinical screening of novel drugs targeting RA. The model is highly reproducible and has a high incidence rate with synchronised onset and progression, which strengthens its potential.
In immunotherapy, dendritic cells (DCs) can be used as powerful antigen-presenting cells to enhance or suppress antigenspecific immunity upon in vivo transfer in mice or humans. However, to generate sufficient numbers of DCs, most protocols include an ex vivo culture step, wherein the cells are exposed to heterologous serum and/or antigenic stimuli. In mouse models of virus infection and virus-induced autoimmunity, we tested how heterologous serum affects the immunomodulatory capacity of immature DCs generated in the presence of IL-10 by comparing fetal bovine serum (FBS)-or normal mouse serum (NMS)-supplemented DC cultures. We show that FBSexposed DCs induce a systemic immune deviation characterized by reduction of virus-specific T cells, delayed viral clearance, and enhanced systemic production of interleukin 4 (IL-4), IL-5, and IL-10 to FBS-derived antigens, including bovine serum albumin (BSA). By contrast, DCs generated in NMS-supplemented cultures modulated immunity and autoimmunity in an antigen-specific fashion. These cells did not induce systemic IL-4, IL-5, or IL-10 production and inhibited generation of virus-specific T cells or autoimmunity only if pulsed with a viral antigen. These data underscore the importance of using autologous serum-derived immature DCs in preclinical animal studies to accurately assess their immunomodulatory potential in future human therapeutic settings, where application of FBS is not feasible. IntroductionDendritic cells (DCs) are potent antigen-presenting cells important for induction of both tolerance and immunity, 1-3 and animal studies have sought to use these features to induce either antigen-specific immunity, antigen-specific tolerance, or antitumor immunity. 4,5 Methods have been developed to generate large numbers of DCs in vitro from spleen or bone marrow precursors in mice or from monocytes in humans. [6][7][8][9] Most of these protocols involve the use of growth factors such as granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), which can specifically promote DC generation. Other and less well-characterized factors are provided by, for example, heterologous fetal bovine serum (FBS), which has been used extensively.By nature, DCs sample their environment by antigen uptake, 10,11 and antigens are subsequently presented on major histocompatibility complex class I (MHC-I) and MHC-II molecules. Thus, in our opinion it is quite likely that DCs cultured in medium containing exogenous proteins will process and present not only an experimental antigen (eg, an antigenic peptide), but also antigens derived from heterologous proteins (eg, bovine serum albumin [BSA] from FBS). In addition, DCs rapidly respond to a variety of maturation and modulatory stimuli and it is not clear if and how a culture step in heterologous serum might affect subsequent DC function upon in vivo transfer.Nevertheless, DCs propagated or cultured in FBS-containing medium in vitro have been used in a number of different in vivo protocols, including attempts to immuni...
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