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...
Mucosal tissues contain large numbers of memory CD4+ T cells that, through T-cell receptor-dependent interactions with antigen-presenting cells, are believed to have a key role in barrier defense and maintenance of tissue integrity. Here we identify a major subset of memory CD4+ T cells at barrier surfaces that coexpress interleukin-18 receptor alpha (IL-18Rα) and death receptor-3 (DR3), and display innate lymphocyte functionality. The cytokines IL-15 or the DR3 ligand tumor necrosis factor (TNF)-like cytokine 1A (TL1a) induced memory IL-18Rα+DR3+CD4+ T cells to produce interferon-γ, TNF-α, IL-6, IL-5, IL-13, granulocyte–macrophage colony-stimulating factor (GM-CSF), and IL-22 in the presence of IL-12/IL-18. TL1a synergized with IL-15 to enhance this response, while suppressing IL-15-induced IL-10 production. TL1a- and IL-15-mediated cytokine induction required the presence of IL-18, whereas induction of IL-5, IL-13, GM-CSF, and IL-22 was IL-12 independent. IL-18Rα+DR3+CD4+ T cells with similar functionality were present in human skin, nasal polyps, and, in particular, the intestine, where in chronic inflammation they localized with IL-18-producing cells in lymphoid aggregates. Collectively, these results suggest that human memory IL-18Rα+DR3+ CD4+ T cells may contribute to antigen-independent innate responses at barrier surfaces.
To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitro؊activated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3-4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4 ؉ fraction, as purified CD4 ؉ T-cells prevented the accelerated diabetes. When CD25 ؉ cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. CD25ϩ cells prevent Th1-and Th2-induced colitis as well as Leishmania major infection (10), control active experimental autoimmune encephalomyelitis (11), and ameliorate established colitis (12).In the NOD mouse model of human type 1 diabetes, regulatory T-cells were first defined as a subpopulation of cells expressing CD4 ϩ CD45RB low or CD4 ϩ CD62L ϩ (13-15). Later, these regulatory T-cells were shown to coexpress CD62L and CD25 (16).In the other spontaneous model of type 1 diabetes, the DP-BB, regulatory cells have not been characterized with regard to their expression of CD25. Previously, spontaneous disease was prevented by a single transfer of spleen cells (17) and the prevention was enhanced by enrichment for CD4 ϩ cells (18). However, transfer of leukocytes from normal rats has so far been successful only if the transfer takes place before the initiation of insulitis (i.e., at about age 1 month) (17,19); transfers conducted at 2 months accelerate diabetes (19). Protection is dependent on the engraftment of the ART2 ϩ (formerly RT6) cells (20); in the control strain, the DR-BB, depletion of these induces diabetes (21). We recently showed that accelerated diabetes induced by the transfer of in vitro phorbol myristate acetate (PMA) plus ionomycinϪactivated unfractionated splenocytes from acutely diabetic donors is delayed by transfer of DR-BBϪderived leukocytes (22).In this study, we proposed to prevent diabetes using a new rat model where diabetes onset was accelerated by transfer of pure, in vitro PMA plus ionomycinϪactivated T-cells; that is, without activated antigen-presenting cells as were used in our previous study (22). Using preactivated diabetogenic T-cells in the current study allowed us to explore a situation that is relevant to understanding how such already activated T-cells can be controlled in patients with ongoing -cell destruction. Establishing the conditions required for preventing diabetes by cotransferring various leukocytes from healthy DR-BB rats allowed us to narrow a powerful regulatory potential to CD4 ϩ CD25 ϩ T-cells. This new model in rats is well suited to testing preventive s...
The objective of this study was to explore the relationship between serum levels of β2-microglobulin (β2MG), which some studies suggest reflect disease activity in systemic lupus erythematosus (SLE), and various clinical and immunological markers of disease activity in SLE. Twenty-six SLE patients and 10 healthy controls were included. Disease activity was assessed by: SLEDAI, 24 hr-proteinuria, circulating levels of complement C3, anti-double-stranded DNA (anti-dsDNA), β2MG and various pro-inflammatory and anti-inflammatory cytokines (IL-6, IL-8, IL-10, IL-18) measured with a multiplex assay, IFN-α assessed with a reporter gene assay, and a combined expression score of 12 IFN-α inducible genes in peripheral blood mononuclear cells. Median serum levels of β2MG were significantly higher in SLE patients vs controls (2.8 mg/L, range: 1.1-21.6 and 1.2 mg/L, range: 0.9-1.7, respectively, p < 0.001). β2MG was correlated with SLEDAI score (R = 0.68, p < 0.001), 24 hr-proteinuria (R = 0.64, p < 0.001), and complement C3 (R = -0.52, p = 0.007). The cytokines were significantly correlated with β2MG: IL-6 (R = 0.45, p = 0.02), IL-8 (R = 0.75, p < 0.001), IL-10 (R = 0.67, p < 0.001) and IL-18 (R = 0.71, p < 0.001) as were serum IFN-α (R = 0.45, p = 0.02) and the IFN-α inducible gene-score (R = 0.51, p = 0.01). The results support that β2MG may serve as a marker of disease activity in SLE. The correlations with the measured cytokines indicate that increased β2MG in SLE reflects immunological activity.
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