Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by diverse cellular and biochemical aberrations, including decreased production of IL-2. Here we show that nuclear extracts from unstimulated SLE T cells, unlike extracts from normal T cells, express increased amounts of phosphorylated cAMP-responsive element modulator (p-CREM) that binds the −180 site of the IL-2 promoter. Nuclear extracts from stimulated normal T cells display increased binding of phosphorylated cAMP-responsive element binding protein (p-CREB) to the −180 site of the IL-2 promoter, whereas nuclear extracts from stimulated SLE T cells display primarily p-CREM and decreased p-CREB binding. In SLE T cells, p-CREM bound to the transcriptional coactivators, CREB binding protein and p300. Increased expression of p-CREM correlated with decreased production of IL-2. The transcription of a reporter gene driven by the −180 site was enhanced in normal T cells, but was suppressed in SLE T cells. These experiments demonstrate that transcriptional repression is responsible for the decreased production of IL-2 by SLE T cells.

IntroductionA subpopulation of T cells, termed regulatory T cells (Tregs), have been described in humans and animal models; these cells are important because they suppress autoreactive T cells by direct cell contact. [1][2][3][4] Phenotypically, Treg are characterized by cell surface expression of the proteins CD4 and CD25 and by intracellular expression of the transcription factor FOXP3. Only CD4 ϩ CD25hi ϩ FOXP3 ϩ T cells express suppressor functions. FOXP3 is a member of the forkhead/winged-helix family of transcription regulators (FOXP1-4). In humans, mutations in FOXP3 result in an autoimmune syndrome termed immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. 5 The transcription factor NFAT1 induces FOXP3 expression by binding to its promoter. 6 In addition, FOXP3 expresses its repressive effects on cytokine expression and its activating effects on CD25 by cooperation with NFAT1. 7,8 Acquired aplastic anemia is characterized by destruction of hematopoietic stem cells by cytotoxic T lymphocytes. 9 Hematopoietic response and hematologic recovery after successful immunosuppressive treatment represent the most powerful evidence that this rare and complex disease is immune-mediated. 10 Increased T-bet protein levels in T cells probably are responsible for the increased interferon-␥ levels and the Th1 polarization in patients with aplastic anemia. 11 We show that Tregs were decreased in acquired aplastic anemia, as in other autoimmune diseases. [12][13][14][15][16] All patients examined had low levels of FOXP3. Mechanistically, FOXP3 down-regulation appeared to be mediated by the transcription factor NFAT1. Patients, materials, and methods Patients and control subjectsInformed consent was acquired according to protocols approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute (NHLBI) and was obtained in accordance with the Declaration of Helsinki for all patients (n ϭ 20; age range, 13-52 years) with acquired aplastic anemia examined (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article) and healthy volunteers (n ϭ 14; age range, 18-55 years). Lymphocyte isolation, flow cytometry, and immunoblotsCD4, CD25, and FOXP3 expression was examined in peripheral blood mononuclear cells (PBMC) by 3-color flow cytometry as previously described 11 using an APC-antihuman FOXP3 staining kit (eBioscience, San Diego, CA). 15 Immunoblot experiments were performed as previously described 11 (Document S1). Quantitative real-time polymerase chain reactionFOXP3 gene expression was measured in CD4 ϩ CD25 ϩ T cells as previously described. 15 All polymerase chain reaction assays were performed in duplicate and reported as the mean. Confocal microscopy and T-cell transfectionsNFAT1 and FOXP3 expression was examined by confocal microscopy as previously described 17 (Document S1). Transfections were performed 18,19 using a GFP-wild-type NFAT1 plasmid 20 (a gift from Dr. A. Rao, Harvard University, Cambridge, MA) and examined ...

Objective. Dkk-1 is an inhibitory molecule that regulates the Wnt pathway, which controls osteoblastogenesis. This study was undertaken to explore the potential role of Dkk-1 in ankylosing spondylitis (AS), a prototypical bone-forming disease.Methods. Serum Dkk-1 levels were measured in 45 patients with AS, 45 patients with rheumatoid arthritis (RA), 15 patients with psoriatic arthritis (PsA), and 50 healthy subjects by sandwich enzyme-linked immunosorbent assay (ELISA). A functional ELISA was used to assess the binding of Dkk-1 to its receptor (lowdensity lipoprotein receptor-related protein 6). Furthermore, we studied the effect of sera from patients with AS and healthy subjects on the activity of the Wnt pathway in the Jurkat T cell model, with and without a neutralizing anti-Dkk-1 monoclonal antibody, by Western immunoblotting.Results. Serum Dkk-1 levels were significantly increased in patients with AS (mean ؎ SEM 2,730 ؎ 135.1 pg/ml) as compared with normal subjects (P ‫؍‬ 0.040), patients with RA (P ‫؍‬ 0.020), and patients with PsA (P ‫؍‬ 0.049). Patients with AS receiving anti-tumor necrosis factor ␣ (anti-TNF␣) treatment had significantly higher serum Dkk-1 levels than patients with AS not receiving such treatment (P ‫؍‬ 0.007). Patients with AS studied serially prior to and following anti-TNF␣ administration exhibited a significant increase in serum Dkk-1 levels (P ‫؍‬ 0.020), in contrast to patients with RA, who exhibited a dramatic decrease (P < 0.001). Jurkat cells treated with serum from AS patients exhibited increased Wnt signaling compared with cells treated with control serum. In that system, Dkk-1 blockade significantly enhanced Wnt signaling in control serum-treated, but not AS serum-treated, Jurkat T cells.Conclusion. Our findings indicate that in patients with AS, circulating bone formation-promoting factors functionally prevail. This can be at least partially attributed to decreased Dkk-1-mediated inhibition.

Systemic lupus erythematosus (SLE) T cells express high levels of cAMP response element modulator (CREM)that binds to the IL-2 promoter and represses the transcription of the IL-2 gene. This study was designed to identify pathways that lead to increased binding of CREM to the IL-2 promoter in SLE T cells. Ca 2+ /calmodulin-dependent kinase IV (CaMKIV) was found to be increased in the nucleus of SLE T cells and to be involved in the overexpression of CREM and its binding to the IL-2 promoter. Treatment of normal T cells with SLE serum resulted in increased expression of CREM protein, increased binding of CREM to the IL-2 promoter, and decreased IL-2 promoter activity and IL-2 production. This process was abolished when a dominant inactive form of CaMKIV was expressed in normal T cells. The effect of SLE serum resided within the IgG fraction and was specifically attributed to anti-TCR/CD3 autoantibodies. This study identifies CaMKIV as being responsible for the increased expression of CREM and the decreased production of IL-2 in SLE T cells and demonstrates that anti-TCR/CD3 antibodies present in SLE sera can account for the increased expression of CREM and the suppression of IL-2 production.

IntroductionAplastic anemia, the paradigm of bone marrow failure syndromes, is characterized by peripheral blood pancytopenia and an empty bone marrow. 1 In most cases, aplastic anemia is an immunemediated disease with active destruction of hematopoietic cells by T lymphocytes. 2 The aberrant immune response may be triggered by drugs, virus, or chemical exposure, but in the majority of cases there is no obvious etiologic factor. 3,4 The clinical observations that most patients respond to immunosuppressive treatment with cyclosporine and antithymocyte globulin-based regimens 5,6 is the most powerful evidence for the pivotal role of the immune system in the pathophysiology of aplastic anemia. Excessive production of interferon-␥ (IFN-␥), tumor necrosis factor (TNF), and interleukin-2 (IL-2) from patients' T cells suggests that the hematopoietic cells are destroyed through a Th1 T-cell response. [7][8][9][10][11] This Th1 "shift" in aplastic anemia results in both Fas-mediated cell death and inhibition of hematopoietic stem cell proliferation. 2,12 Oligoclonal expansion of cytotoxic T lymphocytes (CTLs) correlates with disease activity. 13,14 In an animal model of aplastic anemia, injection of alloreactive lymphocytes results in bone marrow failure, but pancytopenia can be prevented with anti-IFN-␥ and anti-TNF monoclonal antibody. 15 IFN-␥, the hallmark cytokine of the Th1 immune response, is produced primarily by T cells and natural killer (NK) cells. Following activation, naive T cells differentiate into Th1 CD4 ϩ and cytotoxic CD8 ϩ cells that secrete IFN-␥ and other cytokines, and Th2 CD4 ϩ cells that produce IL-4 and other cytokines. Two transcription factors are responsible for the shift of CD4 ϩ T cells into the Th1 or Th2 phenotype: T-bet for Th1 and GATA-3 for Th2. 16,17 IFN-␥ is also produced when T cells are stimulated with IL-12 or IL-18 secreted by antigen-presenting cells (APCs). Regulation of IFN-␥ production occurs primarily at the level of transcription. 18 The proximal site of the IFN-␥ gene (Ϫ75 to Ϫ45 bp of the IFN-␥ promoter) is a binding site for nuclear factor for activated T cells (NFATs), AP-1, ATF, and CREB transcription factors. 19,20 In the proximal IFN-␥ promoter site, a half T-box sequence allows T-bet binding, resulting in increased IFN-␥ production. 21 T-bet is a member of the T-box family of transcription factors. 22 This family contains a highly conserved DNA binding domain, the T-box. T-box binds to a specific sequence in the promoter of different genes. T-bet is found in Th1 but not in Th2 cells and is the key regulator of Th1 development and function. 16,23 Mice lacking T-bet fail to develop Th1 cells and are driven toward Th2-mediated disease. 24 Overexpression of T-bet in Th2 cells results in loss of the Th2 phenotype and increased production of IFN-␥. 16 Activated T cells result in increased T-bet expression, which induces IL-12R␤2 expression. 25 T-bet also positively regulates its own expression through an autoregulator loop involving Hlx, a homeobox gene. 26 T-cell engagem...