ABSTRACT
The pattern recognition receptor, RAGE, has been shown to be involved in adaptive immune responses but its role on the components of these responses is not well understood. We have studied the effects of a small molecule inhibitor of RAGE and the deletion of the receptor (RAGE-/- mice) on T cell responses involved in autoimmunity and allograft rejection. Syngeneic islet graft and islet allograft rejection was reduced in NOD and B6 mice treated with TTP488, a small molecule RAGE inhibitor (p < 0.001). RAGE-/- mice with streptozotocin-induced diabetes showed delayed rejection of islet allografts compared with wild type (WT) mice (p < 0.02). This response in vivo correlated with reduced proliferative responses of RAGE-/- T cells in MLRs and in WT T cells cultured with TTP488. Overall T cell proliferation following activation with anti-CD3 and anti-CD28 mAbs were similar in RAGE-/- and WT cells, but RAGE-/- T cells did not respond to costimulation with anti-CD28 mAb. Furthermore, culture supernatants from cultures with anti-CD3 and anti-CD28 mAbs showed higher levels of IL-10, IL-5, and TNF-alpha with RAGE-/- compared with WT T cells, and WT T cells showed reduced production of IFN-gamma in the presence of TTP488, suggesting that RAGE may be important in the differentiation of T cell subjects. Indeed, by real-time PCR, we found higher levels of RAGE mRNA expression on clonal T cells activated under Th1 differentiating conditions. We conclude that activation of RAGE on T cells is involved in early events that lead to differentiation of Th1(+) T cells.
Subject(s)
Cell Differentiation/immunology , Lymphocyte Activation/immunology , Receptors, Immunologic/metabolism , T-Lymphocyte Subsets/enzymology , T-Lymphocyte Subsets/immunology , Animals , Cell Differentiation/genetics , Cell Proliferation , Cells, Cultured , Diabetes Mellitus, Type 1/enzymology , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/immunology , Glycation End Products, Advanced/metabolism , Graft Survival/drug effects , Graft Survival/immunology , Islets of Langerhans Transplantation/immunology , Islets of Langerhans Transplantation/pathology , Ligands , Lymphocyte Activation/genetics , Lymphocyte Culture Test, Mixed , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mice, Transgenic , Receptor for Advanced Glycation End Products , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/deficiency , Receptors, Immunologic/genetics , T-Lymphocyte Subsets/pathology , Th1 Cells/enzymology , Th1 Cells/immunology , Th1 Cells/pathology , Th2 Cells/enzymology , Th2 Cells/immunology , Th2 Cells/pathologyABSTRACT
A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays.
