ABSTRACT
Systemic lupus erythematosus (SLE) is an autoimmune disease resulting from dysregulation of the immune system. Interleukin-6 (IL-6) is a multifunctional cytokine produced by macrophages, monocytes and T and B cells. It stimulates B-cell differentiation/maturation, immunoglobulin secretion, and T-cell functions. Elevated levels of IL-6 in serum, urine and renal glomeruli were detected in patients with active SLE and in murine models of SLE. Our study investigated the role of IL-6 in an SLE-like disease in New Zealand Black/White (NZB/W) F1 mice by administration of an anti-murine IL-6 monoclonal antibody (mAb). Intraperitoneal administration of the anti-IL-6 mAb suppressed the production of anti-dsDNA autoantibody. B-cell proliferation induced by anti-IgM and anti-CD40 was lower in the anti-IL-6 mAb-treated mice, ex vivo studies demonstrated that anti-IL-6 mAb treatment inhibited anti-dsDNA production. Anti-CD3-induced T-cell proliferation and mixed lymphocyte reactions were inhibited by anti-IL-6 mAb treatment, indicating a partial down-regulation of T cells. Histological analysis showed that treatment with anti-IL-6 mAb prevented the development of severe kidney disease. These results suggest that treatment with anti-IL-6 mAb has a beneficial effect on autoimmunity in murine SLE and that autoreactive B cells may be the primary target for anti-IL-6 mAb treatment; its effect on autoreactive T cells is also indicated.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Autoimmune Diseases/therapy , Interleukin-6/antagonists & inhibitors , Lupus Erythematosus, Systemic/therapy , Animals , Antibodies, Antinuclear/biosynthesis , Autoimmune Diseases/blood , Autoimmune Diseases/immunology , B-Lymphocytes/immunology , DNA/immunology , Disease Models, Animal , Disease Progression , Interleukin-6/blood , Interleukin-6/immunology , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/immunology , Lupus Nephritis/immunology , Lupus Nephritis/pathology , Lupus Nephritis/prevention & control , Lymphocyte Activation/immunology , Mice , Mice, Inbred NZB , STAT3 Transcription Factor/metabolism , Serum Amyloid A Protein/analysis , Severity of Illness Index , T-Lymphocyte Subsets/immunologyABSTRACT
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.
