ABSTRACT
TGF-ß1 is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-ß1 as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-ß1 inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. Murine splenocytes were differentiated into Th17 cells, and the effect of the TGF-ßRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. Flow cytometry analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction. SB-505124 potently reduced murine Th17 differentiation by decreasing Il17a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants. In vivo, SB-505124 reduced Th17 numbers, while increased numbers of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction. Blocking TGF-ß1 signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, local SB-505124 treatment does not suppress experimental arthritis.
Subject(s)
Arthritis, Experimental/metabolism , Cytokines/metabolism , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Benzodioxoles/pharmacology , Cell Differentiation/drug effects , Female , Humans , Imidazoles/pharmacology , Interleukin-17/metabolism , Mice , Mice, Inbred C57BL , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Pyridines/pharmacology , Signal Transduction , T-Lymphocytes, Regulatory/drug effects , Th17 Cells/drug effects , Tissue Culture Techniques/methodsABSTRACT
OBJECTIVE: To investigate the ways testosterone influences the murine bone marrow-derived macrophages (BMMs) and how testosterone affects the function of BMMs after bound to their membrane surface. METHODS: BMMs were cultured in vitro, their total RNA and proteins isolated, and the expression of intracellular androgen receptor (AR) detected through RT-PCR and Western blotting. The binding site of testosterone (T) to the membrane surface of BMMs was observed by confocal laser scanning microscopy after T-BSA-FITC incubation. Moreover, the intracellular Ca2+ was tested by Fura-2 method, and the influence of ionic currents on BMMs plasma membrane induced by testosterone was examined by the whole cell patch-clamp. RESULTS: RT-PCR and Western blotting failed to detect intracellular ARs in BMMs, but confocal laser scanning microscopy showed testosterone to be bound to the membrane surface of BMMs by impermeable T-BSA-FITC, inducing a rapid rise in the intracellular free Ca2+ concentration ([Ca2+]i) of Fura-2 loaded BMMs, predominantly due to the influx of extracellular Ca2+ through Ni2+ -blockable Ca2+ channels in the plasma membrane. Similarly, the patch-clamp technique revealed T-induced calcium influx in BMMs. CONCLUSION: It is reasonable to assume that the testosterone receptor exists on the plasma membranes, and testosterone act through unconventional plasma membrane receptors, induce Ca2+ influx and a rapid rise in the intracellular Ca2+ concentration, and influence the function of BMMs.
