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Checkpoint Kinase

Full serum-free culture medium did not contain detectable amounts of GC

Full serum-free culture medium did not contain detectable amounts of GC. Statistics Estimation of statistical differences between groups was carried out using the unpaired Students t-test or two-way ANOVA test, where appropriate. detectable. Irrespective of their maturation stage, T cells that produced GC in this manner undergo autonomous cell death as this was blocked when glucocorticoid receptor-deficient T cells were treated with GC metabolites. These results indicate that both immature and mature T cells possess the capacity to undergo apoptosis in response to intrinsically generated GC. Consequently, positive selection of thymocytes, as well as survival of peripheral T cells may depend on TCR-induced escape of otherwise HSD11B1-driven autonomous T-cell death. Glucocorticoids (GC) are steroid hormones primarily produced in the adrenal cortex in response to emotional, physical and immunological stress. Corticosterone, the predominant GC in mice, and its human homolog cortisol, have numerous effects on diverse processes such as metabolic activity, immune function and behavior.1 GC bind to their receptor, the glucocorticoid receptor (GR), which reduces the expression of many pro-inflammatory cytokines and it is generally assumed that this explains the potent anti-inflammatory and immunosuppressive properties of GC.2 The thymus is the key immunological organ for the maturation of T cells in mammals. Elevation of GC due to chronic stress or experimental administration causes involution of the thymus due to the fact that GC are strong inducers of apoptosis in thymocytes and have a critical role in their development and function. Immature double-negative (DN) thymocytes (CD4?CD8?) proliferate and differentiate in the thymus to generate double-positive (DP) CD4+CD8+ cells. Most of these DP cells undergo apoptosis; the surviving differentiate into single-positive (SP) CD4+ or CD8+ cells that migrate to peripheral lymphoid tissues.3, 4 Positive selection of developing thymocytes for progression from the DP to the SP stage requires low to moderate avidity TCR-mediated interactions with self-peptide/MHC ligands.5, 6 GC have been proposed to be essential for the selection of immunocompetent T cells.7 The mutual antagonism hypothesis proposes that a quantitative balance between TCR and GR signaling determines the fate of a developing thymocyte. GC thereby promote positive selection by antagonizing negative selection signals.8, 9, 10, 11 In contrast, TCR signaling increasingly reverses GC-induced apoptosis12 as GSK598809 thymocyte development progresses.13 While the main source of GC are the adrenals, evidence accumulated over the last two decades that GC are also synthesized in other organs including the brain, intestinal tract, skin and thymus (both epithelial and immune cells).14, 15 Accordingly, these organs express the steroidogenic enzymes necessary for the synthesis of GC which apparently act in an autocrine or paracrine fashion.3 Overexpression of GR in the T-cell lineage leads to a reduced number of thymocytes in adrenalectomized mice, suggesting that non-adrenal-derived GC could exert a negative effect on thymocyte development.16 In the mouse thymus, however, there is considerable controversy about the cellular origin of GC synthesis. The presence of key enzymes for GC synthesis has been extensively described GSK598809 in thymic epithelial cells (TEC10, 17). On the other hand, some studies show the ability of thymocytes to synthesize GC.18, 19 Disagreement exists also on whether the expression KITH_HHV1 antibody of GC-synthesizing enzymes is dependent on T-cell activation status.20, 21 Of note, corticosterone can also be produced from the inactive metabolite 11-dehydrocorticosterone (11-DHC) via the reductase activity of HSD11B1, which is expressed by murine CD4+ and CD8+ lymphocytes.22 In GSK598809 thymocytes, has been shown to be expressed at substantial levels20 and also to be functionally active.23 Along similar lines, we aimed to investigate the quantitative contribution of either GC synthesis or conversion of 11-DHC to T-cell-derived corticosterone and tested whether this hormone displays intracrine activity. We performed a detailed analysis of the expression and activity of steroidogenic enzymes in mouse thymus and spleen, throughout T-cell development. Based on our findings, we can refute a significant role for CYP11B1 in GC synthesis, suggesting that neither thymocytes nor splenocytes synthesize significant amounts of GC In contrast, HSD11B1 converts inactive 11-DHC into active corticosterone that can induce subsequent thymocyte and T-cell death. Our findings highlight an underappreciated T-cell autonomous mechanism that can affect the T-cell selection process and contribute to the tolerizing effects and immune suppressive function of glucocorticoids. Results Expression analysis of glucocorticoid metabolic enzymes across T-cell development To date it is unclear which cell type(s) of.