Corticosteriod Receptor Agonists Protect T Cells From Apoptosis

Objective: Sepsis is a major public health concern with high morbidity and mortality in the U.S. More recently, sepsis has been viewed as a state of immune dysregulation with excessive activation early followed later by immune-suppression, characterized by secondary infections. This critical late phase has been associated with increased effector T-cell apoptosis. Studies in mice have demonstrated extensive T-cell apoptosis during septic shock and improved outcome with adoptive T-cell transfer or the administration of caspase inhibitors, a numbers of studies have shown an inverse correlation between lymphocyte count and survival. It was clear that activation-induced T cell apoptosis is caused by the upregulation of Fas ligand(Fas L) expression and its subsequent interaction with Fas(CD95). The binding of Fas ligand(Fas L) to its receptor Fas(CD95) activates the caspase cascade and triggers extrinsic T-cell apoptosis. Caspases including caspases-8, caspases-6 or caspases-9 and downstream caspase- 3 cleave target proteins that lead to DNA fragmentation and programmed cell death.Corticosteroids(GCs) therapy has been use in varied doses for sepsis for more than half a century, with no clear benefit on mortality and serious unwanted effects. Stress-dose corticosteroid regimens that target both glucocorticoid receptor(GR) and mineralocorticoid receptors(MR) reverse hypotension in septic shock, but have a variable impact on survival. Lack of a survival benefit has been attributed to secondary infections and other adverse effects mediated through GR. Conversely, the role of mineralocorticoids(MCs) activity in the risks/benefits of corticosteroid therapy in septic shock is largely unknown. The effects of glucocorticoids on lymphocytes are dramatic. Corticosteroids induce apoptosis on immature thymocytes(positive selection and negative selection), mature splenic T cells, pre-B lymphoma cells, and several leukemic cell lines through caspase-dependent manner. Conversely, Glucocorticoids protect activation-induced cell death via down-regulating CD95 ligand gene expression through GR(in a DNA binding dependent manner) in T cells. A negative GR element found at position-990 in the Fas L promoter binds GR in vitro as as in the chromatin context. Fas L promoter contains bingding sites for several transcription factors including NF- κB. Negative glucorticoid response element(n GRE) overlaps with a known NF-κB binding site.GR down-regulates Fas L promoter by competing with NF- κB for binding to the common response element.Glucocorticoids(GCs) may address the hyper-inflammatory early phase of sepsis by trans-repressing inflammatory gene transcription and the late immune suppression phase by inhibiting activation-induced T-cell apoptosis via the down-regulation of Fas L. Closely related to GR, MR also expressed on T cells, but less so in phagocytic leukocytes and therefore may regulate T-cell function while preserving antibacterial mechanisms. In this study we set out to explore the effects Dexamethasone(DEX), a GR agonist, and deoxycorticosterone(DOC), a MR agonist on activation-induced Fas L expression and apoptosis in human peripheral blood-T cells and Jurkat cells.Methods: Human primary T cells were incubated with PHA or SEB in the absence or presence of DEX or DOC, apoptosis were analyzed by flow cytometry using Annexin V/PI staining. The caspase-8, caspase-9/6 and caspase-3 activities were measure by fluorescence method, IL-2 production was measured by ELISA, and IL-2 and Fas L expression level were measured via q RT-PCR. Fas L-driven reporter assays and NF-κB-driven reporter assays in PHA plus PMA-stimulated Jurkat cells were used to explore the effect of DEX and DOC on Fas L transpcription. Then dominant negative mutant MR were transfected into Jurkat cells, DEX and DOC effect on NF-κB and Fas L reporter activity and Fas L gene expression were measured. Further, using the proximal NF-κB/AP-1 composite sequence from the human IL-2 promoter and the putative NF-κB/GRE Fas L composite sequence from the Fas L promoter, EMSAs were performed with Jurkat cell nuclear extracts. Next, to further confirm that both DEX and DOC suppression of IL-2 and Fas L was primarily mediated via a transrepression mechanism, cycloheximide, a protein synthesis inhibitor was used to block any induction of anti-inflammatory genes. Finally human wild type MR and mutant MR(K624A) were over expressed in Jurkat cells simultaneously transfectted with the wild type Fas L reporter construct to test the MR specificity of DOC.Results: Either hytohemagglutinin(PHA) or Staphylococcus aureus enterotoxin B(SEB) can trigger apoptosis in human primary T cell, which were blocked by both DEX and DOC. PHA activated caspase-3,8,9/6 activity in human primary T cells, while both DEX or DOC inhibited those responses. These inhibitory effects of DEX and DOC on PHAinduced caspase-3 activation were dose-dependent. PHA induced IL-2 production in human primary lymphocytes. Both DEX and DOC significantly blocked this response. PHA plus PMA similarly stimulated IL-2 production in Jurkat cells, which was also suppressed by DEX and DOC. PHA plus PMA inhanced Fas L and IL-2 m RNA in Jurkat cells. Both DEX and DOC significantly blocked these responses. Dual specificity phosphatase 1(DUSP1) and glucocorticoid-induced leucine zipper(GILZ) were strongly induced by DEX in primary human lymphocytes, but either unaffected or weakly induced by DOC, respectively. DEX and DOC significantly inhibited NF-κB driven reporter gene activity, which was also were dose-dependent. DEX and DOC inhibited wild type Fas L promoter activity, but had no significant effect on the mutant Fas L promoter reporter construct containing a disrupted mutant NF-κB binding site.Conclusions: DEX, a selective glucocorticoid agonist, and DOC, a selective mineralocorticoid agonist, both block apoptosis in human primary lymphocytes. DEX and DOC inhibit PHA-induced caspase activity in human primary lymphocytes. DEX and DOC block IL-2 and Fas L expression in human primary lymphocytes and Jurkat cells. DEX and DOC differently affect anti-inflammatory genes, but similarily block NF-κB and Fas L promoter driven gene transcription. DEX and DOC inhibition of IL-2 and Fas L expression is consistent with transrepression. DOC suppression of NF-κB signaling and Fas L induction are MR dependent. Selected MR ligands may be useful in blocking the immune dysregulation and possibly have a better risk/bnefit profile than GR agonists in the treatment of sepsis and other immune disorders.