engleski

The effect of immunosuppressant cyclosporin A and dexamethasone on T-lymphocyte activation, cell-cycle and apoptosis

Aim. Immunosuppressants are used for the prevention and treatment of graft rejection, graft-versus-host disease and autoimmune disorders. We investigated the effects of commonly used immunosuppressants, cyclosporin A (CsA) and dexamethasone (DEX), on T-lymphocyte activation, cell-cycle progression and apoptosis in the in vitro model of mitogen-stimulated T-lymphocyte leukemic Jurkat cell-line and separated peripheral blood mononuclear cells (PBMC). Methods. Jurkat cells or PBMC were pretreated with different doses of CsA and DEX for 1 hour at 37 °C, activated with previously-optimized doses of mitogen phytohemagglutinin (2.5 and 0.625  g/ml for Jurkat and 5 and 20  g/ml for PBMC), and cultured for 24, 48 or 72 hrs. The separate effects of each immunosuppressant, as well as their additive effect were analyzed. Activation, cell-cycle and apoptosis were measured using fluorescent labeling, followed by flow-cytometric analysis. The expression of CD69 (24 hrs), an early cell-surface activation marker, was analyzed by flow cytometry, and propidium-iodide staining was used for cell-cycle analysis (48 hrs) and nick-translation assay for the detection of apoptosis (72 hrs). Results. Expression of CD69 decreased in a dose-dependant manner after immunosuppressant-pretreatment. Cell-cycle analysis revealed an inverse correlation between the number of cells in G2/M-phase and the dose of CsA and DEX. Decrease in activation, as well as in the number of cells in G2/M-phase, was higher when cells were treated with CsA than with DEX, and a more pronounced additive effect was detected in the cultures of PBMC than Jurkat cells. Cell apoptosis significantly increased with CsA- and DEX-pretreatmen and the additive effect on cell apoptosis was observed with the combination of CsA and DEX. Conclusion. Our results suggest that both CsA and DEX inhibit T-lymphocyte activation and block cells in G0/G1-phase of the cell-cycle, with CsA having a stronger effect. Additive effect of CsA and DEX may have an important role in immunosuppressive combination therapy which aims at minimizing side effects of any single drug. Further investigations need to be performed to analyze the effects of immunosuppressants on Jurkat cells and PBMC, by approaching other aspects of the immune response, such as cytokine expression, lymphocyte subpopulation polarization, intracellular drug interactions, and transcriptional factors’ activation. Finally, the effects should be confirmed by the analysis of PBMC from patients receiving immunosuppressive treatment.

Jurkat cells; Cyclosporin A; Glucocorticoids; Activation; Apoptosis; Cell-cycle

Rad je kao poster prezentiran i na skupu Fourth European-American School in Forensic Genetics and Mayo Clinic Course in Advanced Molecular and Cellular Medicine, održanom od 05.-09-09.2005., Dubrivnik, Hrvatska ; objavljen u knjizi sažetaka ; Zagreb : International Society of Applied Biological Sciences, 2005. ; str. 146-146.