Curcumin blocks cyclosporine A-resistant CD28 costimulatory pathway of human T-cell proliferation

Introduction. Curcumin (Cur) is a phenolic component of common spice, turmeric. We have reported earlier that it possesses antineoplastic and immunosuppressive properties in vitro. It has been reported that cyclosporine A (CyA), a commonly used immunosuppressant does not inhibit CD28 costimulatory pathway of T-cell activation. We hypothesized that Cur, a tyrosine kinase inhibitor, would block CyA-resistant CD28 costimulatory pathway of human T cell proliferation. Materials and methods. Human T- lymphocytes were isolated from healthy donors using gradient centrifugation and rosetting techniques. In four separate experiments T-cells were plated in triplicate in 96-well plates at a density of 2X105 cells/well. These cells were stimulated with 0.5 ng/ml phorbol myristate acetate (PMA) + 0.5 (g/ml anti-CD28 antibody (PMA. CD28 group) or 2.5 μg/ml PHA (PHA group). Cur or CyA at varying concentrations (0.31, 0.625, 1.25, 2.5, 5, or 10 μg/ml and 1.25, 2.5, 5, 10, 20, or 250 ng/ml, respectively) was added and cellular proliferation was measured by the uptake of [³H]thymidine and is reported (mean cpm/well(SD). Cells from the PMA-CD28 group that were treated with either curcumin or 0.4% DMSO (vehicle control for curcumin) were studied for evidence of apoptosis by staining with viable dyes MC540 and Hoechst 33342 and subsequently analyzed in the cell sorter. Results. Cur caused a concentration-dependent inhibition of T-cell proliferation in the PMA-CD28 group (from 32775 ± 3084 to 66 ± 42 at 5.0 μg/ml of cur) and PHA group (from 50956 ± 5747 to 24 ± 12 at 5.0 μg/ml) with a calculated ED50 of 3.5 and 7.7, μM respectively. CyA inhibited T-cell proliferation in the PHA group with a calculated ED50 of 2.7 ng/ml but failed to block PMA + anti. CD28-stimulated T-cell proliferation even at 250 ng/ml. PMA-CD28 group cells treated with 10 μg/ml curcumin showed a significantly increased apoptosis as compared to control (0.4% DMSO). Conclusion. Since Cur blocks the CyA- resistant PMA + anti-CD28 pathway of T-cell proliferation, it may have novel adjuvant immunosuppressive properties.