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 an d immunosuppressive properties in vitro. It has been reported that cyc losporine 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 costi mulatory pathway of human T cell proliferation. Materials and methods. Human T-lymphocytes were isolated from healthy donors using gradient centrifugation and resetting techniques. In four separate experiments T-cells were plated in triplicate in 96-well plates at a density of 2X 105 cells/well. These cells were stimulated with 0.5 ng/ml phorbol myr istate acetate (PMA) + 0.5 (g/ml anti-CD28 antibody (PMA-CD28 group) o r 2.5 mu g/ml PHA (PHA group). Cur or CyA at varying concentrations (0 .31, 0.625, 1.25, 2.5, 5, or 10 mu g/ml and 1.25, 2.5, 5, 10, 20, or 2 50 ng/ml, respectively) was added and cellular proliferation was measu red by the uptake of [H-3]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 conce ntration-dependent inhibition of T-cell proliferation in the PMA-CD28 group (from 32775 +/- 3084 to 66 +/- 42 at 5.0 mu g/ml of curl and PHA group (from 50956 +/- 5747 to 24 +/- 12 at 5.0 mu g/ml) with a calcul ated ED50 of 3.5 and 7.7, mu M respectively, CyA inhibited T-cell prol iferation in the PHA group with a calculated ED50 of 2.7 ng/ml but fai led to block PMA + anti-CD28-stimulated T-cell proliferation even at 2 50 ng/ml. PMA-CD28 group cells treated with 10 mu g/ml curcumin showed a significantly increased apoptosis as compared to control (0.4% DMSO ). Conclusion. Since Cur blocks the CyA-resistant PMA + anti-CD28 path way of T-cell proliferation, it may have novel adjuvant immunosuppress ive properties. (C) 1998 Academic Press.