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


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 microgram/ml PHA (PHA group). Cur or CyA at varying concentrations (0.31, 0.625, 1.25, 2.5, 5, or 10 microgram/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 microgram/ml of cur) and PHA group (from 50956 +/- 5747 to 24 +/- 12 at 5.0 microgram/ml) with a calculated ED50 of 3.5 and 7.7, microM 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 microgram/ml curcumin showed a significantly increased apoptosis as compared to control (0.4% DMSO).


CONCLUSIONS
Since Cur blocks the CyA-resistant PMA + anti-CD28 pathway of T-cell proliferation, it may have novel adjuvant immunosuppressive properties.
Ranjan
D
D
Department of Surgery, University of Kentucky, Lexington, Kentucky, 40536, USA.
Johnston
T D
TD
Wu
G
G
Elliott
L
L
Bondada
S
S
Nagabhushan
M
M
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology; D018106:CD28 Antigens / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D003474:Curcumin / Q000494:pharmacology*; D016572:Cyclosporine / Q000494:pharmacology; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D018106:CD28 Antigens; D002273:Carcinogens; D007166:Immunosuppressive Agents; D016572:Cyclosporine; D003474:Curcumin; D013755:Tetradecanoylphorbol Acetate