FIGURE 5.

The effect of EGCG on CD3-mediated AP-1 DNA binding and IL-2 cytokine release. A, gel-shift assay. Nuclear extracts were obtained from cells pretreated with different concentrations of EGCG (0, 4, or 8 μm) for 1 h and then exposed to mouse IgG₁κ monoclonal immunoglobulin isotype control (2 μg/ml) or mouse anti-human CD3 (2 μg/ml) for 24 h. The nuclear extracts were loaded onto a 5% polyacrymide gel and probed with γ-³²P-labeled AP-1. Specific binding of nuclear proteins to the AP-1-responsive element was analyzed by the electrophoretic mobility shift assay. B and C, IL-2 secretion was measured from cells pretreated for 1 h with various concentrations of EGCG and then exposed to mouse IgG₁κ monoclonal immunoglobulin isotype control (2 μg/ml) or mouse anti-human CD3 (2 μg/ml) for 24 (B) or 48 h (C). IL-2 production was measured in supernatant fractions by an enzyme-linked immunosorbent assay according to the manufacturer's instructions. Data are represented as the average of triplicate samples from three independent experiments. The asterisks indicate a significant change relative to P116.cl39 cell stimulated with CD3 (^*, p < 0.005; ^**, p < 0.001).