Role of Protein Kinase C and Nox2-Derived Reactive Oxygen Species Formation in the Activation and Maturation of Dendritic Cells by Phorbol Ester and Lipopolysaccharide

Figure 4

Analysis of the effects of apocynin treatment on the formation of ROS, maturation, and the T cell stimulatory capacity of BM-DCs upon activation by PDBu or LPS. (a) BM-DCs (1 × 10⁶ cells/well) were stimulated with PDBu (10 μM) and additionally treated with different concentrations of apocynin (100–1000 μM) or with the potent inhibitor of Nox2 activity nebivolol (100 μM), and ROS generation was measured by L-012 chemiluminescence at 10 min after stimulation. (b) ROS formation after stimulation of BM-DCs (1 × 10⁶ cells/well) with LPS (50 μg/ml) and cotreatment with apocynin (100–300 μM) was measured by L-012 chemiluminescence at 40 min after stimulation. (c) Maturation of BM-DCs (expression of MHCII and CD86 at high level) was analyzed after stimulation with LPS (1.5 μg/ml) or PDBu (10 μM) and treatment with apocynin (150–300 μM) after 24 h via flow cytometry. (d) T cell stimulatory capacity after apocynin treatment was compared in a proliferation assay (see legend to Figure 3). Data are mean ± SEM of 8 (a, b) and 3 (c) experiments. (d) One representative experiment of two is shown. (a, b) versus Ctr (w/o PDBu or LPS); versus PDBu or LPS treated sample; (c) ; versus unstimulated; versus LPS. (d) ; versus unstimulated; versus PDBu.

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