Oxidized β₂-GPI induced maturation of human monocyte-derived DCs. Five-day human DCs were stimulated with or without ox-β₂-GPI (10 μg/mL), H₂O₂-β₂-GPI (10 μg/mL), αTOC-β₂-GPI (10 μg/mL), and LPS (100 ng/mL). Expression of surface molecules was analyzed by flow cytometric analysis as described in “Materials and methods.” Phenotypic maturation of DCs was detected by the appearance of CD83 and by the expression of costimulatory molecules. (A) H₂O₂-β₂-GPI and ox-β₂-GPI induced the appearance of CD83 after 18 hours of culture (□) and (B) induced a significant up-regulation of CD80 (□), CD86 (▪), HLA-DR (▦), and CD40 (▧) expression at 48 hours of culture. Pretreatment of β₂-GPI with the antioxidant α-tocopherol (5 μM) prevented the phenotypic maturation of DCs, whereas pretreatment with polymyxin B (pol B) did not. Pretreatment of LPS with the antioxidant α-tocopherol left LPS-induced phenotypic maturation unchanged. Adding metal chelators (DTPA) also inhibited DC maturation (the appearance of CD83), suggesting that metals probably catalyze β₂-GPI oxidative modification (A, right). In contrast, protease inhibitors failed to inhibit β₂-GPI-induced DC maturation. Control protein H₂O₂-HSA failed to induce DC phenotypic maturation (A, right). Results are expressed as mean ± SD of the positive cell percentages (A) and of the mean fluorescence intensity (B) of 11 independent experiments. Significant differences are indicated (^*P < .001; ^**P = .043 by Student t test; ^***P < .002 by Wilcoxon nonparametric test). Samples were analyzed on a FACScan cytofluorimeter using CELLQuest software (Becton Dickinson, Pharmingen Biosciences).