OPN induces terminal DC differentiation with up-regulated expression of MHC class II, costimulatory, and adhesion molecules. DCs (1 × 106) were cultured in the presence or absence of either OPN or LPS from days 5 to 7 of culture. Cells were stained with antibodies against HLA-DR, CD40, CD80, CD86, CD44, and CD54 (A) and with antibodies against CD1a and CD14 to determine their DC phenotype (B) and were analyzed by FACS after 24 and 48 hours of stimulation. FACS analysis of 1 representative of 5 experiments is shown. The dotted line on the left in panels A and B represents isotype control staining of unstimulated DCs. (C) To demonstrate that the OPN added for DC activation induces HLA-DR expression independent of the endogenous OPN, DCs from day 5 (▪) were cultured for 24 hours in 100% (▩) or 50% (▨) DC-conditioned medium or in fresh medium without GM-CSF or IL-4 (0%; □), with (+) or without (–) exogenous OPN. Corrected mean fluorescence (cMFI) of HLA-DR expression from 1 representative of 3 independent experiments is given.
