OPN induces the emigration of human Langerhans cells from the epidermis and their activation with up-regulated expression of MHC class II molecules. Split thickness sheets of human skin (2 cm²) were floated on c-RPMI supplemented with (closed symbols) or without (open symbols) OPN (0.5 μg/mL). Sheets were removed after 24 hours. Emigrated cells were counted microscopically following lyses of contaminating erythrocytes. Cells were double stained by FITC-labeled antibodies against CD1a- and PE-labeled antibodies against HLA-DR. (A) The relative number of LCs was determined by flow cytometry by positive CD1a staining. The absolute number of emigrated LCs per cm² of skin was determined by the following formula: percentage of CD1a⁺ cells × microscopically counted cells/cm² of skin. Data are expressed as absolute number of LCs that had emigrated from 1 cm² skin. The mean of 6 independent experiments ± SEM is shown. Each pair of closed and corresponding open symbols represents data from the same donor. In 5 of 6 donors, OPN stimulated LC migration. When calculating the percentage of OPN-induced migration for each donor and performing a Wilcoxon signed rank test, a P value of .094 was obtained for this set of experiments. (B) In FACS analysis CD1a and HLA-DR, double-stained emigrated cells were gated for CD1a expression and HLA-DR expression of CD1a⁺ cells was analyzed. The dotted curves give the isotype control staining. Shown is 1 representative experiment of 5, each with tissues from different donors.