The maturation of DCs triggered by PR3 occurs through PAR-2. (A) Immature DCs were pretreated with SAM-11 at 10 μg/mL alone or with PR3 (10 μg/mL) for 24 hours. The cell surface expression of CD40 and CD83 was analyzed by flow cytometry. (B) DCs were stimulated with 100 μM PAR-2 agonist peptide (SLIKV-NH2, PAR-2AP) or a control peptide (100 μg/mL) for 48 hours and then were stained for CD80, CD83, CD86, and HLA-DR and analyzed by flow cytometry. The histograms show fluorescence intensity on gated cells. The thin lines represent staining with isotype-matched irrelevant mAb. (C) Effect of PLC inhibition on the expression of maturation marker on DCs. Immature DCs were treated with 400 nM U73122 or the control compound U73343 (mock inhibitor) 1 hour before exposure to PAR-2AP or PR3 for 24 hours. The expression of the DC maturation marker CD83 was analyzed by flow cytometry. Open curves indicate isotype Ab control; shaded areas, CD83. Tracings are representative of 3 distinct experiments.