Subtilisin serine protease activity and signaling through PAR-2 are both essential for the development of Th2 responses. C57BL/6 mice were s.c. sensitized with active or inactivated form of the enzyme, 10 μM of PMSF for subtilisin or 50 nM E-64 for papain, on days 0 and 7 and i.n. challenged with the active enzyme on days 14 and 21. Samples were harvested 24 h after the last i.n. administration, on day 22. BAL (A) total and (B) differential cell counts. (C) Total serum IgE. (D) Representative lung sections (200×). C57BL/6 WT or PAR-2-/- mice were sensitized with s.c. subtilisin on days 0 and 7 and i.n. challenged on days 14 and 21. (E, left) Total number of cells from BAL recovered on day 22. (E, right) BAL neutrophil and eosinophil numbers from WT or PAR-2-/- mice by morphological analysis. (F) Total serum IgE. *Significant differences when compared with sensitization to active protease (A-C) or with WT group (E, F). Data are mean ± SEM and are representative of three independent experiments (n=5).