Tryptase ϵ/uPA-mediated extravasation of smooth-muscle cells in a Matrigel cell-invasion assay. The ability of normal human bronchial smooth muscle cells to migrate through a basement membrane–like extracellular matrix was evaluated in the presence or absence of tryptase ϵ and uPA. Smooth muscle cells were added to the top wells of each chamber. Nothing else was added in panel A. Pro-uPA alone or with inactive C90A/D92E tryptase ϵ or active tryptase ϵ was added to panels B, C, and D, respectively. FCS-enriched culture medium was added to each bottom chamber as a source of chemotactic factors. Shown are the Masson trichrome⁺ smooth muscle cells (red) that migrated through each Matrigel-coated membrane. Similar results were obtained in a second experiment. Pro-uPA–expressing smooth muscle cells (SMCs) were able to invade the Matrigel when exposed to approximately 100 nM enzymatically active tryptase ϵ alone (data not shown) but not its inactive C90A/D92 mutant. However, a better response was obtained if a small amount of exogenous pro-uPA was present, presumably because the tryptase ϵ–generated exogenous uPA can overwhelm the amount of PAI-1 present in the 10% FCS and conditioned medium that counteracts the chemotactic response.