Figure 3

Host and fungal proteases regulate the expression and function of PARs. (a) Calcium mobilization in HEK293 cells pretreated (arrows) with elastase E+catepsin G (HE/CatG, each at 600 nM) or supernatants from degranulated WT (20 ng ml⁻¹ of protease), tlr2^−/− or tlr4^−/− PMNs exposed to Candida (upper panel) or Aspergillus (lower panel), and stimulated (arrows) with thrombin or trypsin, 4 min later. The results, representative of three experiments, are expressed as 340/380 nm ratio. (b) Surface expression of PAR₁ or PAR₂ in HEK293 cells unexposed (none) or exposed to thrombin or trypsin alone or after preincubation with supernatants from WT PMNs exposed to Candida (upper panel) or Aspergillus (lower panel). (−), cells stained with irrelevant Ab. Numbers refer to the percentage of positive cells over total cells analyzed. (c) Calcium mobilization in HEK293 cells pretreated with Aspergillus or Candida culture supernatants (Sup, 50 μl containing 50 ng of protease activity) and stimulated with thrombin (3 U ml⁻¹) or trypsin (100 nM), 4 min later. For inhibitors, culture supernatants were preincubated with the serine protease inhibitor, phenylmethylsulfonyl fluoride, and the cathepsin B inhibitor, leupeptin. Data are the mean±s.e. from two independent experiments. Bars are s.e. *P<0.05, thrombin- or trypsin-stimulated vs. unstimulated cells; **P<0.05, trypsin+Aspergillus-Sup vs. trypsin alone. ***P<0.05, trypsin+Aspergillus-Sup, with and without inhibitors. Ab, antibody; PAR, protease-activated receptor; PMN, polymorphonuclear neutrophil; WT, wild type.

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