Figure 2: PAR2 stimulation induced neutrophil degranulation in a Ca2+-dependent manner and upregulated ROS production. Neutrophils were treated as described in Material and Methods Section. (a) After stimulation with PAR2 agonist and IFNγ, the concentration of MPO and elastase was quantified in cytochalasin B primed neutrophils. (b) Comparing MPO levels in cytochalasin B primed neutrophils that were either pretreated with agonists (b/a-stimulation) or not showed a reduction in PAR2 agonist stimulated neutrophils. Concomitant stimulation with PAR2-agonist and IFNγ induced similar MPO levels in both pretreated and nonpretreated cells. (c, d) Neutrophils were loaded with Fura-2 AM (30 min), washed, and then PAR2 agonist was added, and calcium mobilization was investigated. The availability of extracellular Ca2+ led to increased intracellular calcium levels after PAR2 agonist application. 2-APB almost completely blocked intracellular Ca2+ fluxes, independent of extracellular Ca2+. (e) Pretreatment of neutrophils with 2-APB prevented PAR2 agonist induced elastase release. (f) Changes in ROS levels were measured using a fluorescent substance (CM-H2DCFDA) that was added 30 min before the stimulation was stopped (see Material and Methods Section). Only at early time points, PAR2 agonist elevated ROS level as measured by changes of the MFI. IFNγ did not induce ROS upregulation. For student’s t-test: #,*; **. The symbol * marks the significance as compared to control and the symbol # as compared to IFNγ sample.