Granzyme K Activates PAR-1 Figure 3. PAR-1 neutralization reduces GrK-induced IL-6, IL-8 and MCP-1 production. Cells were incubated with the PAR-1 neutralizing antibody (ATAP-2; 5 mg/ml) PAR-2 neutralizing antibody (Sam11L; 25 mg/ml) or mouse isotype IgG control antibody for 30 min prior to incubation with GrK (200 nM) for 24 h. Supernatants from cell cultures were collected and analyzed for IL-6, IL-8 and MCP-1 production by ELISA. Cell counts were used to normalized to cell numbers. Data are expressed as % of media control (DMEM+0.0001% NaN3) 6 SEM from three separate experiments run in triplicate. + p,0.05 when compared to media control; ** p,0.05 when compared to GrK (200 nM) treatment. doi:10.1371/journal.pone.0021484.g003 (in the absence of perforin or any other delivery agent) on cell viability using the WST-1 assay. As demonstrated in Figure 1, HFLs treated with 100 and 200 nM of GrK exhibited similar cell viability to HFLs incubated in media alone (Figure 1). GrK induces IL-6, IL-8 and MCP-1 production in HFLs HFLs stimulated with GrK at concentrations ranging from 10– 300 nM (in the absence of perforin or any other delivery agent) exhibited a dose-dependent increase in IL-6 and IL-8 protein production in cell culture supernatants at 24 h (Figures 2 A and B). Incubation with GrK also induced the secretion of MCP-1 though this release did not appear to be dose-dependent (Figure 2 C). Enzymatic activity was required to stimulate cytokine and chemokine production as heat-inactivated GrK (100 nM) did not induce IL-6, IL-8 or MCP-1 production. Time course experiments using GrK (200 nM) demonstrated that maximal release of IL-6, IL-8 and MCP-1 was detected in supernatants at 24 h (data not shown). Induction of IL-6, IL-8 and MCP-1 in HFLs occurs through activation of PAR-1 Previous studies have established that HFL express PARs-1-4 [30,31,32]. Given its trypsin-like activity, we examined whether extracellular GrK was inducing the release of pro-inflammatory molecules through the cleavage and subsequent activation of PARs. To address this, we used antibodies to specifically block PAR-1 and PAR-2 cleavage sites, using a PAR-1 neutralizing antibody as described previously [33,34,35]. As shown in Figure 3 A–C, incubation with the PAR-1 neutralizing antibody (ATAP-2) prior to GrK treatment markedly diminished IL-6, IL-8 and MCP-1 production, whereas incubation with the PAR-2 (Sam11L) neutralizing antibody had no significant impact on these events. In the current study, we did not specifically neutralize PAR-3 or PAR-4 because we observed no cytokine production following treatment with the potent PAR-4 agonist peptide GYPGQV (data not shown). While PAR-3 and PAR-4 can be proteolytically activated by thrombin, evidence suggests that PAR-3 acts as a cofactor for PAR-4 at low concentrations of thrombin and is a non-signaling receptor [36]. Furthermore, we observed near complete ablation of cytokine production using the PAR-1 neutralizing antibody suggesting from a functional standpoint that most of the biological activity observed following GrK treatment is the result of PAR-1 activation, but does not completely rule out a potential role for PAR-3 or PAR-4. PAR-1 receptor desensitization reduces GrK-induced IL-6, IL-8 and MCP-1 production To further verify the specificity of GrK-mediated PAR-1 activation, we used the high affinity ligand for PAR-1, thrombin (2.5 U/ml) to desensitize PAR-1 prior to exposure to GrK. As PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21484