Cell culture medium Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and PBS were obtained from Invitrogen (Carlsbad, CA, USA). Thrombin, ERK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB202190, and the antibiotics (penicillin and streptomycin) were obtained from Sigma (St. Loius, MO, USA). Granzyme K was obtained from Axxora (Burlington, ON Canada), and the PAR-1 (ATAP-2 cat no: sc-13503) and PAR-2 (Sam11L cat no: sc-13504) neutralizing antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Mouse IgG (Abcam cat no 37355) was used as an isotype control. Antibodies: Phospho-p44/42 (Cell Signaling Technology (clone E10) cat no: 9106s), total p44/42 (Cell Signaling Technology (clone 137F5) cat no: 4695), phospho-p38 MAPK (Cell Signaling Technology (clone 28B10) cat no: 9216), total p38 MAPK (Cell Signaling Technology cat no: 9212s) β-tubulin (Millipore (clone AA2) cat no: 05-661) was purchased from Millipore, goat anti-mouse IRD 700 and goat IRD anti-rabbit 800 were purchased from LI-COR Biotechnology (Lincoln, Nebraska). IL-6, IL-8 and MCP-1 Duoset ELISAs were purchased from R&D Technologies (Burlington, ON, Canada). Dimethyl sulphoxide (DMSO) was purchased from Sigma (St. Loius, MO, USA) and sodium azide was purchased from sigma (St. Louis, MO, USA). Cell lysis solution, protease inhibitor cocktail and phosphatase inhibitor cocktail were purchased from Sigma (St. Loius, MO, USA). BioRad protein reagent and nitrocellulose membranes were purchased from Bio-Rad (Mississauga, ON, Canada).

Human fetal lung fibroblasts (HFL) were purchased from ATTC (Cat. No CCL-153). Cells were maintained in DMEM supplemented with 10% (vol/vol) FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin, and kept in a humidified atmosphere with 5% CO₂. All experiments were performed between passages 5–11. Cells were routinely passaged every 4–5 d. For all experiments, cells were grown to confluency and serum-starved in DMEM with 0.1% FBS for 24 h before treatment.

IL-6, IL-8 and MCP-1 levels were determined using human IL-6, IL-8 or MCP-1 ELISAs, according to according to manufacturer's protocols. Briefly, cells were seeded in 12-well culture dishes (1×10⁶ per well), grown to 90% confluency and serum-starved in DMEM (0.01% FBS) over-night. Cells were then incubated with GrK (10–300 nM), in the absence of any delivery/cell permeabilizing agent (ie. perforin, streptolysin O), or Thrombin (2.5 U/ml), at the concentrations and time points indicated for each experiment in DMEM containing 1% FBS. Supernatants were collected for analysis by ELISA and the remaining cells were trypsinized and counted using a hemocytometer. For inhibitor studies, cells were pre-treated with the ERK inhibitor (U0126; 10 µM) or the p38 MAPK inhibitor (SB202190; 10 µM) or neutralizing antibodies for 45 min prior to stimulation with GrK (200 nM) or thrombin (2.5 U). Cells treated with vehicle alone, heat-inactivated GrK (100 nM, heat-inactivated at 85°C for 5 min) or mouse IgG (5 µg/ml) isotype served as baseline controls.

HFL were seeded in 6-well culture dishes at 2×10⁶ cells/well, grown to desired confluency, serum-starved over night and then treated with 10 µM U0126 or 10 µM SB202910 for 45 min prior to treatment with 200 nM of GrK or 2.5 U/ml of Thrombin. After 10 min, 30 min, and 2 h, supernatants were collected for IL-6, IL-8 and MCP-1 determination and remaining cells harvested for Western blot analysis of the levels of the downstream products for phospho-p44/42 (ERK1/2) and phospho-p38 MAPK.

Briefly, cells were rinsed with ice-cold Dulbecco's phosphate-buffered saline (dPBS) and lysed in 100 µl of cell lysis buffer containing proteinase inhibitor cocktail and phosphatase-inhibitor cocktail followed by scraping with a cell scraper. Cell debris was removed by centrifugation (12,000× g for 15 min) and protein was quantified by the Bradford method (BioRad) using bovine serum albumin as standard. Samples containing equal amounts of total cell protein were separated by 10% SDS-PAGE, and transferred onto a nitrocellulose membrane using the Bio-Rad wet transfer system at 100 v for 1 h. The membranes were blocked with 2.5% skim milk in TBST (50 mM Tris, pH 7.6, 0.15 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Membranes were then incubated with antibodies against phospho-p44/42 MAPK (1∶2000), total p44/42 MAPK (1∶1000), phospho-p38 MAPK (1∶2000), p38 MAPK (1∶1000) over night at 4°C with gentle shaking. After several washes in TBST, membranes were incubated with goat anti-mouse IRD 700 and goat IRD anti-rabbit 800 for 1 h at room temperature. Flourescent signal was imaged using the Li-COR Odyssey Infrared imaging system (Li-COR biosciences). Densitometry was used to quantify all bands. Membranes were then re-probed with mAb β-tubulin (1∶5000) for 1 h at room temperature and incubated with goat anti-mouse IRD 800 for 45 min. The relative levels of phospho-p44/42 MAPK or phospho-p38MAPK are expressed as the ratio to β- tubulin.

For PAR-1 receptor desensitization studies, cells were pre-treated with 2.5 U/ml of thrombin for 10 min. Media was removed and cells were washed three times with PBS. For IL-6, IL-8 and MCP-1 measurements, fresh media containing GrK (200 nM) or thrombin (2.5 U/ml) was then added cells for a 24 h incubation period and supernatants were collected and screened as described above. In addition to measuring potential changes in cytokine and chemokine production, thrombin-induced PAR-1 activation will induce ERK1/2 phosphorylation. Therefore, we screened for changes in p44/42 (ERK1/2) activation following receptor de-sensitization in cells were pre-treated with thrombin. Here cells were pre-treated with thrombin (2.5 U/ml) for 10 min, media was removed and cells were washed three times with PBS. Cells were then treated with either GrK (200 nM) or thrombin (2.5 U/ml) for 10 min. Cell lysates were then collected as described above and screened for phospho-p44/42 activation as described.

As extracellular GrA and GrB have been shown to induce cell detachment, we examined the impact of extracellular GrK on cell viability and proliferation in HFLs. Cell viability was measured using the WST-1 assay (Roche, Ltd. Missasauga, ON, Canada) according to manufacturer's instructions. Here, 96-well culture dishes were seeded with 1×10⁴ cells and incubated with media alone or GrK (100 nM, 200 nM) for 24 h. Cells were then incubated with 1∶10 dilution of WST-1 reagent for 3 h and absorbance was read at 450 nM. Cell proliferation was assessed using cell counts. Cells were seeded in 12-well culture dishes and incubated with varying concentrations of GrK for 48 h, with or without ATAP-2 (5 µg/ml). Cells were then trypsinized, centrifuged for 5 min 100× g, re-suspended in 1 ml PBS, diluted 1∶1 in 0.4% Trypan Blue and counted using a hemocytometer. Cells treated with ATAP-2 were incubated for 30 min prior to treatment with GrK.

All data are expressed as mean +/− SEM for three separate experiments. Statistical analysis was performed using One-Way ANOVA followed by Dunnets post hoc analysis for multiple group comparisons. Differences were considered significant at p<0.05.

As extracellular GrA and GrB may induce cell death through the cleavage of extracellular matrix proteins and the subsequent induction of anoikis, we examined the impact of extracellular GrK (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).

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).

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.

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 shown in Figure 4 (A–C), an initial proteolytic activation of PAR-1 by thrombin prior to incubation with GrK decreased IL-6, IL-8 and MCP-1 production. This was further confirmed by examination of ERK1/2 phosphorylation where pre-treatment (PT) with thrombin decreased the level of phosphorylation elicited by subsequent application of GrK (Figure 4 D).

It has previously been reported that activation of PAR-1 leads to the activation of ERK1/2 and p38 MAPK in several cell types, including fibroblasts ^[37], ^[38], ^[39]. Thus, we next performed experiments to determine if GrK activation of PAR-1 induced ERK1/2 or p38MAPK phosphorylation in HFL. Figure 4 A–D demonstrates that incubation with GrK (200 nM) induced phosphorylation of both ERK1/2 and p38MAPK. Phosphorylation of ERK1/2 was maximal 10 min after stimulation with GrK and gradually declined to control levels by 2 h incubation (Figure 5 A and B). GrK also induced p38 MAPK phosphorylation with a similar kinetic profile (Figure 5 C and D).

Using specific pharmacological inhibitors, we assessed the relative roles of ERK1/2 and p38 MAPK activation on the synthesis and release of GrK- and thrombin-induced IL-6, IL-8 and MCP-1 in HFL. As shown in Figure 5, pre-treatment of cells with the MEK1/2 inhibitor U0126 inhibited GrK-induced IL-6 and IL-8 release, and thrombin-induced IL-6, IL-8 and MCP-1 release back to control levels. While a reduction was noted in GrK-induced MCP-1 levels, levels were not significantly different from controls (Figure 6 C). In contrast, inhibition of p38 MAPK with SB202910 (10 µM) had no significant impact on GrK-induced IL-6, IL-8 or MCP-1 production but did significantly reduce thrombin-induced IL-6, IL-8 and MCP-1 levels, suggesting that although GrK can activate p38 MAPKs in HFL, p38 may not be involved in mediating PAR-1 induced IL-6, IL-8 or MCP-1 production.

Because fibroblasts are a major component of connective tissues and fibroblasts proliferation is an important process in wound healing, we examined the functional consequences of GrK-induced signaling on fibroblast viability and proliferation. As can be seen in Figure 7, GrK stimulated proliferation in a concentration dependent manner (A). No significant difference was detected between controls and GrK-treated cells prior to 48 h (data not shown). GrK (200 nM)-induced proliferation was inhibited by ATAP-2 (5 µg/ml) (black bars) suggesting that GrK-induced proliferation is occurring through a PAR-1 mediated mechanism (B).