Extracellular granzyme K mediates endothelial activation through the cleavage of protease‐activated receptor‐1

Abstract

Granzymes are a family of serine proteases that were once thought to function exclusively as mediators of cytotoxic lymphocyte‐induced target cell death. However, non‐apoptotic roles for granzymes, including granzyme K (GzK), have been proposed. As recent studies have observed elevated levels of GzK in the plasma of patients diagnosed with clinical sepsis, we hypothesized that extracellular GzK induces a proinflammatory response in endothelial cells. In the present study, extracellular GzK proteolytically activated protease‐activated receptor‐1 leading to increased interleukin 6 and monocyte chemotactic protein 1 production in endothelial cells. Enhanced expression of intercellular adhesion molecule 1 along with an increased capacity for adherence of THP‐1 cells was also observed. Characterization of downstream pathways implicated the mitogen‐activated protein kinase p38 pathway for intercellular adhesion molecule 1 expression, and both the p38 and the extracellular signal‐regulated protein kinases 1 and 2 pathways in cytokine production. GzK also increased tumour necrosis factor α‐induced inflammatory adhesion molecule expression. Furthermore, the physiological inhibitor of GzK, inter‐α‐inhibitor protein, significantly inhibited GzK activity in vitro. In summary, extracellular GzK promotes a proinflammatory response in endothelial cells.

Granzyme K Expressed by Classically Activated Macrophages Contributes to Inflammation and Impaired Remodeling

“…Because classically activated macrophages secrete GzmK (Figure 1e), we investigated the downstream effects of GzmK in vitro in human HaCaTs (keratinocytes) and primary human skin fibroblasts, the predominating cell types in skin. Addition of recombinant human GzmK (rhGzmK) to cells ( 100 nmol/L) showed no detectable cytotoxicity up to 48 hours in culture (see Supplementary Figure S2 online), as previously reported in endothelial cells (Sharma et al, 2016) and lung fibroblasts (Cooper et al, 2011). Using an electric cellsubstrate impedance sensing wound healing assay, rhGzmK exhibited a dose-dependent and reproducible impairment of wound closure in HaCaTs, which was approximately 50% slower compared with untreated controls (Figure 4a).…”

“…Proinflammatory IL-6, essential for timely wound healing, is involved in generating acute phase responses, inflammation, and lymphocyte differentiation (McFarland-Mancini et al, 2010). GzmK-mediated IL-6 secretion occurs in endothelial cells (Sharma et al, 2016), and our data showed GzmKmediated IL-6 secretion from cultured HaCaTs and skin fibroblasts, releasing similar quantities from each, and both operating through PAR-1. Indeed, in GzmK e/e mouse burns at day 3 after injury, IL-6 expression was reduced compared with equivalent WT samples.…”

“…GzmK induces PAR-1emediated proinflammatory cytokine release from keratinocytes, skin fibroblasts, and classically activated macrophages rhGzmK induces proinflammatory cytokine expression in both endothelial cells and lung fibroblasts, functioning through a PAR-1emediated pathway (Cooper et al, 2011;Sharma et al, 2016). Studies were therefore performed to determine whether GzmK exposure in HaCaTs and skin fibroblasts induced proinflammatory cytokine expression in a similar fashion, thus providing mechanistic details regarding GzmK's role in burn wound repair.…”

“…Endothelial cells cultured with rhGzmK show increased MCP-1, ICAM-1, and VCAM-1 expression (Sharma et al, 2016); thus, gene expression of each was quantified in mouse burn wounds. MCP-1 (P ¼ 0.035), ICAM-1 (P ¼ 0.0049), and VCAM-1 (P ¼ 0.0017) expressions in GzmK e/e mice at day 3 after injury were significantly reduced compared with those in WT mice (Figure 5b).…”

“…GzmK e/e mice exhibit reduced foot swelling in response to Chikungunya virus infection (Wilson et al, 2017). Exposure of cultured lung fibroblasts and endothelial cells to GzmK stimulates proinflammatory cytokine release that is dependent on PAR-1 activation (Cooper et al, 2011;Sharma et al, 2016). GzmK also induces IL-1b production in macrophages (Joeckel et al, 2011).…”