The role of extracellular Granzyme b in cytotoxic Lymphocyte migration

Granzyme B (GrB) is a known intracellular cytotoxin that has also been shown to cleave extracellular substrates. GrB is detectable in extracellular fluids under normal, healthy conditions and its levels increase in many disease states that are associated with cytotoxic lymphocyte (eL) hyperactivity. Until now, the role of extracellular GrB has gone unexplained. Among the known extracellular substrates of GrB are several ECM proteins, cleavage of which leads to cell detachment and potential anoikis. Based on the known expression and substrates of GrB, it was hypothesised that extracellular GrB is derived from CLs, which release GrB to degrade extracellular matrix proteins and facilitate cell migration into target tissues. This study showed that zymogen and active forms of GrB are constitutively secreted from CLs, by both conventional and non-conventional pathways. It was subsequently shown that in the absence of GrB, or upon perturbation of its secretion/activity, CLs exhibit a cellautonomous defect in migration through basement membranes in vitro, and in homing to sites of inflammation and infection in vivo. No such defect was evident during interstitial migration. Using a proteomics-iJased approach, it was also demonstrated that GrB cleaves several components of the basement membrane in the context of a complex matrix. The proposed model requires GrB to be released by CLs upon exit from the vasculature (extravasation) and entry to target tissues to break down the basement membrane lining each compartment. This process was previously believed to be predominantly mediated by matrix metalloproteinases, but the studies here reveal the significant contribution made by GrB, and highlight the broader nature of granzyme function.