Granzyme B, a cytotoxic immune protease, induces apoptosis of smooth muscle cells in the absence of perforin

Abstract

The granzyme/perforin pathway is a key mediator of the cytotoxic immune response to allogeneic tissue/cells. Granzyme B (GrB), the most potent granzyme, localizes to apoptotic smooth muscle cells in human transplant vascular disease (TVD). In this report we examine the effects of GrB on smooth muscle cells (SMC) in vitro. Human coronary artery SMC were cultured to 70% confluence, serum starved, and treated with varying doses of purified GrB for 24h. Surprisingly, GrB alone induced morphological changes that included cell rounding and surface detachment. MTS assay indicated that GrB alone dose-dependently reduced cell viability. There was a significant reduction in cell viability that began at 1.25μg/mL GrB and reached a minimum of 65% of untreated control at 5μg/mL GrB. Hoechst nuclear staining and Annexin V labeling of SMC treated with GrB (2.5μg/mL) demonstrated that the rounded SMC were apoptotic, and Western analysis indicated that GrB activated both caspase-3 and -8. The addition of z-VAD.fmk (multi-caspase inhibitor) or z-AAD.cmk (GrB inihibitor) to SMC prior to treatment with GrB completely inhibited cell death. Further, addition of fluorscently labeled GrB showed that this protease is internalized by SMC. However, although the addition of 20mM soluble mannose-6-phosphate (M6P) inhibited GrB internalization, it did not inhibit cell death. These novel results suggest that GrB may contribute to the pathogenesis of TVD by inducing apoptosis of SMC, at least in part, through a cytotoxic pathway that is independent of perforin. A potential mechanism could involve proteolysis of extracellular and/or plasmalemmal proteins.