Granzyme B Induces Smooth Muscle Cell Apoptosis in the Absence of Perforin

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Abstract
Objective— T cell-induced cytotoxicity, of which granzyme B is a key mediator, is believed to contribute to the pathogenesis of inflammatory vascular diseases. In this report, we investigate the mechanism of granzyme B-induced smooth muscle cell (SMC) death. Methods and Results— The addition of purified granzyme B alone to cultured SMCs caused a significant reduction in cell viability. Chromatin condensation, phosphatidylserine externalization, and membrane blebbing were observed, indicating that the mechanism of granzyme B-induced SMC death was through apoptosis. Activated splenocytes from perforin-knockout mice induced SMC death through a granzyme B-mediated pathway. Inhibition of the proteolytic activities of caspases and granzyme B prevented granzyme B-induced SMC death, whereas attenuation of granzyme B internalization with mannose-6-phosphate (M6P) did not. Further, granzyme B induced the cleavage of several SMC extracellular proteins, including fibronectin, and reduced focal adhesion kinase phosphorylation. Conclusions— These results indicate that granzyme B can induce apoptosis of SMCs in the absence of perforin by cleaving extracellular proteins, such as fibronectin. We studied the mechanisms of granzyme B-induced smooth muscle cell (SMC) death. In the absence of perforin, granzyme B induced SMC apoptosis. Internalization of granzyme B was not required for SMC death, and this protease induced the cleavage of several extracellular proteins, including fibronectin.