Granzyme B In Sub-epidermal Blistering
David J. Granville, Valerio Russo, Theo Klein, Nick Carr, Richard Crawford, Chris M. Overall.
University of British Columbia, Vancouver, BC, Canada.
INTRODUCTION: In healthy skin, the epidermis and dermis are anchored together at the dermal-epidermal junction (DEJ), a specialized basement membrane pivotal for skin integrity and function. However, in sub-epidermal bullous conditions, the DEJ is compromised resulting in DEJ disruption and separation resulting in blistering. Although the etiology of these conditions can vary, they all involve epidermal detachment leading to increased risk of infection and reduced quality of life. Granzyme B (GzmB) is a pro-apoptotic serine protease secreted by immune cells that can cleave extracellular matrix proteins. Although previous studies have observed abundant levels of GzmB in the DEJ, a non-apoptotic role in blistering has never been considered. The present study suggests that GzmB cleaves key DEJ proteins leading to epidermal detachment and blistering.
METHODS: Cleavage assays and TAILS analysis were performed to identify and confirm novel GzmB substrates in skin. Collagen VII, α6β4 integrin and Collagen XVII were identified as substrates. To investigate whether the aforementioned substrates are cleaved in vivo, immunostaining for GzmB and ECM substrates were performed on sub-epidermal blistering diseases (Bullous pemphigoid, dermatitis herpetiformis, SJS/TEN, and epidermolysis bullosa). Finally, sections of human skin were exposed to GzmB to investigate whether GzmB could induce DEJ disruption.
RESULTS: Cleavage of ECM substrates was confirmed by western blotting and ATOMS was utilized to identify cleavage sites. Ex vivo studies indicated that GzmB could induce DEJ separation; a process that was inhibited by GzmB inhibition. Excitingly, all sub-epidermal blistering conditions exhibited increased GzmB and reduced Collagen VII, alpha6beta4 integrin and Collagen XVII specifically in the area proximal to DEJ disruption and blistering.
CONCLUSIONS: GzmB may be a common causative link in sub-epidermal blistering and thus could be used as a broad approach to preventing blistering in such conditions.
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