Abstract
In healthy skin, epidermis and dermis are anchored together at the dermal-epidermal junction (DEJ), a specialized basement membrane pivotal for skin integrity and function. However, increased inflammation in the DEJ is associated with the disruption and separation of this junction and sub-epidermal blistering. Granzyme B (GzmB) is a serine protease secreted by immune cells. Dysregulated inflammation may lead to increased GzmB accumulation and proteolysis in the extracellular milieu. Although elevated GzmB is observed at the level of the DEJ in inflammatory and blistering skin conditions, the present study is the first to explore GzmB in the context of DEJ degradation in autoimmune sub-epidermal blistering. In the present study, GzmB induced separation of the DEJ in healthy human skin. Subsequently, α6/β4 integrin, collagen VII, and collagen XVII were identified as extracellular substrates for GzmB through western blot, and specific cleavage sites were identified by mass spectrometry. In human bullous pemphigoid, dermatitis herpetiformis, and epidermolysis bullosa acquisita, GzmB was elevated at the DEJ when compared to healthy samples, while α6/β4 integrin, collagen VII, and collagen XVII were reduced or absent in the area of blistering. In summary, our results suggest that regardless of the initial causation of sub-epidermal blistering, GzmB activity is a common final pathway that could be amenable to a single targeted treatment approach.
Highlights
Blistering is a hallmark of many dermatological conditions, and can manifest itself with varying degrees of severity, but is typically characterized by erosions or fluid filled elevations from the skin surface caused by disruption of the cell to cell attachment in different layers of the epidermis, or detachment of the epidermis from dermis
As Granzyme B (GzmB) accumulates on both sides of the dermal-epidermal junction (DEJ), including the dermis which is devoid of keratinocytes, and given the established potential of this enzyme to cleave multiple extracellular matrix (ECM) proteins, we hypothesized that GzmB compromises DEJ integrity and function through cleavage of key basement membrane components α6/β4 integrin, collagen VII, and collagen XVII, directly contributing to epidermal detachment and blistering through extracellular mechanisms
We used western blot and amino-terminal oriented mass spectrometry (ATOMS) to show for the first time that α6/ β4 integrin and collagen VII, which are key components of the DEJ, are cleaved by GzmB in key regions for their anchoring functions
Summary
Blistering is a hallmark of many dermatological conditions, and can manifest itself with varying degrees of severity, but is typically characterized by erosions or fluid filled elevations from the skin surface caused by disruption of the cell to cell attachment in different layers of the epidermis, or detachment of the epidermis from dermis. Such as bullous pemphigoid, dermatitis herpetiformis and epidermolysis bullosa acquisita (EBA), auto-antibodies targeting components of the dermal-epidermal junction (DEJ) lead to the disruption of this basement membrane and consequent detachment of the epidermis[2]. As GzmB accumulates on both sides of the DEJ, including the dermis which is devoid of keratinocytes, and given the established potential of this enzyme to cleave multiple ECM proteins, we hypothesized that GzmB compromises DEJ integrity and function through cleavage of key basement membrane components α6/β4 integrin, collagen VII, and collagen XVII, directly contributing to epidermal detachment and blistering through extracellular mechanisms. This work demonstrates for the first time a role for extracellular GzmB in the blistering process that goes beyond a cytotoxic effect on basal keratinocytes
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