Indirect Immunohistochemistry and Immunoelectron Microscopy Distribution of Eight Epidermal-Dermal Junction Epitopes in the Pig and in Isolated Perfused Skin Treated with Bis (2-Chloroethyl) Sulfide

Abstract

Sulfur mustard (bis [2-chloroethyl] sulfide, HD) is a potent cutaneous vesicant that causes gross blisters by separation of the epidermal-dermal junction (EDJ). The EDJ of the skin is a highly specialized and complex structure composed of several components and plays a major role in the integrity of the skin. The isolated perfused porcine skin flap (IPPSF) was dosed with 0.2 mg/ml (n = 4), 5.0 mg/ml (n = 4), and 10.0 mg/ml (n = 5) HD or ethanol (n = 4) for 8 hr (dose-response study) and 10.0 mg/ml HD or ethanol for 1, 3, 5, and 8 hr (n = 4/treatment) (time-response study). Successful EDJ mapping was carried out in normal pig skin (NPS), ethanol-treated IPPSFs, and HD-treated IPPSFs using the following antibodies: laminin, type IV collagen, fibronectin, GB3 (Nicein), bullous pemphigoid (BP), and epidermolysis bullosa acquisita (EBA). Two mouse anti-human monoclonal antibodies, L3d and 19-DEJ-l (Uncein), did not cross-react with the EDJ of the pig. Antibody staining in NPS, ranging from very intense for laminin and type IV collagen to weak for fibronectin, was generally more discrete than in the IPPSF. No differences in staining were noted between the ethanol and nonblistered areas of the HD-treated IPPSFs. In HD-blistered areas, BP stained only the epidermal hemidesmosomes, and laminin, fibronectin, and GB3 stained primarily the dermis with fragments attached to the basal pole of the stratum basale cells, while type IV collagen and EBA stained only the dermis. Mapping of these epitopes determined that the precise plane of EDJ separation in the HD-treated skin occurred beneath the hemidesmosomes within the upper portion of the lamina lucida. The conservation of human epitopes in the EDJ of the pig further emphasizes the similarities between human skin and pig skin. Therefore, pig skin and the IPPSF may be used to study HD-induced vesication and blistering diseases.

Therapeutic potential of a non-steroidal bifunctional anti-inflammatory and anti-cholinergic agent against skin injury induced by sulfur mustard

“…SM has also been shown to cause microvesication in mouse skin, with focal epidermal staining of laminin, resulting from the separation of the DEJ. This suggests a precise cleavage plane of the DEJ within the lamina lucida (Monteiro-Riviere and Inman, 1995). Laminin 332 has been reported to be upregulated and expressed at the migrating leading edge of SM wounded mouse skin (Chang et al , 2009).…”

Use of methyl salicylate as a simulant to predict the percutaneous absorption of sulfur mustard

“…The IPPSF is a viable and vascularized in vitro skin model that has been utilized successfully to model the mechanism of percutaneous chemical absorption [11][12][13] and to extrapolate these results to humans. [14][15][16] Of particular relevance to the present work are the pathogenesis and cutaneous toxicity of HD vesication [17][18][19][20][21][22] and the nature of the cutaneous immune response 23 using the IPPSF model. The purpose of the present study was to characterize the dermal absorption and penetration profile of MS in the IPPSF model, to compare this with the previously conducted HD studies and to assess whether methyl salicylate was a valid simulant for HD relative to cutaneous absorption and disposition.…”

Free‐floating cryostat sections for immunoelectron microscopy: Bridging the gap from light to electron microscopy

“…This technique has been used extensively for mapping the distribution of epidermal-dermal junction proteins in various inherited bullous diseases, including bullous pemphigoid (Bedane et al, 1997) and dystrophic epidermolysis bullosa (Bruckner-Tuderman et al, 1989). Recently, immunoelectron microscopy was employed to study basement membrane proteins in sulfur mustard-induced cutaneous vesicating lesions (Monteiro-Riviere and Inman, 1995;Petrali and Oglesby-Megee, 1997). A persistent concern of immunoelectron microscopy is the preservation of ultrastructural morphology while simultaneously maintaining the immunoreactivity of proteins of interest.…”