Immunohistochemical characterization of the basement membrane epitopes in bis(2-chloroethyl) sulfide-induced toxicity in mouse ear skin.

Sulfur mustard (bis(2-chloroethyl) sulfide (HD)), a potent cutaneous vesicant and bifunctional alkylating agent, produces significant time-dependent histopathological changes in the skin of the mouse. The right ears of male CD1 mice were exposed topically to 5.0 microl of 195 mM (0.16 mg) HD in dichloromethane and harvested at 6, 12, 18 and 24 h. The left ear control was dosed with 5.0 microl of dichloromethane. In all controls and HD-treated mouse ear, moderate immunofluorescence staining was seen at the epidermal-dermal junction with bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA) and laminin (Lam), and light staining was observed with bullous pemphigoid 180 (BP180), fibronectin (Fn) and type IV collagen (Coll IV). Mouse anti-human monoclonal antibodies for GB3, L3d and 19-DEJ-1 (Uncein) did not cross-react. In microvesicles, BP, BP180 and Fn showed areas of light focal epidermal staining and homogeneous dermal staining, and EBA, Lam and Coll IV showed moderate dermal staining. Both BP and Fn exhibited weak, inconsistent staining with time. Immunoelectron microscopy (IEM) revealed similar results, with an increase in cell damage from 6 to 24 h, which corresponded to a decrease in staining intensity. Cell proliferation, expressed as the growth fraction of proliferating cell nuclear antigen (PCNA), showed an increase in cell damage. The growth fraction was lower in the inner ear and showed time-dependent differences. The immunofluorescence and IEM results indicate that HD causes an undulating inconsistent separation in the uppermost lamina lucida with focal cleavage into the lower portion of the basal keratinocytes just above the plasma membrane. Although this pattern of separation differs from other in vivo models in which the split occurs exclusively within the lamina lucida, this should not preclude its role as a screening model to study the effects and development of specific prophylactic and therapeutic strategies.