We have used a new approach to identify early events in SM-induced injury mediated by SM by exposing three-dimensional (3-D), bioengineered, human skin generated in vitro and in vivo to determine the morphologic, apoptotic, ultrastructural and basement membrane alterations that were linked to dermal-epidermal separation. Skin equivalent cultures were generated and exposed to SM both before and after transplantation to nude mice. Skin equivalents transplanted to nude mice generated normal human skin that was exposed to SM vapor for 5,8,10 or 12 minutes and removed 6 or 24 hours after exposure. Distinct prevesication (6h) and post-vesication (24h) phases of SM-induced tissue damage were identified. Prevesication injury was restricted to focal groups of basal keratinocytes that were found to undergo apoptotic cell death in a dose-independent manner. Immunoreactivity for basement membrane proteins and structured basement membrane were preserved during this prevesication stage, suggesting that intact basement membrane may be associated with the initiating events in SM damage. Dermal-epidermal separation was accompanied by the presence of microvesicles at the basement membrane interface 24h after SM exposure. This change was accompanied by the dose-dependent induction of apoptosis of basal and suprabasal keratinocytes and partial loss of basement membrane ultrastructure and immunoreactivity. In vitro skin equivalents, grown in the absence of structured basement membrane, demonstrated similar dermal-epidermal separation when exposed to media containing 150 ug/ml SM for 7 minutes. However, skin equivalents grown in the presence of intact basement membrane were resistant to SM-induced damage at this SM dose, suggesting that basement membrane proteins could protect basal keratinocytes from SM-induced apoptosis. To further explore the role of basement membrane proteins in keratinocyte resistance to SM-induced damage, we directly studied the role of laminin 5 in this process. Primary keratinocytes harvested from patients with the blistering skin disease Junctional Epidermolysis Bullosa (JEB), that lack a functional gamma 2 chain of laminin 5 and are not able to adhere to basement membrane, were transduced with retroviral vectors designed to restore laminin 5-mediated adhesion. We found that only JEB cells in which laminin 5 adhesive function was restored (F-GAL) were resistant to apoptosis when exposed to SM (150 ug/ml), thereby implicating laminin 5-mediated attachment as being important in limiting SM damage. These studies provide important proof of concept that in vitro and in vivo tissue models mimic many of the tissue alterations previously found in animal models of SM injury. Our findings show that adhesion to HOME 1 basement membrane proteins enables subsets of keratinocytes to resist SM damage.