Sulfur mustard [bis-2-chloroethyl sulfide] is a vesicating agent first used as a weapon of war in WWI. It causes debilitating blisters at the epidermal-dermal junction and involves molecules that are also disrupted by junctional epidermolysis bullosa (JEB) and other blistering skin diseases. Despite its recurring use in global conflicts, there is still no completely effective treatment. We have shown by imaging human keratinocytes in cell culture and in intact epidermal tissues that the basal cells of skin contain well-organized molecules (keratins K5/K14, α6β4 integrin, laminin 5 and α3β1 integrin) that are early targets of sulfur mustard. Disruption and collapse of these molecules is coincident with nuclear displacement, loss of functional asymmetry, and loss of polarized mobility. The progression of this pathology precedes basal cell detachment by 8-24 h, a time equivalent to the “clinical latent phase” that defines the extant period between agent exposure and vesication. Our images indicate that disruption of adhesion-complex molecules also impairs cytoskeletal proteins and the integration of structures required for signal transduction and tissue repair. We have recently developed an optical system to test this hypothesis, i.e., to determine whether and how the early disruption of target molecules alters signal transduction. This environmentally controlled on-line system provides a nexus for real-time correlation of imaged lesions with DNA microarray analysis, and for using multiphoton microscopy to facilitate development of more effective treatment strategies.
Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a chemical warfare agent that produces persistent, incapacitating blisters of the skin. The lesions inducing vesication remain elusive, and there is no completely effective treatment. Using mulitphoton microscopy and immunofluorescent staining, we found that exposing human epidermal keratinocytes (HEK) and intact epidermis to SM (400 μm for 5 min) caused progressive collapse of the keratin (K5/K14) cytoskeleton and depletion of α6β integrins. We now report that SM causes concomitant disruption nad collapse of the basal cell's α3β1-integrin receptors. At 1 h postexposure, images of Alexa488-conjugated HEK/α3β1 integrins showed almost complete withdrawal and disappearance of retraction fibers and a progressive loss of polarized mobility. With stero imaging, in vitro expression of this SM effect was characterized by collapse and abutment of adjacent cell membranes. At 2 h postexposure, there was an average 13% dorso-ventral collapse of HEK membranes that paralleled progressive collapse of the K5/K14 cytoskeleton. α3β1 integrin, like α6β4 integrin, is a regulator of cytoskeletal assembly, a receptor for laminin 5 and a mediator of HEK attachment to the basement membrane. Our images indicate that SM disrupts these receptors. We suggest that the progressive disruption destabilizes and potentiates blistering of the epidermal-dermal junction.
It is well known that topical exposure to sulfur mustard (SM) produces persistent, incapacitating blisters of the skin. However, the primary lesions effecting epidermal-dermal separation and disabling of mechanisms for cutaneous repair remain uncertain. Immunofluorescent staining plus multiphoton imaging of human epidermal tissues and keratinocytes exposed to SM (400 μM x 5 min)have revealed that SM disrupts adhesion-complex molecules which are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Images of keratin-14 showed early, progressive, postexposure collapse of the K5/K14 cytoskeleton that resulted in ventral displacement of the nuclei beneath its collapsing filaments. This effectively corrupted the dynamic filament assemblies that link basal-cell nuclei to the extracellular matrix via α6β4-integrin and laminin-5. At 1 h postexposure, there was disruption in the surface organization of α6β4 integrins, associated displacement of laminin-5 anchoring sites and a concomitant loss of functional asymmetry. Accordingly, our multiphoton images are providing compelling evidence that SM induces prevesicating lesions that disrupt the receptor-ligand organization and cytoskeletal systems required for maintaining dermal-epidermal attachment, signal transduction, and polarized mobility.
Topical exposure to sulfur mustard (HD), a known theat agent, produces persistent and debilitating cutaneous blisters. The blisters occur at the dermal-epidermal junction following a dose-dependent latent period of 8-24 h, however, the primary lesions causing vesication remain uncertain. Immunofluorescent images reveal that a 5-min exposure to 400 (mu) M HD disrupts molecules that are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Using keratinocyte cultures and fluorochomes conjugated to two different keratin-14 (K14) antibodies (clones CKB1 and LL002), results have shown a statistically significant (p<0.1) 1-h decrease of 29.2% in expression of the CKB1 epitope, a nearly complete loss of CKB1 expression within 2 h, and progressive cytoskeletal (K14) collapse without loss in expression of the LL002 epitope. With human epidermal tissues, multi-photon images of (alpha) 6 integrin and laminin 5 showed disruptive changes in the cell-surface organization and integrity of these adhesion molecules. At 1 H postexposure, analyses showed a statistically significant (p<0.1) decrease of 27.3% in (alpha) 6 integrin emissions, and a 32% decrease in laminin 5 volume. Multi-photon imaging indicates that molecules essential for epidermal-dermal attachment are early targets in the alkylating events leading to HD-induced vesication.
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