Multiphoton microscopy: an optical approach to understanding and resolving sulfur mustard lesions

Abstract

Sulfur mustard (SM; 2,2(')-dichloroethyl sulfide) is a percutaneous alkylating agent first used as a chemical weapon at Ypres, Belgium, in World War I. Despite its well-documented history, the primary lesions effecting dermal-epidermal separation and latent onset of incapacitating blisters remain poorly understood. By immunofluorescent imaging of human epidermal keratinocytes (HEK) and epidermal tissues exposed to SM (400 microM for 5 min), we have amassed unequivocal evidence that SM disrupts adhesion complex molecules, which are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Images of keratin 14 (K14) in control cells showed tentlike filament networks linking the HEK's basolateral anchoring sites to the dorsal surface of its nuclei. Images from 6-h postexposure profiles revealed early disruption (</=1 h) and progressive collapse of the K14 cytoskeleton. Collapse involved focal erosions, loss of functional asymmetry, and displacement of nuclei beneath a mat of jumbled filaments. In complementary studies, 1-h images showed statistically significant (p<0.01) decreases of 25 to 30% in emissions from labeled alpha(6)beta(4) integrin and laminin 5, plus disruption of their receptor-ligand organization. Results indicate that SM alkylation destabilizes dermal-epidermal attachments and potentiates vesication by disrupting adhesion complex molecules and associated signaling mechanisms required for their maintenance and repair.

Multiphoton imaging the disruptive nature of sulfur mustard lesions

“…By 1 and 2 h after exposure, most HEK had lost their central polarized stress fibers, and were enveloped in an oval ring of basolateral actin filaments indicating a loss of polarized mobility. HEK continued to show lamellipodia and filopodia during that 2-h period; however, previous studies have indicated a concurrent loss of 6 4 and 3 1 integrins from these processes 1,2,16 . Displacement of the integrin receptors and disorganization of the actin cytoskeleton precludes the maintenance of their normal signaling pathways and associated repair mechanisms.…”

“…We know from the casualties of WW I and other global conflicts that SM blisters form at the epidermal-dermal junction of skin. In studies of the molecular lesions induced by SM, we reported an early onset and progressive disruption of the keratins (K5/K14), integrins ( 6 4 , 3 1 ) and laminin-5 ligand of the basal cell's adhesion complex 1,2 . Those same molecules are targets of EB-type blistering skin diseases [3][4][5] , which share a common phenotype with SM blisters.…”

Molecular determination of laminin‐5 degradation: a biomarker for mustard gas exposure diagnosis and its mechanism of action

“…(14) suggested that the SM alkylation effect in skin causes significant reduction of laminin‐5, destabilizes epidermal–dermal attachment, and potentiates vesication by disrupting adhesion complex molecules. A multiphoton microscopy study showed statistically significant decrease of 25% to 30% in emissions from labelled α6β4 integrin and laminin‐5, plus disruption of their receptor–ligand organization (26). These results indicated that a part of the mechanism of action of SM is a direct effect on the BMZ.…”

Treatment of keratin intermediate filaments with sulfur mustard analogs

“…One hypothesis for vesicant action is the activation of an endogenous protease by SM [7][8][9][10] and an endogenous inhibitor of this protease has been identified [11,12]. Other investigators have analyzed the effects of SM on basal cell adhesion complex molecules [13][14][15][16][17], hypothesizing alkylation of basal cell adhesion complex molecules results in failure of the complex [16,18], leading to blister formation, similar to inherited skin blistering diseases [19].…”