Robert J. Werrlein’s research while affiliated with United States Army Medical Research Institute of Chemical Defense and other places

of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

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, alpha6beta4 integrin, laminin 5 and alpha3beta1 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 multiphoton microscopy and immunofluorescent staining, we found that exposing human epidermal keratinocytes (HEK) and intact epidermis to SM (400 muM for 5 min) caused progressive collapse of the keratin (K5/K14) cytoskeleton and depletion of alpha6beta4 integrins.1 We now report that SM causes concomitant disruption and collapse of the basal cell's alpha3beta1-integrin receptors. At 1 h postexposure, images of Alexa488-conjugated HEK/alpha3beta1 integrins showed almost complete withdrawal and disappearance of retraction fibers and a progressive loss of polarized mobility. With stereo 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. Alpha3beta1 integrin, like alpha6beta4 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 muM 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 alpha6beta4-integrin and laminin-5. At 1 h postexposure, there was disruption in the surface organization of alpha6beta4 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.

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.

Sulfur mustard [HD, di(2 chloroethyl) sulfide] is a blistering agent that has been used by warring nations for more than 80 years. There is still no completely effective treatment for its vesicating lesions, and that presents a fundamental question. How is skin altered by sulfur mustard so that structural bonds between basal cells and basement membrane are selectively disrupted? We have postulated that, like blistering diseases of the skin, HD may potentiate dermalepidermal separations by altering molecules of the basal cells adhesion complex (Werrlein and Madren-Whalley, 2000). Accordingly, we are using confocal and multiphoton laser scanning techniques in an attempt to determine whether there are detectable postexposure changes in the interactive attachment molecules.

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

Among the most intriguing questions about sulfur mustard (di(2‐chloroethyl) sulfide) is why basal cells are the primary targets of its vesicating lesions. To investigate this problem, replicate cultures of human epidermal keratinocytes (HEK) were grown from normal skin and exposed to 400 μM sulfur mustard (HD) for 5 min. Using fluorescein isothiocyanate (FITC)‐conjugated antibodies, confocal laser microscopy and image analyses, we found that in early passages, sham‐treated HEK maintained in a 0.15 mM Ca²⁺ medium continued to express keratins K5 and K14 as well as α6β4‐integrin. Both K5 and K14 are intermediate filaments characteristic of basal cells and linked with attachment mechanisms effecting epidermolysis bullosa simplex, a family of blistering skin diseases. Acute exposure to HD caused a statistically significant (P < 0.01) 30.74% decrease in K14 fluorescence within 1 h of exposure. Within 2 h of exposure, K14 fluorescence decreased to near‐zero values. The loss in expression of K14 was progressive and occurred well before the expected appearance of in vivo blisters, which have a dose‐dependent, clinical latent phase of 8–24 h. Acute exposure to HD also caused a statistically significant (P < 0.002) decrease in expression of β4, an integrin which has been associated with junctional epidermolysis bullosa (JEB). Disruption of K14 and α6β4‐integrin may be early events in the HD injury pathway; however, they had no immediate or obvious effect on cell to substrate attachment. Published in 2000 by John Wiley & Sons, Ltd.

Among the most intriguing questions about sulfur mustard (di(2-chloroethyl) sulfide) is why basal cells are the primary targets of its vesicating lesions. To investigate this problem, replicate cultures of human epidermal keratinocytes (HEK) were grown from normal skin and exposed to 400 microM sulfur mustard (HD) for 5 min. Using fluorescein isothiocyanate (FITC)-conjugated antibodies, confocal laser microscopy and image analyses, we found that in early passages, sham-treated HEK maintained in a 0.15 mM Ca2+ medium continued to express keratins K5 and K14 as well as alpha6beta4-integrin. Both K5 and K14 are intermediate filaments characteristic of basal cells and linked with attachment mechanisms effecting epidermolysis bullosa simplex, a family of blistering skin diseases. Acute exposure to HD caused a statistically significant (P < 0.01) 30.74% decrease in K14 fluorescence within 1 h of exposure. Within 2 h of exposure, K14 fluorescence decreased to near-zero values. The loss in expression of K14 was progressive and occurred well before the expected appearance of in vivo blisters, which have a dose-dependent, clinical latent phase of 8-24 h. Acute exposure to HD also caused a statistically significant (P < 0.002) decrease in expression of beta4, an integrin which has been associated with junctional epidermolysis bullosa (JEB). Disruption of K14 and alpha6beta4-integrin may be early events in the HD injury pathway; however, they had no immediate or obvious effect on cell to substrate attachment.

One of the most fascinating questions about sulfur mustard (HD) injuries is the unanswered issue of why basal cells are specific targets for vesicating lesions. What mechanism sets basal cells apart from all other cells of the epidermis and causes their interface with the basement membrane to be the primary site for blister formation? In recent studies with confocal microscopy, Werrlein et al. (1) reported that increased extracellular calcium affected the morphology and in vitro response of human epidermal keratinocytes (HEK) to HD. The image changes observed were consistent with discoveries by Hennings et al. (2–4) that small increases in extracellular calcium (as little as 0.01 mM) can cause fast-acting, dramatic responses in the differentiation of keratinocytes. Kruszewski et al. (5) demonstrated that keratinocytes can respond to small incremental calcium changes (of 0.1–0.12 mM) within 60 s, but noted that the capacity for transient response to calcium was lost as keratinocytes differentiated. Others have demonstrated that calcium-induced terminal differentiation can alter cell-surface receptors (6) and epidermal protein markers (7,8). Complementary studies (9–11) have demonstrated that calcium gradients exist in the epidermis of mice and humans, with the lowest calcium concentrations residing at the basal layer. Each has determined that basal cells exist in an environment that is relatively low in calcium when compared to keratinocytes located in the suprabasal layers of the epidermis. Absolute calcium concentrations within the gradient have not been determined. It is known, however, that epidermal gradients can be disrupted by certain chemicals, e.g., acetone and lovastatin (12),by diseases like psoriasis (13,14), and by essential fatty acid deficiencies (12). Based on the evidence accumulated, we have proposed that exposure to HD may disrupt extracellular calcium gradients or programmed responses to calcium, which would be especially disruptive to basal cells because they are more sensitive to changes in calcium concentration than are differentiated keratinocytes. To test this hypothesis, we have initiated development of primary and first-passage keratinocyte cultures from human skin resections that will allow us to use confocal microscopy to determine how calcium concentration affects HEK proliferation, differentiation, and basal cell response(s) to sulfur mustard toxicity. The system will subsequently be used for testing the efficacy of prophylactic and therapeutic compounds.