Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7 days. This was associated with a 2-15 fold increase in expression of the proinflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor α at time points up to 7 days post-SM exposure. Marked increases (20-1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.

Time‐related changes in the histopathology of mouse skin occur after topical exposure to the vesicant agent, bis(2‐chloroethyl) sulfide (SM) using the mouse ear vesicant model (MEVM). Some of these changes include increasing edema, detachment of the epidermis from the dermis, and upregulation of laminin‐332. Endoplasmic reticulum stress response (ESR) was observed by confocal microscopy. Microarray analysis and RT‐PCR experiments indicated there was upregulation of several heat shock proteins and folding chaperones such glucose‐regulated protein 78 and GRP94 in the ER. It confirmed the confocal observations and suggested that ER stress occurs very quickly in the MEVM. The accumulation of laminin γ2 protein, one of the chains of laminin‐332, in the ER was visualized by confocal microscopy and co localized with the ER chaperones. This accumulation appeared specifically in the migrating, but not proliferating cells. These observations are consistent with recent reports that laminin‐332 is found in migrating, transformed epithelial cells that have left the cell cycle, but not in proliferating cells. These results suggest that laminin γ2 is misfolded after SM treatment, resulting in decreased secretion and reduced overall amounts of laminin‐332 in the extracellular matrix. This would explain the observation that there is a delayed wound healing response evident in this wound model.Grant Funding SourceES005022, ES004738, EY09056, and the NIH CounterACT Program through NIAMS U54AR055073

Chemical vesicants such as sulfur mustard (SM) can produce severe tissue damage. A time‐course study using mouse ear skin exposed to a low dose (0.08 mg) of SM resulted in excessive keratinocyte hyperplasia about three days after exposure. The abnormal cell proliferation was reminiscent of that observed in several types of cancer and potential oncogenic hyperproliferative signal molecules were examined to determine if the two systems were comparable. Immunohistochemical, Western blotting and RT‐PCR studies were performed and markers of skin injury and repair were examined for various time‐points. The protein, p16‐INK4a, which triggers either growth arrest or apoptosis was hyperexpressed in the hyperplasia areas of injured skin. The p16‐INK4a co‐expressed with laminin γ2, a polypeptide that promotes epithelial sheet migration over a wound bed. The endoplasmic reticulum stress‐regulated gene, GADD153/CHOP had a similar expression pattern to that of p16‐INK4a. In contrast, both keratin 5, an intermediate filament protein of basal epithelial cells and Ki67, a marker of cells in the growth stage of the cell cycle had a much more restricted distribution. Understanding the basic mechanism of action of vesicant‐induced skin injury and repair should help to identify new targets of potential medical countermeasures against chemical vesicants. This work is supported by ES005022, EY09056, and AR055073.Grant Funding Source: ES005022, EY09056, and AR055073.