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.

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Rabbit skeletal myosin rod contains two tryptophan residues per chain (four per coiled-coil) that are located about 50 and 175 A from the N-terminus of the light meromyosin (LMM) region of rod. We have characterized the local polarity, excited-state photophysics, solvent accessibility, and rotational dynamics of these tryptophans in myosin rod filaments at 125 mM KCl using steady-state and time-resolved fluorescence techniques. The fluorescence decays were described using a complex bimodal distribution with a discrete long-lifetime component of 5.44 ns (amplitude of 0.51) and a Gaussian distribution of short lifetimes with mean of 0.105 ns and width of 2.15 ns (amplitude 0.49). The discrete long-lifetime species was efficiently quenched by the neutral quencher acrylamide with a bimolecular collision constant (kq) of 0.85 x 10(9) M-1 s-1. The emission spectrum, lifetime distribution, and quenching behavior of the tryptophans in myosin rod monomers (at 0.5 M KCl) were quite similar. Time-resolved anisotropy decays of the rod monomers and filaments exhibited nearly identical double-exponential decays to a constant. Each had a fast, subnanosecond component (amplitude 0.07), probably corresponding to fast wobble of the tryptophans on the coiled-coil surface, a slower, approximately 6-ns component (amplitude approximately 0.04), corresponding to an unidentified internal, segmental mode of motion of the coiled-coil, and a constant (r infinity) of 0.15.(ABSTRACT TRUNCATED AT 250 WORDS)

Laminin-332 is a basement membrane protein composed of three genetically distinct polypeptide chains that actively promote both skin epidermal cell adhesion and migration. Proteolytic fragments of the laminin γ2 chain stimulate migration and scattering of keratinocytes and cancer cells. Sulfur mustard (SM) is a bifunctional alkylating agent that induces separation of basal keratinocytes from the dermal-epidermal junction and invokes a strong inflammatory response leading to delayed wound repair. In the present studies, the role of laminin γ2 in SM-induced skin injury and wound repair was investigated using the mouse ear vesicant model. We found that laminin γ2 chain mRNA was preferentially upregulated in mouse ear skin exposed to SM. In situ hybridization confirmed overexpression of laminin γ2 transcript. Western blot analysis showed increased protein expression of the full-length proform of laminin γ2 and smaller processed fragments of laminin γ2 in skin exposed to SM. Dual immunofluorescence labeling indicated that laminin γ2 fragments are prevalent in suprabasal keratinocytes behind the leading edge in areas of hyperplasia in injured skin. In addition, co-expression of laminin γ2 and the senescent marker, p16 INK4a was found to overlap with the hyperplastic migratory epithelial sheet. This observation is similar to hypermotile keratinocytes reported in invasive carcinoma cells. Overall, our studies indicate that laminin γ2 is preferentially expressed in skin post SM exposure and that protein expression appears to become progressively more fragmented. The laminin γ2 fragments may play a role in regulating SM-induced skin wound repair. Anat