Expression of cytokines and chemokines in mouse skin treated with sulfur mustard

Abstract

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.

Keywords: Dermal toxicity; Immuno-multiplex assays; Inflammatory cells infiltration; Inflammatory mediators; Sulfur mustard; Vesicants.

Fig. 1.

Structural alterations in mouse ear skin following exposure to SM. Hematoxylin and Eosin (H&E) stained histological sections of control and SM-treated mouse ear skin. Panels A-C, control, naïve tissue (Ctl); panels D-F: mouse ear skin 24 h post-SM exposure; panels G-I: mouse ear skin 72 h post-SM exposure; panels J-L: 168 h post-SM exposure. Images shown in panels A, D, G, and J are lower magnifications (20× objective; scale bar = 50 μm); ventral side ear skin shown in panels B, E, H, and K are higher magnifications (40× objective; scale bar = 20 μm); dorsal side ear skin shown in panels C, F, I, and L (40× objective; scale bar = 20 μm). DEJ: dermal-epidermal junction; N: necrosis; H: hyperplasia; I: inflammatory cell infiltrate. Ventral is exposed side (panels B, E, H, and K) and dorsal unexposed side (panels C, F, I, and L).

Fig. 2.

Myeloperoxidase (MPO) staining of mouse ear skin post-SM exposure. Tissue sections were stained using an antibody to MPO. Panel A: Control, naive (Ctl); panel B: mouse ear skin 24 h post-SM exposure; panel C: 72 h mouse ear skin post-SM exposure; panel D: mouse ear skin 168 h post-SM exposure. Sections were stained with DAB and counterstained with hematoxylin (scale bar = 20 μm). Bottom left inserts of Fig. 2B and C show higher magnifications of MPO positive cells, dark staining indicates MPO expression in the cells (100× objective, scale bar = 10 μm).

Fig. 3.

Accumulation of macrophages in mouse ear skin post-SM exposure. Tissue sections were stained for macrophages using antibody to F4/80. Panel A, Control, naive (Ctl); panel B: mouse ear skin 24 h post-SM exposure; panel C: mouse ear skin 72 h post-SM exposure (insert showed enlarged image of an F4/80+ cell); panel D: mouse ear skin 168 h post-SM exposure. Sections were stained with DAB and counterstained with hematoxylin (Scale bar = 20 μm).

Fig. 4.

Effect of SM on accumulation of macrophages in mouse ear skin. Tissue sections were stained for macrophages using antibody to F4/80. Total macrophage counts for the ventral (inner, exposed side) and dorsal side (outer, unexposed side) of the ear mouse skin, and the whole ear were determined. At least three animals and three different slides per group were analyzed for F4/ 80+ cells. Data are presented as mean ± S.E. (macrophage counts per mm²). All data were analyzed using one-way Analysis of Variance (ANOVA) followed by Dunnett’s test or non-pair student t-test. Macrophages counts at 72 h (red bar) and 168 h (purple bar) post-SM in ventral and dorsal ear skin are significantly increased when compared to macrophages counts in the tissues in naïve unexposed control and at 24 h (blue bar) post-SM (*p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

Expression of mRNA for MMP8 (neutrophil collagenase) and MMP12 (macrophage elastase) in mouse ear skin post-SM exposure. Fold changes were analyzed using one-way Analysis of Variance (ANOVA) followed by Dunnett’s test or non-pair student t-test. Data are expressed as fold changes over time and are presented as the mean ± SE (n = 10); a p value of < 0.05 was considered statistically significant and marked with *, when compared to unexposed, naïve, control samples.

Fig. 6.

Cytokine/chemokine mRNA expression in mouse ear skin post-SM exposure. Real-time PCR with Taqman gene expression assays was used to quantify mRNA in tissue sections. Data are expressed as fold changes over time and presented as the mean ± SE (n = 10). Fold changes were analyzed using one-way Analysis of Variance (ANOVA) followed by Dunnett’s test or non-pair student t-test; a p value of < 0.05 was considered statistically significant and marked with *, when compared to unexposed, naïve, control samples. Control skin (blue bars), mouse ear skin 6 h post-SM (red bars), mouse ear skin 24 h post-SM exposure (green bars), mouse ear skin 72 h post-SM (purple bars); mouse ear skin168 h post-SM (light blue bars). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.

Cytokine/chemokine protein expression in mouse ear skin post-SM exposure. Lysates from pooled samples prepared from mouse ears were analyzed using a mouse cytokine/chemokine Milliplex kit (Millipore Inc., Billerica, MA). Data are expressed as pg of cytokines/chemokines per mg total lysate protein and are presented as the mean ± SE, (CV < 15%), n = 3. Data were analyzed using one-way Analysis of Variance (ANOVA) followed by Dunnett’s test or non-pair student t-test when compared to control samples; a p value of < 0.05 was considered statistically significant and marked with *. Control (blue bars), mouse ear skin 6 h post-SM (red bars), mouse ear skin 12 h post-SM (green bars); mouse ear skin 24 h post-SM (purple bars), mouse ear skin 72 h post-SM (light blue bars), mouse ear skin 168 h post-SM (orange bars). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)