Modulation of sulfur mustard induced cell death in human epidermal keratinocytes using IL-10 and TNF-alpha.

We compared the effects of overexpressing a tightly regulated anti-inflammatory cytokine, interleukin 10 (IL-10), and the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on sulfur mustard induced cytotoxicity in human epidermal keratinocytes. Both cytokines were overexpressed when compared with the cells transfected with the empty vector as determined by quantitative ELISA. Cells overexpressing interleukin 10 suppressed the pro-inflammatory cytokines interleukin 8 and interleukin 6 following exposure to 50-300 microM sulfur mustard. These cells exhibited delayed onset of sulfur mustard induced cell death. On the other hand, cells overexpressing tumor necrosis factor alpha induced a sustained elevation in both interleukin 6 and 8 expression following exposure to 50-300 microM sulfur mustard. These cells were sensitized to the effects of sulfur mustard that resulted in an increased sulfur mustard induced cell death. Normal human epidermal keratinocytes treated with sulfur mustard exhibited elevated levels of tumor necrosis factor alpha expression and increased activity of nuclear factor kappa B. Gene array data indicated that cells overexpressing interleukin 10 induced several genes that are involved in growth promotion and cell-fate determination. We, therefore, identify IL-10 and TNF-alpha signal transduction pathways and their components as possible candidates for early therapeutic intervention against sulfur mustard induced cell injury.

Immortalized human keratinocytes: A model system to study the efficacy of therapeutic drugs in response to the chemical warfare agent sulfur mustard (HD)

Cytokines have been established as biomarkers to detect exposure of cells to chemical warfare agents such as sulfur mustard (2,2'-dichlorodiethyl sulfide, HD). In this study cultured normal and SV40 immortalized human epidermal keratinocyte (NHEK/IHEK) cells were compared as potential model systems to measure the efficacy of therapeutic drugs against HD. Immortalized human epidermal keratinocytes resemble their primary cell counterparts but have the advantage of being carried through long-term culture. Immortalized cells also provide consistency and durability and are less costly than primary keratinocytes. Immunoassay studies were performed to examine the response of these two cell lines to HD. We found that both normal and immortalized NHEKs secreted the pro-inflammatory mediator interleukin-8 (IL-8) when exposed to HD. However, a major difference was observed between the NHEK cell line 6207 and IHEK cell line 425. IHEK cell line 425 produced higher levels of Interleuken-8 then those of its normal counterpart cell line 6207. This observation is significant since therapeutic drugs such as ibuprofen, which depress cytokine production, may not allow these biomarkers to be detected efficiently in experimental analysis of certain NHEK cell lines. The fact that Il-8 production higher in cell line 425 cell makes this in vitro model a potential screening tool to study the efficacy of drugs that suppress production of cytokine markers.

Regulation of 1-alpha, 25-dihydroxyvitamin D3 on interleukin-6 and interleukin-8 induced by sulfur mustard (HD) on human skin cells.

The regulatory effects of the active form of vitamin D, 1-alpha, 25-dihydroxyvitamin D3 (1-alpha, 25 (OH)2D3) were assessed on the cytokine and chemokine secretion induced by sulfur mustard on human skin fibroblasts and human epidermal keratinocytes. Stimulation of human skin fibroblasts with sulfur mustard (10(-4) M for 24 hr at 37 degrees ) resulted in approximately a 5 times increase in the secretion of interleukin-6 and over a 10 times increase for interleukin-8, which was inhibited by 1-alpha, 25 (OH)2D3, at

A rapid colorimetric assay for sulfur mustard cytotoxicity using isolated human peripheral blood lymphocytes and keratinocytes.

Sulfur mustard (SM) is a potent vesicating agent that has pronounced cytotoxic effects as well as mutagenic, carcinogenic, and radiomimetic properties. Isolated human peripheral blood lymphocytes (PBLs) and human epidermal keratinocytes (HEKs) have been used as in vitro models for determining SM-induced cytotoxicity. A recently developed colorimetric assay (the CellTiter 96 AQ ueous Non-radioactive Cell Proliferation Assay) was assessed using both of the in vitro models described above. Using 24- or 96-well microplates, reproducible (+/- 10%) SM dose/response curves for both types of human cells were obtained using a spectrophotometric microplate reader set at 490 nm. After a 4-h incubation time, as many as 96 sample wells could be measured within 45 s using this commonly available equipment. Multiple plates of samples can be run immediately. This technique may facilitate cytotoxicity investigations of new candidate compounds for both prophylaxis of and therapy for SM intoxication.