Cytokines can be used as biomarkers to detect exposure of cells to chemical toxins.  Sulfur mustard (2,2'-dichlorodiethyl sulfide, HD) is a chemical toxin that has been used as a Chemical warfare agent. In this study cultured human epidermal keratinocyte cells were used as a potential model system to measure how effective therapeutic drugs are against chemical toxins such as HD.  We compared two human epidermal keratinocyte cell lines Simian Virus 40 (SV40) immortalized human epidermal keratinocytes (IHEK) were compared normal human epidermal keratinocytes (NHEK) to see which model system produced better results. IHEKs resemble NHEKs 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 interleukin-8 (IL-8) when exposed to HD. IL-8 is released by a variety of cell types in response to an inflammatory stimulus however, a major difference was observed between the NHEK cell line 6207 and IHEK cell line 425. The immortalized cell line produced higher levels of Interleuken-8 then those of its normal counterpart. 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 was higher in cell line 425 makes this in vitro model a potential screening tool to study the efficacy of drugs that suppress production of cytokine markers.

Introduction

Epithelial cells are the initial target of a variety of toxic chemicals, including the chemical warfare agent Sulfur Mustard.  The first time that HD was used as a chemical warfare agent was on the 12^th of July 1917, the German Army fired artillery rounds filled with mustard at British troops near Ypres, Belgium (Prentiss, 1937). These soldiers experienced a wide variety of symptoms, with most of the clinical manifestations afflicting the eyes, airways and the skin. Through the years HD has been allegedly used as a chemical weapon and as recently as 1988 employed by the government of Iraq on its own citizen population (Sidell and Franz, 1997). Research has been conducted on the effects of Sulfur Mustard since the early twenties, but as of yet there is no known prophylaxis or therapy (Papirmeister et al. 1991; Petrali and Oglesby-Megee, 1997).

HD has been studied by using several different in vitro systems including; cultivated human fibroblast, cells derived from tumors and normal epithelial human keratinocytes (NHEK). These systems have provided insight into the metabolic and cellular reactions of chemical toxins, but are not without experimental challenges. NHEK cells rapidly undergo senescence are expensive and come from a variety of different donors. Cells transformed with Simian Virus 40 (SV40) have been established as immortalized cell cultures that exhibit an indefinite growth potential (Steinberg and Defendi, 1979; Defendi et al. 1982; Steinberg and Defendi, 1983; Morris et al. 1985). These Immortalized human epithelial keratinocytes (IHEK) can be carried through long term culture, are cost effective and come from the same donor. SV40 transformed cell lines have been used as a tool for studying the effects of both mutagenic and nongenotoxic chemicals and are an established toxicological model (Steinberg et al. 1999).

Cytokines are used as biomarkers to detect exposure of cells to chemical agents such (Arroyo et al, 1999). Therapeutic non-steroidal anti-inflammatory drugs (NASID) such as ibuprofen (IB), have been shownto inhibit the normal expression ofinflammatory cytokines. If cytokines are not detected efficiently in NHEKs then a model where these cellular markers can be measured at toxicologically important concentrations must be established (Konstan et al. 1995; Stuhlmeier et al. 1999; Scheuren et al. 1998). SV40 INEKs that produce higher concentrations of cytokines have been used as model cell lines to study the expression of cytokines such as IL-8 (Gerritsma et al. 1998; Chodosh et al. 2000; Petit-Frère et al. 2000; Walsh et al. 2001).We compared a normal cell line to an immortalized cell line that secreted higher levels of the IL-8, so as to measure the efficacy of possible therapeutic intervention against the effects of exposure to HD.

Results

Analysis of NHEK/IHEK cells exposed to different concentrations of HD revealed similar viability profiles at concentrations between 25 to 100 µM, but differed at higher concentrations of sulfur mustard, above 100 µM (Figure 1). These observations are consistent with the fact that exposure of human epithelial cell lines to a concentration of HD that is between 1-100 µM is considered physiologically significant. Cell death occurs rapidly at concentrations over 100 µM.

At concentrations below 100 µM HD, when the secretion of IL-8 in response to the chemical warfare agent was analyzed, we found that immortalized cell line 425 produced higher quantities of the cytokine then did its normal counterpart. When we compared these two cell lines at higher doses of HD, above 100 µM, we observed an increase in the expression of IL-8 in the normal cell line that was not observed in the immortalized cells. In fact at 200 µL of HD we see a drop in the concentration of IL-8 in the SV40 transformed cells not observed in the normal cell line (Figure 2).

When normal cells were treated with ibuprofen there were no detectible amounts of IL-8 either in the absence or presence of HD. But when SV40 transformed cell line 425 was treated with the anti-inflammatory agent ibuprofen the concentration levels of IL-8 went from 1.87 µg/mL to 0.319 µg/mL, this is a 6-fold decrease in the production of the cytokine. We also observed a decrease in the secretion IL-8 in 425 cells that were exposed to HD concentrations that ranged from 25-100 µM (Figure 3).

Concluding Remarks

Results obtained in experiments using the non-steroidal anti-inflammatory drug, ibuprofen, appeared to indicate that the amount of IL-8 secretion is an important factor in evaluating the efficacy of this drug in reducing the inflammatory response. Studies conducted with NHEK in the presence of Ibuprofen did not yield any detectible concentrations of IL-8 both in the cells exposed and not exposed to sulfur mustard. In contrast, IL-8 levels in HD-treated IHEK cells in replicate experiments were dramatically suppressed by IB treatment. When SV40 transformed cell line 425 was treated with the anti-inflammatory agent ibuprofen the concentration levels of IL-8 went from 1.87 µg/mL to 0.319 µg/mL, this is a 6-fold decrease in the production of the cytokine. But it is important to note that IL-8 concentrations were still detectable in the 425 cell line but not in the normal cell line while using the same concentration of the prophylaxis ibuprofen. We also observed a decrease in the secretion IL-8 in 425 cell's that were exposed to HD concentrations that ranged from 25-100 µM in the presence of ibuprofen.

These preliminary results support the idea that the SV40 immortalized epidermal cells can be used as a useful, and inexpensive, alternative in vitro model system to test inflammatory processes stemming from cutaneous vesicant injury.

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