Lipopolysaccharide enhances the cytotoxicity of 2-chloroethyl ethyl sulfide
© Stone et al; licensee BioMed Central Ltd. 2003
Received: 2 October 2002
Accepted: 6 January 2003
Published: 6 January 2003
The bacterial endotoxin, lipopolysaccharide (LPS), is a well-characterized inflammatory factor found in the cell wall of Gram-negative bacteria. In this investigation, we studied the cytotoxic interaction between 2-chloroethyl ethyl sulfide (CEES or ClCH2CH2SCH2CH3) and LPS using murine RAW264.7 macrophages. CEES is a sulfur vesicating agent and is an analog of 2,2'-dichlorodiethyl sulfide (sulfur mustard). LPS is a ubiquitous natural agent found in the environment. The ability of LPS and other inflammatory agents (such as TNF-alpha and IL-1beta) to modulate the toxicity of CEES is likely to be an important factor in the design of effective treatments.
RAW 264.7 macrophages stimulated with LPS were found to be more susceptible to the cytotoxic effect of CEES than unstimulated macrophages. Very low levels of LPS (20 ng/ml) dramatically enhanced the toxicity of CEES at concentrations greater than 400 μM. The cytotoxic interaction between LPS and CEES reached a maximum 12 hours after exposure. In addition, we found that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) as well as phorbol myristate acetate (PMA) also enhanced the cytotoxic effects of CEES but to a lesser extent than LPS.
Our in vitro results suggest the possibility that LPS and inflammatory cytokines could enhance the toxicity of sulfur mustard. Since LPS is a ubiquitous agent in the natural environment, its presence is likely to be an important variable influencing the cytotoxicity of sulfur mustard toxicity. We have initiated further experiments to determine the molecular mechanism whereby the inflammatory process influences sulfur mustard cytotoxicity.
In this investigation, we explored the potential cytotoxic interaction between LPS and CEES using a murine macrophage cell line (RAW264.7). CEES is a monofunctional analog of sulfur mustard (bis-2-(chloroethyl)sulfide) which a bifunctional vesicant and a chemical warfare agent. Both bis-2-(chloroethyl)sulfide) and CEES are known to provoke acute inflammatory responses in skin [1–3]. The resulting skin blistering is thought to involve the stimulation of specific protease(s) . Apoptosis is now considered a possible molecular mechanism whereby CEES induces cytotoxicity [5, 6].
LPS is a major component of the cell wall of gram-negative bacteria and is known to trigger a variety of inflammatory reactions in macrophages and other cells having CD14 receptors [7, 8]. In particular, LPS is known to stimulate the macrophage secretion of nitric oxide  and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) . For this reason, we also determined if TNF-alpha or IL-1-beta were capable of enhancing the cytotoxic effects of CEES.
LPS stimulation of macrophages is known to involve the activation of protein phosphorylation by kinases as well as the activation of nuclear transcription factors such as NF-kappaB [10–13]. The activation of protein kinase C (PKC) by diacylglycerol is also a key event in LPS macrophage activation . In vitro experiments have shown that the secretion of TNF-alpha and IL-1-beta by LPS-stimulated monocytes is dependent upon PKC activation [13, 14]. In this study, we also determined if phorbol myristate acetate (PMA) activation of PKC also enhanced CEES toxicity. Evidence suggests that LPS [15, 16] as well as TNF-alpha  stimulate the production of free radicals by macrophages. Our long-term goal is to understand the molecular mechanism of sulfur mustard toxicity and to determine if free radical production plays an important role in this toxicity.
In our experiments, cytotoxicity was measured by a decrease in the optical density by the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay or by an increase in the fluorescence of propidium iodide (PI). The MTT assay is based on reduction of MTT by actively growing cells to produce a blue formazan product with absorbance at 575 nm. A low MTT absorbance indicates cell death. The PI assay differentiates between live and dead cells. Cells that have lost membrane integrity cannot exclude PI, which emits a red fluorescence after binding to cellular DNA or double stranded RNA. A high PI% indicates cell death.
Cytotoxic interaction between LPS and CEES as measured by the MTT assay
Time course for the cytotoxic interaction between LPS and CEES as measured by the propidium iodide (PI) assay
TNF-alpha and IL-1beta also enhance the cytotoxic effect of CEES
IL-1β Enhances the Cytotoxicity of CEES to RAW264.7 Macrophages (24 hours)1
0.0 ± 0.0a
10.6 ± 2.1a
10.2 ± 4.1a
64.8 ± 16.9b
120.8 ± 2.3c
111.1 ± 3.9c
1.61 ± 0.1b
1.20 ± 0.04a
1.45 ± 0.03b
1.13 ± 0.16a
0.25 ± 0.07c
0.74 ± 0.07d
TNF-α Enhances the Cytotoxicity of CEES to RAW264.7 Macrophages (24 hours)1
0.0 ± 0.0a
0.8 ± 0.5a
3.3 ± 1.3a
15.6 ± 4.1b
62.6 ± 1.4c
34.1 ± 7.6b
1.68 ± 0.08a
1.61 ± 0.04a
1.55 ± 0.08a
1.37 ± 0.09a
0.32 ± 0.05b
1.08 ± 0.18c
PMA Enhances the Cytotoxicity of CEES to RAW264.7 Macrophages (24 hours)1
0.0 ± 0.0 a
2.6 ± 1.03a
1.8 ± 0.96a
18.7 ± 6.23b
59.1 ± 6.63c
41.4 ± 5.0 d
1.50 ± 0.03a
1.56 ± 0.06a
1.71 ± 0.03a
1.46 ± 0.10 b
1.07 ± 0.11c
1.36 ± 0.04 b
We have found that inflammatory agents such as LPS, TNF-alpha, IL-1beta, and PMA amplify the cytotoxicity of CEES to RAW264.7 macrophages. There is evidence suggesting that CEES can modulate levels of inflammatory cytokines but this information is controversial and inconsistent. Cultured monocytes exposed to CEES show a transient increase in TNF-alpha . Similarly, cultured normal human keratinocytes exposed to CEES show a transient increase in both TNF-alpha and IL-1beta [19, 20]. In contrast to the results of Arroyo et al. [18–20], Ricketts et al.  found that IL-1beta or TNF-alpha protein did not increase in sulfur mustard-exposed mouse skin. Blaha et al. , using an in vitro human skin model, found that CEES treatment resulted in a decreased level of IL-1alpha.
Sabourin et al.  also addressed this issue and studied the in vivo temporal sequence of inflammatory cytokine gene expression in sulfur mustard exposed mouse skin. These investigators found an increase in IL-1beta mRNA levels after 3 hours that dramatically increased between 6–24 hours post exposure . Moreover, immunohistochemical studies showed an increase in tissue levels of IL-1beta . The in vitro results reported here support a role for inflammatory cytokines in the mechanism and kinetics of CEES toxicity. Our unique findings with regards to LPS are significant because they demonstrate that this bacterial endotoxin enhances CEES toxicity even when present at extremely low levels, i.e., nanograms of LPS per ml.
LPS is ubiquitous and is present in serum, tap water, and dust. Military and civilian personnel would, indeed, always have some degree of exposure to environmental LPS, which could increase the toxicity of sulfur mustard. In addition, there is always the possibility of purposeful LPS exposure. Our primary future goals are to understand the general mechanism for the enhanced toxicity of CEES in the presence of inflammatory agents and use this information to develop effective countermeasures. Our experiments with PMA (Table 3) suggest that the activation of protein kinase C may play a key role in molecular mechanism whereby LPS enhances the cytotoxic of CEES.
In experiments that will be reported elsewhere, we have found the antioxidants such as RRR-alpha-tocopherol and N-acetylcysteine are effective in reducing the cytotoxic effects of CEES on LPS-stimulated macrophages. We have also initiated experiments to distinguish the roles of apoptosis and necrosis in the observation reported here.
Our results suggest that that LPS dramatically enhances the toxicity of sulfur mustard. Since LPS is a ubiquitous agent in the natural environment, its presence is likely to be an important variable influencing the cytotoxicity of sulfur mustard toxicity. LPS is known to stimulate the production of inflammatory cytokines such as TNF-alpha and IL-1beta in human monocytes . We, therefore, also determined if these cytokines influenced the cytotoxicity of CEES. The data in Tables 1 and 2 demonstrate that both these cytokines enhance the cytotoxicity of CEES as measured by either the MTT assay or the PI assay. Inhibition of protein kinase C is known to block the secretion of TNF-alpha and IL-1beta  whereas stimulation of protein kinase C promotes TNF-alpha and IL-1beta production . The data reported in Table 3 show that stimulation of protein kinase C activity by PMA also enhances the cytotoxicity of CEES. Collectively, the data reported here provide the basis for future experiments attempting to determine the signal transduction mechanisms whereby inflammatory agents enhance sulfur mustard cytotoxicity.
LPS (Escherichia Coli Serotype 0111:B4) and PMA (Phorbol 12-Myristate 13-Acetate) were purchased from Sigma Chemical Co. St. Louis, MO. Murine IL-1-beta was obtained from Research Diagnostic Inc. (Flanders, NJ). r-TNF-alpha (human) was obtained from Promega Co (Madison, WI).
Cell culture and treatments
RAW264.7 murine macrophage-like cells (American Type Culture Collection, Rockville, MD) were cultured at 37°C in a humidified incubator (95% air with 5% CO2) in RPMI-1640 medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (GiBcoBRL Grand Island, NY). Adherent cells were subcultured over night in 96 well Costar tissue culture plates in serum-free RPMI with 0.1% BSA (RPMI-0.1%BSA).
MTT assay for determination of cell viability
This assay for cell viability is based on the reduction of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase in viable cells to produce a purple formazan product. Schweitzer et al.  have shown that this assay provides linearity between optical density and cell number. This assay was performed by a slight modification of the method described by Wasserman et al. [24, 25]. Briefly, at the end of each experiment, cells cultured in 96 well plates (with 100 μl of medium per well) were incubated with MTT (20 μl of 5 μg/ml per well) at 37°C for 4 hours. The formazan product was solubilized by addition of 100 μl of dimethyl sulfoxide (DMSO) and 100 μl of 10% SDS (in 0.01 M HCl) for 16 hours at 37°C. The dehydrogenase activity was expressed as the absorbance (read with a Molecular Devices microplate reader) of the formazan product at 575 nm.
Dye exclusion assay (PI assay)
This assay uses propidium iodide (PI) dye to differentiate live and dead cell [26–28]. Cells that have lost membrane integrity cannot exclude PI, which emits a red fluorescence after binding to cellular DNA or double stranded RNA. After each experiment, cells were washed with PBS and incubated with 200 μl of RPMI medium with 5 μg/ml PI for five minutes at room temperature. PI fluorescence was measured using a Fluostar Galaxy microplate reader using an excitation wavelength of 485 nm and an emission wavelength of 650 nm. We expressed the results as the percent increase in the fluorescence of treated cells over the fluorescence of control cells (no treatments), i.e., 100 × (PItreatment-PIcontrol)/PIcontrol)
Means among treatments were compared by one-way ANOVA followed by the Scheffe test with a significance level of 0.05. In both the Tables and Figures, means not sharing a common letter are significantly different (p < 0.05). Means sharing a common letter are not significantly different. The means and standard deviations of three independent experiments are provided in both the Figures and Tables.
This research was supported by a United States Army Medical Research Command Grant: The Influence of Antioxidant Liposomes on Macrophages Treated with Mustard Gas Analogues, USAMRMC Grant No. 98164001.
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