TY - JOUR
AU1 - Ramos,, Gerardo
AU2 - Limon-Flores, Alberto, Y.
AU3 - Ullrich, Stephen, E.
AB - Abstract Applying jet fuel (JP-8) to the skin of mice induces immune suppression. JP-8–treated keratinocytes secrete prostaglandin E2, which is essential for activating immune suppressive pathways. The molecular pathway leading to the upregulation of the enzyme that controls prostaglandin synthesis, cyclooxygenase (COX)-2, is unclear. Because JP-8 activates oxidative stress and because reactive oxygen species (ROS) turn on nuclear factor kappa B (NF-κβ), which regulates the activity of COX-2, we asked if JP-8–induced ROS and NF-κβ contributes to COX-2 upregulation and immune suppression in vivo. JP-8 induced the production of ROS in keratinocytes as measured with the ROS indicator dye, aminophenyl fluorescein. Fluorescence was diminished in JP-8–treated keratinocytes overexpressing catalase or superoxide dismutase (SOD) genes. JP-8–induced COX-2 expression was also reduced to background in the catalase and SOD transfected cells, or in cultures treated with N-acetylcysteine (NAC). When NAC was injected into JP-8–treated mice, dermal COX-2 expression, and JP-8–induced immune suppression was inhibited. Because ROS activates NF-κβ, we asked if this transcriptional activator played a role in the enhanced COX-2 expression and JP-8–induced immune suppression. When JP-8–treated mice, or JP-8–treated keratinocytes were treated with a selective NF-κβ inhibitor, parthenolide, COX-2 expression, and immune suppression were abrogated. Similarly, when JP-8–treated keratinocytes were treated with small interfering RNA specific for the p65 subunit of NF-κβ, COX-2 upregulation was blocked. These data indicate that ROS and NF-κβ are activated by JP-8, and these pathways are involved in COX-2 expression and the induction of immune suppression by jet fuel. cutaneous, cytokines, siRNA, volatile organic compounds In the early 1990′s the U.S. Air Force began a conversion to a new jet fuel. Jet propulsion (JP)-4 was gradually replaced by JP-8. JP-8 was refined to have a higher flash point, lower vapor pressure and a lower freezing point than JP-4, to provide a fuel that was less combustible and more explosion proof, more resistant to evaporation during storage, and to provide a fuel that performs well at the higher altitudes required for military operations. JP-8 is a multiuse fuel used in jet and turboprop aircraft, helicopters, tanks, and fighting vehicles, trucks that can run on diesel and some Navy ships. It is estimated that 60 billion gallons of jet fuel are used per year, making potential exposure to jet fuel via the inhalation of vapors and aerosols, or direct contact with the skin after accidental spills, a major chemical exposure problem for military and civilian aviation personnel (reviewed by Ritchie et al., 2003). Standard toxicological screening during development suggested that JP-8 caused minimal adverse effects. Gastric gavage with high doses (oral LD50 = 16 g/kg body weight) resulted in decreased body weights, gastric and perianal irritation, and elevated liver enzyme levels (although livers showed normal histology on necropsy). Little to no other morbidity was reported (Mattie et al., 1995). Feeding JP-8 to pregnant rats did not induce fetal malformation (Cooper and Mattie, 1996). JP-8 was not irritating to the eyes and at best, JP-8 was a weak sensitizer after dermal exposure (Kanikkannan et al., 2000; Kinkead et al., 1992). However, when immune function was measured, JP-8 was found to be an immunotoxicant. Short-term exposure (once a day for 7 days; 100 mg/m3) to aerosolized JP-8, the most common route of jet fuel exposure, suppressed cell-mediated immune reactions. The suppressed state persisted for up to 4 weeks postexposure (Harris et al., 1997a,b, 2000, 2002). Indeed, Harris and colleagues suggested that immune function is more sensitive to JP-8-induced damage because immunotoxicity was generally induced with lower doses, and was found before toxic effects were found in other organ systems (Harris et al., 1997b). A good example of this is the suppression of antibody formation found in pups born to JP-8–fed dams, in the absence of any other developmental abnormalities (Keil et al., 2003). The second major route of jet fuel exposure is through the skin. Using a mouse model of dermal exposure, we found that applying JP-8 to the skin induced immune suppression (Ullrich, 1999). Cell-mediated immune reactions are particularly sensitive to the effects of JP-8. Delayed-type hypersensitivity (DTH), contact hypersensitivity, and T-cell proliferation, but not antibody production were suppressed by dermal JP-8 treatment (Ullrich, 1999; Ullrich and Lyons, 2000). Both primary and secondary immune reactions were suppressed by jet fuel application (Ramos et al., 2002). The activation of cytokine production by JP-8 treatment appears to play an important role in the activation of immune suppressive pathways. Blocking prostaglandin E2 (PGE2) production with a selective COX-2 inhibitor, or neutralizing interleukin (IL)-10 activity with a monoclonal antibody, blocked JP-8–induced immune suppression (Ullrich and Lyons, 2000). Harris and colleagues recently reported that PGE2 and IL-10 are also involved in the immune suppression activated by exposure to aerosolized JP-8 (Harris et al., 2007). Recently, we provided evidence that the aromatic compounds found in jet fuel are the agents responsible for inducing COX-2 expression and activating immune suppression (Ramos et al., 2007). What remains unclear, however, are the JP-8–induced molecular events that lead to cytokine production and subsequent immune suppression. Others have shown jet fuel treatment of cells induces reactive oxygen species (ROS) (Rogers et al., 2001), and in a previous study we were able to block jet fuel–induced immune suppression with several ROS scavengers (Ramos et al., 2004). ROS are known to induce NF-κB activation, a ROS sensitive transcription factor with the ability to induce COX-2 expression (Hadjigogos, 2003). In addition, Espinoza et al. (2006) reported that prolonged NF-κB activation in jet fuel–treated rat alveolar epithelial cells results in the upregulation of pro-inflammatory cytokine mRNA expression. In view of the fact that our previous findings have demonstrated a role for immune regulatory cytokines in the cascade of events leading to JP-8-induced immune suppression, the focus of the experiments presented in this paper was to test the hypothesis that jet fuel–induced ROS turns on NF-κβ, which activates COX-2 expression and drives immune suppression. MATERIALS AND METHODS Mice. Specific pathogen-free female C3H/HeNCr (MTV-) mice were obtained from the National Cancer Institute Frederick Cancer Research Facility Animal Production Area (Frederick, MD). The animals were maintained in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International, in accordance with current regulations and standards of the United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at UTMDACC. Within each experiment all mice were age matched. The mice were 8–10 weeks old at the start of each experiment. Cell lines. The spontaneously transformed mouse keratinocyte cell line, PAM 212 (Yuspa et al., 1980), was obtained from Dr Stuart Yuspa (NCI, Bethesda, MD). Wild-type JB6 keratinocyte cells (Colburn et al., 1979), and those overexpressing the catalase (Amstad and Cerutti, 1990), and the superoxide dismutase (SOD) (Amstad et al., 1997) genes were obtained from the American Type Culture Collection (Manassas, VA). The keratinocytes were grown in 5% fetal bovine serum (FBS) MEM with L-glutamine, 4-2-hydroxyethyl piperazine-1-ethanesulfonic acid (HEPES), and non-essential amino acids in 5% CO2. Jet fuel sources. JP-8 (lot # 3509) was acquired from the Operational Toxicology Branch, Air Force Research Laboratory, Wright Patterson Air Force Base, Dayton, OH. Synthetic jet fuel (S-8), which is similar in composition to JP-8, but devoid of aromatic hydrocarbons (http://www.syntroleum.com/Resources_MSDS.aspx), and does not induce immune suppression when applied to the skin (Ramos et al., 2007), is produced from natural gas using the Fischer-Tropsch reaction (Syntroleum Corporation, Tulsa, OK). The Operational Toxicology Branch supplied us with S-8. The fuels were stored and used in a chemical fume hood. Nitrile rubber based gloves (Touch N Tuff, Fisher Scientific Co, Pittsburgh, PA) were used in place of normal latex gloves due to their superior performance in preventing the penetration of the fuels. Delayed-type hypersensitivity. On day 0, the mice were immunized by the subcutaneous injection of 108 formalin-fixed Candida albicans into each flank. Nine days later, the mice were treated with JP-8 or S-8 as described previously (Ramos et al., 2007). Briefly, 300 μl (240 mg JP-8; 227 mg S-8) of the undiluted fuel was applied to the shaved dorsal skin (≈8 cm2) of the animals. The mice were held individually in the hood for 3 h after exposure to prevent cage mates from grooming and ingesting the fuel. After 3 h, all the residual fuel was either absorbed or evaporated and the animals were returned to standard housing in a specific pathogen-free barrier facility. Previous studies indicated that applying the jet fuel on days 6 through 9 postimmunization would suppress the elicitation of DTH (Ramos et al., 2002). One day after jet fuel treatment, the thickness of each hind footpad was measured with an engineer's micrometer (Mitutoyo, Tokyo, Japan) and then challenged by the intrafootpad injection of 50 μl of Candida antigen (Alerchek, Inc., Portland, ME) (for complete details see Nghiem et al., 2002). Eighteen to 24 h after challenge, the thickness of each footpad was remeasured, and the mean footpad swelling for each mouse was calculated (left footpad + right footpad ÷ 2). The background footpad swelling (negative control) was determined in a group of mice that were not immunized but were challenged. The positive control was measured in mice that were immunized and challenged. There were five mice per group; the mean footpad thickness for the group ± the standard error of the mean was calculated. Statistical differences between the positive control and experimental groups was determined using a one-way ANOVA followed by the Dunnett's Multiple comparison test (GraphPad Prism Software V4, GraphPad Inc, San Diego, CA). Quantitative real-time polymerase chain reaction (RT-PCR). Quantitative RT-PCR (Nolan et al., 2006) was performed as described previously (Ramos et al., 2007). Six hours after the final JP-8 treatment the dorsal skin of the mice was removed and snap frozen in liquid nitrogen. The samples were then pulverized with a mortal and pestle. Total RNA was then extracted with Trizol (Invitrogen, Carlsbad, CA). The isolated RNA was then further purified by using the RNeasy RNA cleanup procedure (Qiagen, Valencia, CA). The concentration of the isolated RNA was measured, and 0.5–1.0 μg was converted to cDNA using the Retroscript RT kit (Ambion, Austin, TX). cDNA (25–50 ng) was subjected to real-time PCR (Model ABI Prism 7500, Applied Biosystems, Foster City, CA) using TaqMan Universal PCR mix, and primers and fluorescent probes specific for COX-2 (Mm 00478374-m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Taqman Gene Expression Assay reagents, Applied Biosystems). Threshold cycle (CT) values for COX-2 were normalized to GAPDH using the following equation: 1.8(GAPDH-COx-2) × 10,000), where GAPDH is the CT of the GAPDH control, COX-2 is the CT of COX-2, and 10,000 is an arbitrary factor to bring all values above one. There were three mice in each treatment group; RNA was isolated from each individual mouse. The mean and the standard deviation for each treatment group was calculated and statistical differences between each experimental group were determined by using a one-way ANOVA followed by the Student-Newman-Keuls Multiple comparison test (GraphPad Prism Software V4, San Diego, CA). In some experiments RNA was harvested from keratinocyte cell lines. The keratinocytes were plated in six-well plates (5 × 105/well) and exposed to jet fuel. The undiluted jet fuel (800 mg/ml) was first diluted in ethanol (1:20 mixture jet fuel in ETOH), and then extensively diluted in tissue culture medium, as described by others (Stoica et al., 2001). Initial dose ranging experiments indicated that 100 μg/ml of JP-8 was the highest dose tolerated with minimal cell toxicity, and was used here. Four to 6 h after treatment the cells were harvested in 1 ml of Trizol. Real-time PCR was performed as described above. There were at least three wells in each treatment group; RNA was isolated from each individual well. The mean and the standard deviation for each treatment group was calculated and statistical differences between each experimental group were determined by using a one-way ANOVA followed by the Student-Newman-Keuls Multiple comparison test. Electrophoretic mobility shift assay (EMSA). The oligonucleotide containing the NF-κβ consensus sequence (Promega, Madison, WI) was end-labeled with T4 polynucleotide kinase using [γ32P] ATP and purified with a G-50 column (Harvard Apparatus, Holliston, MA). Nuclear extracts were prepared using lysis buffer (1.0M HEPES pH 7.9; 2.0M KCl; 0.5M ethylenediaminetetraacetic acid [EDTA], pH 8.0; in sterile water). Protease Inhibitor Set III (Calbiochem, San Diego, CA) was added at 1:100 just prior to the lysis procedure. After lysis the nuclear extraction buffer (1.0M HEPES pH 7.9; 5.0M NaCl; 0.5M EDTA, pH 8.0; 0.1M ethylene glycol tetraacetic acid, pH 7.0; in sterile water) was added. The protease inhibitor was added again just prior to nuclear extraction procedure. Protein concentration was determined by the Bradford dye binding procedure (Bio-Rad Laboratories, Hercules, CA). For binding assays, 5 μg of nuclear extract was added to binding buffer (1M HEPES, pH 7.9; 0.5M EDTA pH 8.0; 1.0M dithiotrietol; 100% glycerol; and 1 μg/μl poly[dI-dC]:poly[dI-dC] in sterile water) and incubated with 32P-labeled DNA probe in a final volume of 30 μl for 30 min at room temperature. The DNA-protein complexes were then resolved by electrophoresis on a 6% acrylamide gel in 0.5× Tris borate ethylene diamine tetra acetic acid buffer. The gel was then dried at 80°C for 45 min. Once dried, the gel was placed in a film cassette overnight at −80°C to visualize the NF-κβ signal. To confirm the specificity of the probe, a competition-binding assay for the consensus sequence was performed by using ×100 unlabeled probe. To identify the subunits of NF-κβ responsible for the DNA binding, a supershift was performed by preincubating the extracts with 1 μl of antibodies specific for p50 or p65 (Santa Cruz Biotechnology, Santa Cruz, CA). Intensity of staining was determined by scanning the gel and measuring the intensity of the bands with NIH ImageJ software (http://rsb.info.nih.gov/nih-image/). Flow cytometric analysis for ROS. 1 × 105 JB6 cells were plated in six-well dishes. When the cells reached 80% confluency they were shifted to serum free media. After 1h, 100 μg/ml of JP-8 or S-8 diluted in ethanol, or 50 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) was added to the cells. One hour later, the cells were washed, trypsinized and collected in 4 ml of a 5% FBS/PBS buffer. The cells were washed and resuspended in 300 μl of 2μM dichlorofluoresceindiacetate (DCF-DA) (Sigma-Aldrich) and incubated for 1 h in the dark. The cells were then washed with 5% FBS/PBS buffer and resuspended to a final volume of 300 μl in 5% FBS/PBS buffer for flow cytometric analysis (FACSCalibur, BD Biosciences, San Jose, CA). Dual luciferase assay. A dual luciferase assay was used to determine the effects of jet fuel treatment on COX-2 transcription. The COX-2 promoter luciferase construct was generated from a PCR product comprising the −1012 to +12 nucleotides (relative to start of transcription) of the mouse COX-2 5′ promoter region ligated into the pGL3-luc firefly luciferase vector (Promega, Madison, WI). Dr Menashe Bar-Eli (M. D. Anderson Cancer Center, Houston, TX) provided us with the control β-actin luciferase construct (mouse β-actin promoter upstream of sea pansy luciferase). PAM 212 cells were plated in triplicate at a concentration of 5 × 104 cells per well in 24-well tissue culture plates and incubated overnight at 37°C in 5% CO2. Plasmids containing the COX-2 promoter were mixed with the β-actin sea pansy luciferase control plasmid, the liposomal transfection reagent lipofectamine, base Opti-MEM culture medium, and then incubated at room temperature for 20 min. Two hundred microliters of the firefly/sea pansy mixture was added to the PAM 212 cells. The plates were then incubated at 37°C. After 4 h, the media was replaced with 500 μl of complete MEM with 5% FCS and incubated at 37°C. After 24 h, the cell monolayers were washed and treated with 100 μg/ml jet fuel or TPA as a control and incubated for an additional 6 h. At the end of the incubation, the cells were lysed, and firefly luciferase activity was determined using a luminometer. Immediately following the luciferase measurement the reaction was quenched, the substrate for the sea pansy luciferase was added, and a second reading was taken. The sea pansy luciferase reading was used to normalize transfection efficiency. The luminescence from jet fuel–treated cultures was compared with the luminescence obtained with control cultures (medium) and the stimulation was expressed as fold increase. Small interfering RNA. JB6 cells were plated at a concentration of 1 × 105 in six-well plates. At 40% confluency the cells were treated with 20, 60, and 100 pmol/ml of p65 small interfering RNA (siRNA) specific for the Rel A component of NF-κβ (Dharmacon RNAi Technologies, Waltham, MA) diluted in serum free media (Opti-MEM) and Oligofectamine in the presence of Plus reagent (Invitrogen), as per the manufacturer's instructions. Nontargeting control siRNA was also acquired from Dharmacon. Nontargeting siRNA was used to evaluate sequence independent events that are associated with siRNA delivery and can lead to cell toxicity and affect cell viability. The cells tolerated the delivery of 20, 60, and 100 pmol/ml of siRNA with minimal cellular stress and excellent viability as evidenced by the fact that almost all keratinocytes remained attached to the tissue culture dish after treatment. Four hours post-siRNA treatment tissue culture media supplemented with 15% FBS was added, as per the manufacturer's instructions. Forty-eight h post-siRNA treatment, the cells were washed to remove cell debris and treated with JP-8. After a 4-h incubation at 37°C, the cells were harvested and RNA and protein was isolated. Western analysis, as described previously (Ramos et al., 2007), was used to measure the effect of the siRNA on p65 protein expression and real-time PCR was used to measure COX-2 expression. RESULTS ROS Inhibitors Block Jet Fuel-Induced COX-2 Activation and the Induction of Immune Suppression Previously, we demonstrated that antioxidant treatment ameliorated JP-8–induced immune suppression, suggesting that ROS are involved (Ramos et al., 2002). To directly test this hypothesis we performed the following experiment. Keratinocyte cultures (PAM 212 cells) were treated with JP-8 or S-8. The generation of ROS was measured with a general ROS indicator dye, aminophenyl fluorescein. As shown in Figure 1A, no fluorescence was observed when the keratinocytes were simply cultured in ethanol diluted in tissue culture medium. Fluorescent cells were noted, however, in cultures treated with JP-8 (Fig. 1B), and in cultures treated with the positive control, TPA (Fig. 1C). To further validate these results we treated another keratinocyte cell line (JB6) with JP-8, S-8, or TPA. These cells were then incubated with 2′,7′-dichlorodihydrofluorescein diacetate, a dye that is oxidized into the highly fluorescent product, dichlorofluorescein, by ROS (Brubacher and Bols, 2001). We then measured the mean fluorescence intensity of the staining by flow cytometry (Fig. 1D). We took advantage of the fact that three variants of JB6 are available, the wild-type cells, and cells overexpressing the catalase (CAT) and SOD genes. ROS was significantly induced (p = 0.001 JP-8 vs. medium control) when the wild-type JB6 were exposed to JP-8. Treating the cells with ethanol, the vehicle for this experiment, or S-8 did not significantly increase ROS production above background. As expected, treating the cells with 50 ng/ml of TPA caused a significant increase (p = 0.001 vs. medium control) in ROS production. When the catalase overexpressing JB6 cells (CAT) were treated with TPA the induction of ROS was significantly increased over the medium-only control (p = 0.001 TPA-treated CAT cells vs. medium-treated CAT cells), but the induction of ROS was less than that seen in the wild-type TPA-treated cells. Treatment with JP-8 also induced a significant increase in ROS production over the control (p = 0.001, JP-8–treated CAT cells vs. medium-treated CAT cells) but here again ROS generation in the CAT cells was much less than what was found in the JP-8–treated wild-type cells. Similarly, when the SOD overexpressing cells were treated with TPA, a significant increase in ROS activity was noted versus the medium-only control (p = 0.01 TPA-treated SOD cells vs. medium-treated SOD cells). Note however, when the SOD cells were treated with JP-8, no significant increase in ROS was noted. These data indicate that JP-8 induces ROS, and that when catalase and SOD activity is overexpressed, JP-8–induced ROS production is significantly suppressed. FIG. 1. Open in new tabDownload slide JP-8 treatment activates ROS production. Mouse keratinocytes were cultured in (A) tissue culture medium; (B) JP-8; (C) TPA; and ROS induction was visualized with a ROS indicator dye. (D) Wild-type keratinocytes, or keratinocytes overexpressing CAT or SOD were cultured in tissue culture medium, or treated with TPA, JP-8, or ETOH diluted in tissue culture medium. ROS production was measured by flow cytometry. The data is expressed as the mean fluorescence intensity of triplicate cultures ± the standard deviation. *p = 0.001 versus medium control; †p = 0.01 versus medium control. Representative experiments are shown; these experiments were reproduced independently two to three times and identical results were obtained. FIG. 1. Open in new tabDownload slide JP-8 treatment activates ROS production. Mouse keratinocytes were cultured in (A) tissue culture medium; (B) JP-8; (C) TPA; and ROS induction was visualized with a ROS indicator dye. (D) Wild-type keratinocytes, or keratinocytes overexpressing CAT or SOD were cultured in tissue culture medium, or treated with TPA, JP-8, or ETOH diluted in tissue culture medium. ROS production was measured by flow cytometry. The data is expressed as the mean fluorescence intensity of triplicate cultures ± the standard deviation. *p = 0.001 versus medium control; †p = 0.01 versus medium control. Representative experiments are shown; these experiments were reproduced independently two to three times and identical results were obtained. Next we asked if ROS could activate COX-2 expression. We used the same three keratinocyte cell lines and once again treated them with JP-8, S-8, medium or ethanol diluted in medium. COX-2 mRNA expression was measured by real-time PCR (Fig. 2A). In wild-type cells, JP-8-treatment caused a significant upregulation of COX-2 mRNA expression (p = 0.001 JP-8–treated wild-type cells vs. medium-treated wild-type cells). Treating the wild-type cells with ethanol diluted in medium, or S-8, did not upregulate COX-2 expression in the wild-type cells. When, however, the catalase overexpressing cells, or the SOD overexpressing cells were treated with JP-8, no significant upregulation of COX-2 mRNA was found. Because catalase and SOD scavenge ROS, these data imply that ROS is activating COX-2 expression. To confirm this observation, mice were treated with JP-8, their skin removed and COX-2 expression in the skin was measured (Fig. 2B). As expected, applying JP-8, but not S-8 to the skin of mice upregulates COX-2 expression (p = 0.001 vs. normal skin). When the antioxidant NAC was injected (ip) into the mice 2 h prior to JP-8 treatment, the induction of COX-2 was suppressed. Simply injecting NAC into mice had no effect on COX-2 expression in the skin. FIG. 2. Open in new tabDownload slide COX-2 upregulation and immune suppression are blocked by ROS scavengers. (A) Wild-type keratinocytes, or keratinocytes overexpressing CAT or SOD were cultured in tissue culture medium, or treated with TPA, JP-8, or ETOH diluted in tissue culture medium. COX-2 expression was measured by real-time PCR. (B) Skin samples were obtained from normal mice, mice treated with S-8, mice treated with JP-8, or mice injected with NAC and then treated with JP-8. COX-2 expression was measured by real-time PCR. The data is expressed as arbitrary units relative to GAPDH expression ± standard deviation of triplicate samples. †p = 0.01 versus COX-2 expression found in normal skin. (C) JP-8 activates the transcription of COX-2. Keratinocyte cultures were transfected with a COX-2 reporter construct and treated with ETOH diluted in tissue culture medium, S-8, TPA, JP-8, JP-8 + Vitamin C, JP-8 + Vitamin E, or JP-8 + Parthenolide. Six hours post-jet fuel treatment luciferase activity was determined. The data are expressed as fold induction versus the sea pansy luciferase reading ± standard deviation of triplicate cultures. †p = 0.01 versus ETOH-treated cultures. (D) Reversal of JP-8-induced immune suppression by antioxidant treatment. Mice were injected with various doses of NAC 2 h prior to jet fuel treatment. The positive control refers to mice that were immunized and challenged; the negative control represents the background response in mice that were simply challenged. Data is expressed as mean footpad swelling ± SEM of groups of five mice. †p = 0.01 versus the positive control. *p = 0.001 versus JP-8-treated mice. Representative experiments are shown; these experiments were reproduced independently two to three times and identical results were obtained. FIG. 2. Open in new tabDownload slide COX-2 upregulation and immune suppression are blocked by ROS scavengers. (A) Wild-type keratinocytes, or keratinocytes overexpressing CAT or SOD were cultured in tissue culture medium, or treated with TPA, JP-8, or ETOH diluted in tissue culture medium. COX-2 expression was measured by real-time PCR. (B) Skin samples were obtained from normal mice, mice treated with S-8, mice treated with JP-8, or mice injected with NAC and then treated with JP-8. COX-2 expression was measured by real-time PCR. The data is expressed as arbitrary units relative to GAPDH expression ± standard deviation of triplicate samples. †p = 0.01 versus COX-2 expression found in normal skin. (C) JP-8 activates the transcription of COX-2. Keratinocyte cultures were transfected with a COX-2 reporter construct and treated with ETOH diluted in tissue culture medium, S-8, TPA, JP-8, JP-8 + Vitamin C, JP-8 + Vitamin E, or JP-8 + Parthenolide. Six hours post-jet fuel treatment luciferase activity was determined. The data are expressed as fold induction versus the sea pansy luciferase reading ± standard deviation of triplicate cultures. †p = 0.01 versus ETOH-treated cultures. (D) Reversal of JP-8-induced immune suppression by antioxidant treatment. Mice were injected with various doses of NAC 2 h prior to jet fuel treatment. The positive control refers to mice that were immunized and challenged; the negative control represents the background response in mice that were simply challenged. Data is expressed as mean footpad swelling ± SEM of groups of five mice. †p = 0.01 versus the positive control. *p = 0.001 versus JP-8-treated mice. Representative experiments are shown; these experiments were reproduced independently two to three times and identical results were obtained. Next we measured the effect jet fuel had on the transcription of COX-2 by transfecting a COX-2 promoter construct driving the luciferase gene into cells and measuring its activation by JP-8 (Fig. 2C). Treating the cells with S-8 did not increase COX-2 transcription. Both TPA and JP-8 caused a significant increase (p = 0.01 vs. the ethanol treated control) in COX-2 transcription. Treating the cells with the antioxidants, Vitamin C and Vitamin E, blocked the JP-8–induced increase in COX-2 transcription. This indicates that the increase in ROS drives both COX-2 mRNA expression and COX-2 transcription. In addition, treating the cells with parthenolide (Parth), an NF-κβ inhibitor (Hehner et al., 1999), also blocked COX-2 transcription. Finally we examined the effect that a free radical scavenger had on JP-8-induced immune suppression. Mice were pretreated with NAC (10–1000 nmol; ip injection). Two h later, the mice were treated with JP-8, and the effect NAC had on immune suppression is shown in Figure 2D. As expected, JP-8 treatment caused immune suppression (p = 0.01 vs. positive control). Treating the mice with 100 nmol of NAC totally overcame the immune suppression, as there was no statistical difference in the response found in mice treated with JP-8 + 100 nmol NAC and the positive control (p > 0.05). Treating the mice with 10 and 1000 nmol NAC had a partial restorative effect on immune suppression. The DTH response in mice pretreated with 10 and 1000 nmol NAC was still suppressed (i.e., significantly different from the positive control, p = 0.01), but significantly different from the response found in JP-8 only treated mice (p = 0.001). NAC by itself did not interfere with the immune response, as a substantial DTH reaction was found in mice injected with only NAC. In total, these data indicate that JP-8 treatment, both in vitro and in vivo, induces ROS, which play a role in the induction of COX-2. Blocking the induction of ROS prevents COX-2 activation and prevents the induction of immune suppression. A Role for NF-κβ Activation in JP-8–Induced COX-2 Activation and Immune Suppression A number of transcription factors, including NF-κβ are responsive to ROS, and NF-κB dependent COX-2 induction has been documented (Gloire et al., 2006). Having shown that JP-8 induces ROS in keratinocyte cell lines and mouse skin, we next wanted to determine the role of NF-κβ in COX-2 expression. In the first set of experiments we used EMSA to demonstrate the activation of NF-κβ in the skin following JP-8 application. Mice were treated with 240 mg of JP-8, and 4 h later, epidermal cell suspensions were prepared as described previously (Fukunaga et al., 2008). Nuclear protein extracts were prepared and the binding to a consensus NF-κβ probe was measured (Fig. 3A). Compared with the response found in normal skin, JP-8-treatment caused an increase in NF-κβ activity (lanes 1 vs. 4). The mobility of the NF-κβ band was super-shifted when the complexes were incubated with antibodies to p65 (lane 5) or p50 (lane 6). As a control, some mice were treated with S-8. Compared with normal skin, S-8–induced minimal NF-κβ binding activity (lane 1 vs. 7). The EMSA was repeated in mice injected with an antioxidant (NAC) or the NF-κβ inhibitor parthenolide (Fig. 3B). As before, JP-8 upregulated p65 binding activity and S-8 did not (lane 3 vs. lanes 1 and 5). Incubating the cells with 100-fold excess of the unlabeled consensus NF-κβ probe totally blocked p65-binding activity (lane 3 vs. 4). We also found that treating the mice with NAC or parthenolide decreased p65 binding activity (lane 3 vs. lanes 7 and 9). The gel (Fig. 3B) was then scanned and the relative intensity of each band was quantified using a gel analysis software (Fig. 3C), confirming that NAC or parthenolide treatment decreased the intensity of p65 binding. FIG. 3. Open in new tabDownload slide NF-κβ activation in epidermal cells isolated from JP-8-treated mice. (A) Epidermal cell suspensions were prepared from normal mice (lanes 1–3), JP-8-treated mice (lanes 4–6) and S-8-treated mice (lanes 7–9). Nuclear protein extracts were prepared and DNA binding activity was measured by EMSA. Supershift experiments using antibodies to p65 (lanes 2, 5, 8) and p50 (lanes 3, 6, 9) are shown. (B) Parthenolide and NAC block NF-κβ activation. Epidermal cell suspensions were prepared from normal mice (No TrT, lanes 1–2), JP-8–treated mice (lanes 3–4) and S-8–treated mice (lanes 5–6), mice pretreated with parthenolide before JP-8 treatment (lanes 7–8) or mice pretreated with NAC before JP-8 treatment (lanes 9–10). Some mice were only treated with parthenolide (lanes 11–12) or NAC (lanes 13–14). Nuclear protein extracts were prepared and DNA binding activity was measured by EMSA. Supershift was visualized with antibodies to p65. All even numbered lanes were incubated with 100-fold excess of unlabeled probe. (C) Intensity of staining was determined by scanning the gel and measuring relative intensity with ImageJ. FIG. 3. Open in new tabDownload slide NF-κβ activation in epidermal cells isolated from JP-8-treated mice. (A) Epidermal cell suspensions were prepared from normal mice (lanes 1–3), JP-8-treated mice (lanes 4–6) and S-8-treated mice (lanes 7–9). Nuclear protein extracts were prepared and DNA binding activity was measured by EMSA. Supershift experiments using antibodies to p65 (lanes 2, 5, 8) and p50 (lanes 3, 6, 9) are shown. (B) Parthenolide and NAC block NF-κβ activation. Epidermal cell suspensions were prepared from normal mice (No TrT, lanes 1–2), JP-8–treated mice (lanes 3–4) and S-8–treated mice (lanes 5–6), mice pretreated with parthenolide before JP-8 treatment (lanes 7–8) or mice pretreated with NAC before JP-8 treatment (lanes 9–10). Some mice were only treated with parthenolide (lanes 11–12) or NAC (lanes 13–14). Nuclear protein extracts were prepared and DNA binding activity was measured by EMSA. Supershift was visualized with antibodies to p65. All even numbered lanes were incubated with 100-fold excess of unlabeled probe. (C) Intensity of staining was determined by scanning the gel and measuring relative intensity with ImageJ. We next measured the effect of parthenolide, an NF-κβ inhibitor, on JP-8–induced COX-2 induction and JP-8–induced immune suppression (Fig. 4). As noted above, JP-8 treatment induced COX-2 expression in the skin. Parthenolide, in a dose-dependent fashion, caused a decrease in JP-8-induced COX-2 expression. Parthenolide by itself did not affect COX-2 expression in the skin (Fig. 4A). Parthenolide also blocked the transcription of the COX-2 gene (Fig. 2C). In Figure 4B, the effect of parthenolide on JP-8–induced immune suppression is shown. Treating mice with JP-8–induced immune suppression (p = 0.01 vs. the positive control), and parthenolide, at the two higher doses tested, totally reversed JP-8–induced immune suppression (p > 0.05 vs. the positive control). Finally, we asked if parthenolide, at the doses used here, blocks ROS activity. Keratinocytes that were treated with JP-8 only or JP-8 + parthenolide showed a comparable increase in ROS activity (p = 0.001 vs. ETOH control), and there was no significant difference in the ROS induction between the JP-8 only and JP-8 + parthenolide treated cells (p > 0.05). These data indicate that jet fuel treatments activates NF-κβ activity and that blocking the NF-κβ pathway blocks JP-8-induced COX-2 expression and JP-8–induced immune suppression. They also confirm that parthenolide blocks NF-κβ activation but has no effect on ROS induction in vitro. FIG. 4. Open in new tabDownload slide Parthenolide blocks JP-8-induced COX-2 expression and immune suppression. (A) Skin samples were acquired from normal mice, mice treated with TPA, mice treated with JP-8, mice injected with parthenolide and then treated with JP-8, or mice treated with parthenolide only. COX-2 expression was measured by real-time PCR. The data is expressed as arbitrary units relative to GAPDH expression ± standard deviation of triplicate samples. †p = 0.01 versus COX-2 expression found in normal skin. (B) Parthenolide blocks the induction of immune suppression. Mice were treated with JP-8 only or injected with various dose of parthenolide 2 h prior to JP-8 treatment. The positive control refers to mice that were immunized and challenged; the negative control represents the background response in mice that were simply challenged. Data is expressed as mean footpad swelling ± SEM of groups of five mice. †p = 0.01 versus the positive control. (C) Parthenolide does not interfere with JP-8–induced ROS production. Keratinocytes cultures were treated with treated with ETOH diluted in ethanol, S-8, TPA, JP-8, and JP-8 + Parthenolide. ROS induction was measured by flow cytometry. The data are expressed as the mean fluorescence intensity of triplicate cultures ± the standard deviation. *p = 0.001 versus ETOH control; †p = 0.01 versus ETOH control. Representative experiments are shown; these experiments were reproduced independently two to three times and identical results were obtained. FIG. 4. Open in new tabDownload slide Parthenolide blocks JP-8-induced COX-2 expression and immune suppression. (A) Skin samples were acquired from normal mice, mice treated with TPA, mice treated with JP-8, mice injected with parthenolide and then treated with JP-8, or mice treated with parthenolide only. COX-2 expression was measured by real-time PCR. The data is expressed as arbitrary units relative to GAPDH expression ± standard deviation of triplicate samples. †p = 0.01 versus COX-2 expression found in normal skin. (B) Parthenolide blocks the induction of immune suppression. Mice were treated with JP-8 only or injected with various dose of parthenolide 2 h prior to JP-8 treatment. The positive control refers to mice that were immunized and challenged; the negative control represents the background response in mice that were simply challenged. Data is expressed as mean footpad swelling ± SEM of groups of five mice. †p = 0.01 versus the positive control. (C) Parthenolide does not interfere with JP-8–induced ROS production. Keratinocytes cultures were treated with treated with ETOH diluted in ethanol, S-8, TPA, JP-8, and JP-8 + Parthenolide. ROS induction was measured by flow cytometry. The data are expressed as the mean fluorescence intensity of triplicate cultures ± the standard deviation. *p = 0.001 versus ETOH control; †p = 0.01 versus ETOH control. Representative experiments are shown; these experiments were reproduced independently two to three times and identical results were obtained. Because most inhibitors, such as parthenolide, are selective but not always specific in their mode of action, we decided to confirm the results described above with siRNA (Fig. 5). Keratinocytes (JB6 cells) were transfected with 20–100 pmol of siRNA specific for the Rel A component (p65) of NF-κβ. Western blotting (48-h post-siRNA treatment) confirmed that transfecting the cells with 60 and 100 pmol of p65 siRNA substantially suppressed the expression of the p65 protein (Fig. 5A). The relative intensity of each band was measured, and this analysis indicated that transfecting the cells with 60 pmol of p65 siRNA resulted in over 80% suppression of protein expression. To test whether the p65 knockdown has an effect on jet fuel–induced COX-2 expression, the keratinocytes were transfected with 60 pmol of p65 siRNA and 48 h later, treated with JP-8. Four h after JP-8-treatment the cells were harvested and COX-2 expression was measured by real-time PCR. Treating the cells with nontargeting siRNA, or the p65 specific siRNA in the absence of JP-8, did not induce COX-2 expression (p > 0.05 vs. medium-only control). On the other hand, treating the cells with JP-8 or JP-8 and the nontargeting siRNA, resulted in a substantial increase in COX-2 expression, one that was significantly different (p = 0.01) from the COX-2 expression found in the medium-only treated control. We noted a 58% decease in COX-2 mRNA expression in cells treated with JP-8 and incubated with p65 specific siRNA (p = 0.001 vs. JP-8–treated control). These data further confirm that the NK-κB pathway is involved in the molecular events that lead to JP-8-induced COX-2 expression. FIG. 5. Open in new tabDownload slide p65 specific siRNA blocks JP-8-induced COX-2 upregulation. (A) Keratinocyte cultures were cultured in medium, treated with a nontargeting siRNA construct (NT-siRNA) or treated with different amounts of p65 specific siRNA. Western analysis was performed to document inhibition of p65 protein expression. Equal gel loading was confirmed by staining with an antibody specific for β-actin. (B) The gel was scanned and the relative intensity of each band was analyzed using ImageJ. (C) COX-2 expression in p65 siRNA treated cells. Keratinocytes were treated with medium or JP-8 in the presence or absence of p65 specific siRNA or nontargeting siRNA. COX-2 expression was measured by real-time PCR. The data is expressed as arbitrary units relative to GAPDH expression ± standard deviation of triplicate samples. †p = 0.01 versus COX-2 expression found in medium-only treated cells. ‡p = 0.01 versus JP-8–treated cells. Representative experiments are shown; these experiments were reproduced independently twice and identical results were obtained. FIG. 5. Open in new tabDownload slide p65 specific siRNA blocks JP-8-induced COX-2 upregulation. (A) Keratinocyte cultures were cultured in medium, treated with a nontargeting siRNA construct (NT-siRNA) or treated with different amounts of p65 specific siRNA. Western analysis was performed to document inhibition of p65 protein expression. Equal gel loading was confirmed by staining with an antibody specific for β-actin. (B) The gel was scanned and the relative intensity of each band was analyzed using ImageJ. (C) COX-2 expression in p65 siRNA treated cells. Keratinocytes were treated with medium or JP-8 in the presence or absence of p65 specific siRNA or nontargeting siRNA. COX-2 expression was measured by real-time PCR. The data is expressed as arbitrary units relative to GAPDH expression ± standard deviation of triplicate samples. †p = 0.01 versus COX-2 expression found in medium-only treated cells. ‡p = 0.01 versus JP-8–treated cells. Representative experiments are shown; these experiments were reproduced independently twice and identical results were obtained. DISCUSSION As mentioned above, JP-8 is used by all branches of the US military and is being promoted as the universal military fuel to ease supply and logistics problems in the future (Ritchie et al., 2003). Jet-A is used to fuel commercial airliners. From the point of immune suppression, there is no difference between the immunosuppressive properties, nor the mechanism of action of JP-8 and Jet-A (Ramos et al., 2002, 2004, 2007). This suggests that exposure to jet fuel by military and commercial aviation personnel represent a serious chemical risk exposure problem. Because of its widespread use, total risk avoidance is probably impractical. Instead, we suggest that a better understanding of the mechanisms underlying jet fuel-induced immune suppression is important for the design of rational therapies to overcome jet fuel-induced immunotoxicity. Therefore, the focus of the experiments presented here was to gain a better understanding of the molecular pathways activated by JP-8 that lead to COX-2 expression and immune suppression. We tested the hypothesis that ROS and NF-κβ play an important and essential role in COX-2 activation. We focused on COX-2 induction because our previous work demonstrated that using a selective COX-2 inhibitor to interfere with the enzymatic activity of COX-2 blocked JP-8–induced immune suppression (Ramos et al., 2002; Ullrich and Lyons, 2000). Our findings confirm that treating keratinocytes with JP-8 activates the ROS pathway (Fig. 1). Further when we treated keratinocytes that overexpressed the ROS scavengers, catalase or SOD, ROS induction was significantly depressed. Similarly, when keratinocytes that overexpressed the ROS scavengers were treated with JP-8, COX-2 expression was substantially depressed. We also demonstrated that antioxidant treatment blocked JP-8–induced COX-2 transcription, and mRNA expression in vivo, and blocked JP-8–induced immune suppression (Fig. 2). Because ROS activates NF-κβ, and in light of the importance of this transcriptional activator in COX-2 activation, we also measured the role of NF-κβ in JP-8–induced COX-2 production and immune suppression. Treating mouse skin with JP-8 activated NF-κβ, and prior treatment with parthenolide; an NF-κβ inhibitor blocked its activation (Fig. 3). Moreover, in vivo treatment with parthenolide partially inhibited JP-8–induced COX-2 expression and totally reversed JP-8–induced immune suppression (Fig. 4). Parthenolide treatment also partially blocked the transcription of the COX-2 gene (Fig. 2C). The complete reversal of immunosuppression by parthenolide, despite only a partial effect on COX-2 transcription and expression may be due to effects of parthenolide on pathways other than NF-κβ. For example, parthenolide kills leukemic cells by activating p53 and heme oxygenase-1 (Guzman et al., 2007), and inhibits mast cell degranulation by inhibiting microtubule formation in an NF-κβ–independent manner (Miyata et al., 2008). Alternatively, it is possible that total shut down of COX-2 transcription and expression by parthenolide is not required to block JP-8–induced immunosuppression. For these reasons we confirmed the role of NF-κβ in activating COX-2 in our studies by showing suppression of JP-8-induced COX-2 induction in keratinocytes treated with Rel A (p65)–specific siRNA (Fig. 5). These data indicate that ROS generation and NF-κβ activation play an important role in activating COX-2 expression and immune suppression following dermal JP-8 treatment. On the other hand, we saw no effect of parthenolide, at the doses and conditions used in our experiments, on ROS activation (Fig. 4C). We suggest the following scenario: jet fuel interacts with keratinocytes in the skin and activates ROS production. ROS then activates NF-κB. NF-κB then induces COX-2 mRNA expression and transcription. This leads to the production and secretion of PGE2, which is an essential step in immune suppression. When we use free radical scavengers, such as Vitamin C and NAC to block immune suppression, we are blocking an early step in the pathway, ROS production. When we use parthenolide or p65 siRNA to block NF-κβ activation, we are interfering with a later step in the pathway leading to COX-2 expression. Although we primarily focused on COX-2 expression in these experiments, it is known that other cytokines and immunomodulatory factors, such as PAF are involved in JP-8-induced immune suppression (Ramos et al., 2004). Others have shown that ROS induces PAF (Alappatt et al., 2000) and PAF induces NF-κβ (Ko et al., 2002), so the pathways we identified here are probably involved in PAF production and activation of COX-2 transcription following JP-8 treatment. We found little activation of NF-κβ (Fig. 3B), COX-2, and/or immune suppression (Ramos et al., 2007) when we used synthetic jet fuel (S-8), which is devoid of aromatic hydrocarbons. Others however, have demonstrated that treating cultured human epidermal keratinocytes with S-8 induces the production of the inflammatory cytokines IL-6 and IL-8, and that cytokine production can be inhibited by parthenolide, implying a role for NF-κβ (Inman et al., 2008). We suggest that the differences between our results and those reported by Inman and colleagues could be attributed to a number of factors. First, we measured immune suppression in intact animals, and we measured COX-2 expression and NF-κβ activation in skin samples taken from jet fuel-treated mice, whereas Inman and colleagues used cultured keratinocytes. The differential penetration of aliphatic and aromatic compounds through intact skin (Baynes et al., 2001; McDougal and Robinson, 2002) may contribute to the different results. Second, we directly tested the effect of parthenolide on NF-κβ activation (Fig. 3), whereas Inman et al assumed that parthenolide suppressed NF-κβ activation based on data reported in the literature. The known promiscuity of most inhibitors makes it entirely possible that parthenolide was inhibiting a pathway other than NF-κβ to suppress IL-6 and IL-8 production. This is why the experiments presented in Figure 5, using p65 specific siRNA to confirm that the NF-κβ pathway is actually involved in COX-2 expression are critical to support the conclusions arrived at here. The two most prevalent routes of jet fuel exposure are the skin and the respiratory tract. Interestingly, the features of the immune suppression that occurs after exposure of the skin and the lung are very similar and we suggest that similar mechanisms may be involved. Cell-mediated immune reactions are preferentially suppressed (Harris et al., 2000; Ullrich and Lyons, 2000), a short exposure to JP-8 can result in long-term effects (Harris et al., 1997b; Ullrich and Lyons, 2000), and immune modulatory cytokines such as IL-10 and PGE2 are involved in mediating the immune suppression (Harris et al., 2007; Ramos et al., 2002; Ullrich and Lyons, 2000). Here also, JP-8-induced NF-κβ activation has been reported to play an essential role in jet fuel-induced toxicity via the skin (data reported here) and the lung (Espinoza et al., 2006). JP-8 treatment of lung epithelial cells induces ROS and activates NF-κβ, which was associated with the upregulation of pro-inflammatory cytokines, such as IL-6 and TNF-α. We suggest that NF-κβ inhibitors may be added to the list of agents (selective COX-2 inhibitors, antioxidants, and anti-IL-10) that will block jet fuel-induced toxicity induced by pulmonary and dermal exposure. It should be noted, however, that all of the data demonstrating that JP-8 and Jet-A induce immune suppression comes from animal studies. Selgrade (2007) recently asked if rodent immunotoxicity data is relevant for extrapolating risk to humans. She concluded that immune suppression in rodents is predictive of immune suppression in humans. Although we agree with Selgrade and suggest that the results from the animal studies may be used to predict that jet fuel could be an immunosuppressive agent in humans, the lack of studies demonstrating an effect of jet fuel, either positive or negative, on the human immune system remains a critical gap in our knowledge. Studies to directly address this question are needed and represent the next important step in determining jet fuel toxicity. In summary, our findings indicate that applying jet fuel to the skin causes the production of ROS, which activates NF-κβ, which then activates COX-2, induces the secretion of PGE2, and drives immune suppression. We demonstrate that agents that block this molecular cascade of events prevent JP-8–induced immune suppression in vivo. In view of the fact that total avoidance of JP-8 is probably impossible, these findings present new approaches to preventing JP-8–induced immune suppression after accidental exposure. FUNDING U.S. Air Force Office of Scientific Research grant (FA9550-05-1-402); National Cancer Institute grants (CA131207 and CA112660); USAF Institute of Technology scholarship supported G.R.; and Cancer Center Support Grant from the National Cancer Institute (CA 16672) supported animal and histology facilities at the University of Texas, MD Anderson Cancer Center. The views and opinions expressed here are those of the authors and do not reflect the official policy or position of the United States Air Force. We thank Nasser Kazimi for his help with the animal experiments, and acknowledge with thanks Dr Chengming Zhu (Department of Immunology, UTMDACC), for unlimited access to his supply of γ32P [ATP]. References Alappatt C , Johnson CA , Clay KL , Travers JB . Acute keratinocyte damage stimulates platelet-activating factor production , Arch. Dermatol. Res. , 2000 , vol. 292 (pg. 256 - 259 ) Google Scholar Crossref Search ADS PubMed WorldCat Amstad P , Cerutti P . 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TI - JP-8 Induces Immune Suppression via a Reactive Oxygen Species NF-κβ–Dependent Mechanism
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfn262
DA - 2009-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/jp-8-induces-immune-suppression-via-a-reactive-oxygen-species-nf-t2BiWx0ENb
SP - 100
EP - 109
VL - 108
IS - 1
DP - DeepDyve
ER -