TY - JOUR
AU - Lyons, Heather J.
AB - Abstract Dermal application of JP-8 jet fuel induces immune suppression. Classic delayed-type hypersensitivity as well as the induction of contact hypersensitivity to allergens applied to the shaved skin of JP-8-treated mice is suppressed. In addition, the ability of T cells isolated from JP-8-treated mice to proliferate in vitro is suppressed. Here we focused on further characterizing the immunotoxicity induced by JP-8 exposure and determining the mechanism involved. Suppression of T-cell proliferation was first noted 3 to 4 days after a single JP-8 treatment and lasted for approximately 3 weeks, at which time T-cell proliferation returned to normal. Cellular immune reactions appear to be more susceptible to the immunosuppressive effects of JP-8, as antibody production in JP-8-treated mice was identical to that found in normal controls. The mechanism through which dermal application of JP-8 suppresses cell-mediated immune reactions appears to be via the release of immune biological-response modifiers. Blocking the production of prostaglandin E2 with a selective cyclooxygenase-2 inhibitor abrogated JP-8-induced immune suppression. Neutralizing the activity of interleukin-10 with a highly specific monoclonal antibody also blocked JP-8-induced immune suppression. Furthermore, injecting JP-8-treated mice with recombinant interleukin-12, a cytokine that drives cell-mediated immune reactions in vivo, overcame the immunotoxic effects of JP-8 and restored immune function. These data indicate that immune suppressive cytokines, presumably produced by JP-8-treated epidermal cells, are responsible for immune suppression in JP-8-treated mice and that blocking and/or neutralizing their production in vivo overcomes the immunotoxic effects of JP-8. jet fuel, JP-8, immune suppression, prostaglandin E2, interleukin-10, interleukin-12, cytokines, immunotoxicity During the past decade, the United States Air Force, the air forces of the NATO allies, and the Japanese Self-Defense Forces have been gradually converting to a new jet propellant, JP-8. Commercial and military jet fuels are complex mixtures of hydrocarbons. Because they are produced from petroleum, their exact composition is not rigorously defined, but rather the product is refined to meet a series of performance specifications. JP-8 is refined to have a higher flash point, lower vapor pressure and lower freezing point to provide a safer, less combustible fuel that performs at high altitudes and minimizes evaporative losses during storage and handling. Initial screening suggested that JP-8 exposure induced minimal toxicity (Cooper and Mattie, 1996; Kinkead et al., 1992; Mattie et al.,1995). However, as more and more Air Force bases converted to JP-8 reports of health problems, including nausea, headaches, fatigue, blocked nasal passages, increased incidence of skin irritation and reports of ear infections prompted a systematic review of JP-8-induced toxicity. It is now clear that JP-8 has toxic side effects, particularly pulmonary dysfunction (Hays et al., 1995; Pfaff et al., 1995, 1996) and a subtle effect on neurologic function (Smith et al., 1997). JP-8 is also immunotoxic, and it appears that immune function is very sensitive to its toxic effects. Short-term pulmonary exposure to relatively low doses of aerosolized JP-8 (100 mg/m3 for 7 days) decreased immune organ weights, decreased the number of viable cells recovered, and suppressed the ability of T cells to respond to mitogenic stimulation (Harris et al., 1997b). The effect of JP-8 on immune organ weights was noted 24 h after treatment, and the suppressed T cell mitogenesis persisted for up to 4 weeks post-exposure (Harris et al., 1997a). The immunotoxic effects of JP-8 were noted after dosing schedules that failed to induce pulmonary toxicity, leading Harris et al. (1997a) to suggest that immune function is the most sensitive indicator of JP-8-induced toxicity. Our observation that a single dermal application of JP-8 is immune suppressive supports this hypothesis (Ullrich, 1999). Besides inhalation of aerosolized vapors, the other major route of JP-8 exposure is dermal contact. Dermal application of undiluted JP-8 to mice, either multiple small exposures (50 μl for 5 days) or a single large exposure in excess of 250 μl resulted in immune suppression. The induction of contact hypersensitivity (CHS) was suppressed regardless of whether the contact allergen was applied directly to the jet fuel-treated skin or applied at a distant untreated site. In addition, the ability of splenic T cells from JP-8-treated mice to proliferate in response to plate-bound anti-CD3 monoclonal antibody was significantly suppressed. The mechanism underlying the induction of systemic immune suppression by dermal application of JP-8 is unclear, but the appearance of the immune modulatory cytokine, interleukin (IL)-10 in the serum of JP-8-treated mice (Ullrich, 1999) suggests that immune-suppressive biological-response modifiers may play a role. The focus of the experiments reported here was to test the hypothesis that the systemic immune suppression observed following dermal exposure to JP-8 is driven by immune modulatory cytokines. We concentrated on prostaglandin E2 (PGE2) and IL-10 because they are known to suppress cell-mediated immune reactions and are secreted by epidermal cells. MATERIALS AND METHODS Mice. Specific pathogen-free female C3H/HeNCr (MTV-) mice were obtained from the National Cancer Institute`s 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 Departments of Agriculture and of Health and Human Services, and the National Institutes of Health. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee. Within each experiment, all mice were age- and sex-matched. The mice were 8–10 weeks old at the start of each experiment. Application of JP-8. The jet fuel (lot # 3509) was supplied to us by the Operational Toxicology Branch, Air Force Research Laboratory, Wright Patterson Air Force Base, Dayton, OH. The fuel was stored and used in a chemical fume hood. Nitrile rubber-based gloves (Touch N Tuff, Fisher Scientific Co.) were used in the place of normal latex gloves, due to their superior performance in preventing the penetration of JP-8. The standard protective clothing worn in the SPF-barrier animal facility, Nytex coveralls, surgical bonnets, and masks also afforded protection for the investigators against accidental dermal exposure to JP-8. Different amounts of the undiluted fuel (50 to 300 μl) were applied directly to the dorsal skin 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. Also, the jet fuel was placed high up on the back of each mouse, immediately behind the head, to prevent the animals from grooming themselves and ingesting the fuel. In every experiment a control group treated with acetone was handled in the identical manner. After 3 h all the residual fuel was either absorbed or evaporated, and the animals were returned to standard housing in an SPF barrier facility. T-cell proliferation in vitro. At various times after JP-8 treatment, the mice were killed, their spleens removed, and single cell suspensions prepared. Contaminating erythrocytes were lysed with ammonium chloride (0.83% in 0.01 M Tris–HCl, pH 7.2); and the cells were washed and resuspended in RPMI-1640 supplemented with 5% newborn calf serum (HyClone, Logan, UT). A T-cell enriched population was prepared by passing the spleen cells over a nylon wool column (Julius et al., 1973). The cells were then resuspended in medium supplemented with 10% newborn calf serum, 5 × 10–5 M 2-mercaptoethanol, 100 units/ml streptomycin, 2 mM L-glutamine, 10% sodium pyruvate, 10 mM HEPES buffer, 1 × vitamins and 1 × non-essential amino acids (GIBCO-BRL, Grand Island, NY). The cells (2 × 105/well) were then cultured at 37°C in an atmosphere of 5% CO2, 95% air for 5 days in 96-well tissue culture plates coated with monoclonal anti-CD3 (100 μl of a 10 μg/ml solution overnight prior to culture; PharMingen, San Diego, CA). During the last 18 h of culture, 1 μCi of tritiated thymidine (ICN Radiochemicals, Irvine, CA) was added to each well. The cells were harvested onto glass-fiber filters (Tomtec Harvester, Orange, CT) and the incorporation of the radioisotope into newly synthesized DNA was determined by liquid scintillation counting (1205 Betaplate, LKB Wallac, Gaithersburg, MD). Background responses were determined by culturing the cells in wells devoid of anti-CD3. Cells from each different group were cultured in triplicate. The means and standard deviations of the triplicates were calculated, and statistical differences between controls (acetone-treated) and experimental (JP-8-treated) groups were determined by use of a 2-tailed Student's t-test, with a probability of less than 0.05 considered significant. Each experiment was repeated 3 times. Effect of JP-8 on antibody production in vivo. Various amounts of JP-8 (100 to 300 μl) were applied to the dorsal skins of the mice. Three h later the animals were immunized by subcutaneous injection of 100 μg of keyhole-limpet hemocyanin (KLH) and emulsified in complete Freund's adjuvant (Pierce Immunochemicals, Rockford, IL). One week later the JP-8 application was repeated, and the mice were boosted with 100 μg of KLH emulsified in incomplete Freund's adjuvant. One week after the boost, the mice were deeply anesthetized and exsanguinated, and the serum was collected. An enzyme linked immunoabsorbent assay (ELISA) was used to measure antibody titers (Ullrich and Fidler, 1992). Triplicate serum samples (diluted 1:100, 1:1000, and 1:10,000 in PBS) from the JP-8-treated and acetone-treated controls were added to the wells of a 96-well ELISA dish coated with KLH and incubated at 37°C for one h. After washing, the amount of KLH-specific antibody bound to the antigen was measured by using biotinylated rat anti-mouse IgM, IgG1, and IgG2b (PharMingen, Inc., San Diego, CA) as the detecting antibodies. Generally, 100 μl of a 1:2000 dilution of each antibody was added to the wells and incubated for 90 min at room temperature. After washing, 100 μl of a 1:10,000 dilution of horseradish peroxidase-conjugated streptavidin (Pierce Immunochemicals) was added to each well. Thirty min later the plates were washed, and 100 μl of the substrate (2,2′-azino-bis(3-ethylbenzthiazoline-o-sulfonic acid, Pierce Immunochemicals) was added. Color was allowed to develop for 30 min, at which time the optical density at 410 nm was measured with a Dynatech MR 5000 Microplate reader (Dynatech Labs, Chantilly, VA). Positive-control mice were shaved and treated with acetone before immunization. Negative controls were shaved and treated with acetone but were not immunized. There were 5 mice per group and the serum from each animal was assayed individually. The data represent the mean optical density and standard deviations from the group of 5 mice. Statistically significant differences between the antibody titers in the controls and the JP-8-treated mice were determined by use of the Student's t-test, with a probability of less than 0.05 considered significant. Each experiment was repeated at least 3 times. Contact hypersensitivity (CHS). The dorsal hair of the mice was removed with electric clippers, and a micropipete used to apply the JP-8 directly to the dorsal skin, as described above. Control mice were shaved and treated with acetone. Three h later, the mice were sensitized by painting 50 μl of a 0.3% (w/v) solution of dinitroflurobenzene (DNFB) in acetone onto the shaved abdominal skin. Six days after sensitization the thickness of each ear was measured, recorded, and the mice were challenged by applying 10 μl of a 0.2% solution of DNFB in acetone to each ear. Eighteen to 24 h later, the thickness of each ear was measured again, and a mean ear thickness for each mouse (left ear thickness + right ear thickness ÷ 2) was calculated. The specific ear swelling for each mouse was then calculated as described above. There were 5 mice per group; the data is expressed as specific ear swelling ± the standard deviation. Statistical differences between the controls and experimental groups were determined by use of a 2-tailed Student's t-test, with a probability of less than 0.05 considered significant. Each experiment was repeated at least 3 times. To determine whether prostaglandin E2 or IL-10 production was involved in the induction of immune suppression, we used monoclonal antibodies or specific enzyme inhibitors to neutralize their activity or block their production in vivo. To block PGE2 secretion in vivo, the selective cyclooxygenase-2 (COX-2) inhibitor, SC 236, was employed (a gift from Dr. Peter Isakson, G. D. Searle & Company, St. Louis, MO). SC 236 selectively inhibits the enzymatic action of COX-2, thus preventing the production of PGE2. Because SC 236 has no effect on the enzymatic activity of COX-1, its use is devoid of the side effects (i.e., gastric bleeding) usually associated with in vivo use of non-specific inhibitors of the cyclooxygenase pathway (Seibert et al., 1994). Generally, SC236 was diluted in PBS and 0.1 ml of a 2 μg/ml solution was injected ip 2 h prior to JP-8-treatment. Interleukin-10 activity was neutralized in vivo by injecting the JP-8-treated mice with 100 μg of monoclonal anti-IL-10 (JES5-2A5.11, rat IgG). The hybridoma-secreting anti-IL-10 was provided to us by Dr. Anne O'Garra (DNAX Research Institute, Palo Alto, CA). The hybridoma cells were grown in RPMI-1640 tissue-culture medium supplemented as described above. The supernatants were collected, the IgG fraction was enriched by 33% ammonium sulfate precipitation, and the IgG was purified by passage over protein A/G columns (Pierce Immunochemicals). Protein concentration was determined by use of bicinchoninic acid (BCA protein assay kit, Pierce Immunochemicals). Control rat IgG was purchased from Sigma (St. Louis, MO). Dr. Stanley Wolf, Genetics Institute Inc., Cambridge, MA generously provided us with recombinant IL-12. It was diluted in PBS prior to use. RESULTS Dermal application of JP-8 induces immune suppression. As shown previously, applying JP-8 to mouse skin 3 h prior to immunization suppressed the induction of CHS in vivo (Ullrich, 1999). In all our previous experiments, undiluted JP-8 was applied directly to the skin because this most closely models the situation found in the field where, during the course of their duties, Air Force personnel come into direct contact with undiluted jet fuel. However, since the time of our initial observation, a concern was raised that applying different volumes of the same concentration of JP-8 may introduce a bias into the interpretation of the results. In order to address this concern, JP-8 was diluted in acetone and the same volume of different dilutions of JP-8 (300 μl JP-8/0 acetone; 200 μl JP-8/100 μl acetone; 100 μl JP-8/200 μl acetone; 50 μl JP-8/250 μl acetone) was applied to each mouse. The effect that this treatment had on CHS is found in Table 1. The negative controls for this experiment consisted of measuring the immune response in mice that were treated with 300 μl of acetone but were not immunized with the contact allergen. As expected, minimal ear swelling was found in the negative-control mice. Similarly, painting JP-8 onto the dorsal skin of non-immunized mice had no effect on background inflammation, as there was no statistical difference in the background ear-swelling response in non-immunized mice treated with JP-8 and the negative controls (p > 0.05, Student's t-test). Applying the JP-8/acetone mixture prior to immunization significantly suppressed the induction of CHS in a dose-dependent fashion. Application of 300 μl of undilute JP-8, 200 μl of JP-8 diluted in 100 μl of acetone, or 100 μl of JP-8 diluted in 200 μl of acetone resulted in 47, 39, and 25% immune suppression, respectively. When the mice were treated with 25 μl of JP-8 diluted in 250 μl of acetone, no immune suppression was noted. These data reproduce our previous findings and indicate that the induction of immune suppression is dependent on the concentration of JP-8 applied to the skin, but is independent of the volume. In all future experiments, different volumes of undiluted JP-8 were applied directly to mouse skin, because this more closely mimics human dermal exposure to jet fuel. Minimal time required for induction of immune suppression following a single exposure to JP-8. A time-course study was performed to determine when the immunotoxicity induced by JP-8 develops. Mice were exposed to an immunosuppressive dose of JP-8 (300 μl) and their spleens were harvested 4, 3, 2, and 1 day later. Control mice were treated with acetone 1 day prior to harvest. Splenic T cells were enriched by nylon wool filtration and anti-CD3-driven T-cell proliferation was measured (Fig. 1). Background responses were measured in wells in which the plate-bound anti-CD3 monoclonal antibody was replaced with tissue culture medium. Compared to the proliferation found in the control mice that were treated with acetone one day prior to harvest, the proliferation of T cells from mice treated with JP-8 one or 2 days prior to harvest was not suppressed. Significant (p < 0.001) and substantial (65% suppression 3 days post-JP-8; 97% suppression 4 days post-JP-8) immune suppression was observed in mice that were treated with JP-8 3 to 4 days prior to harvest. These data indicate that the induction of immunotoxicity following a single exposure to JP-8 is a relatively rapid event. Duration of JP-8-induced immune suppression. In the next series of experiments, we wished to determine the duration of immune suppression that results from a single exposure to JP-8. Mice were treated with JP-8 (300 μl) or acetone, and at various times post-exposure (days 4, 8, 18, or 21) they were sacrificed and their spleens were removed, T cells isolated, and the proliferation of the T cells to plate-bound anti-CD3 was measured. Data from this experiment are found in Figure 2. Compared to the proliferative response found in acetone-treated mice, JP-8 treatment significantly suppresses (p < 0.05) T-cell proliferation when the spleen cells are harvested 4, 8, and 18 days post-exposure. No immune suppression was noted, however, when the cells were harvested 21 days post-exposure, as the proliferative response in the JP-8-treated group was identical to that seen in the acetone-treated controls. These data confirm that JP-8-induced immunotoxicity is first apparent 3–4 days post-exposure and persists for approximately 3 weeks. Effect of JP-8 exposure on antibody formation in vivo. In all of our experiments to date, we measured the effect of JP-8 treatment on cell-mediated immune reactions (contact and/or delayed-type hypersensitivity, and T-cell proliferation). Next, we wished to determine if JP-8 exposure suppressed antibody formation in vivo. Mice were exposed to various doses of JP-8 and them immunized with KLH. One week later the mice were boosted with KLH. One week after the boost, the mice were bled and KLH-specific serum antibody titers were measured by ELISA. Data from this experiment are presented in Figure 3. Treating mice with 250 and/or 300 μl of JP-8, doses that completely suppress T-cell proliferation (Figs. 1 and 2) and CHS (Ullrich, 1999) has no effect on antibody formation. We have repeated this experiment 3 times and we never saw any significant suppression of antibody formation. Thus, these findings indicate that cell-mediated immune responses are more sensitive to the immunosuppressive effects of JP-8 than antibody formation. A role for prostaglandin E2 and IL-10 in JP-8-induced systemic immune suppression. In all of the experiments described here, the antigen or contact allergen is applied at a site distant from the site of JP-8 application. This implies that immune suppressive-biological response modifiers are being released by the JP-8-treated epidermal cells and are inducing systemic immune suppression. Because they are produced in the skin, and since they suppress cell-mediated immune reactions, PGE2 and IL-10 are possible candidates. To test this hypothesis, we attempted to neutralize the activity of, or interfere with the production of these cytokines and to determine what effect, if any, this has on JP-8-induced immune suppression. To neutralize IL-10 monoclonal rat-anti-mouse, IL-10 was injected into the JP-8-treated mice (Rivas and Ullrich, 1992, 1994; Ullrich, 1994). To determine whether PGE2 was involved, we injected the JP-8-treated mice with a selective COX-2 inhibitor, SC 236 to determine if blocking the enzymatic conversion of arachidonic acid to PGE2in vivo (Seibert et al., 1994) would affect JP-8-induced immune suppression. Mice were treated with either anti-IL-10 (100 μg/mouse) or SC 236 (0.2 μg/mouse) 2 h prior to JP-8 application. These doses were chosen based on previous work in which similar doses totally blocked the immune suppression induced by ultraviolet radiation (Shreedhar et al.,1998). Three h later the mice were sensitized with a contact allergen (DNFB) and the effect that anti-IL-10 and/or SC 236 had on JP-8-induced immune suppression was measured 6 days later by determining what effect the treatment had on the induction of CHS. Data from this experiment are found in Table 2. Dermal application of 300 μl of JP-8 significantly suppressed the induction of CHS (p = 0.011, versus the positive control, 55% immune suppression). Mice treated with JP-8 and SC 236 generated a CHS reaction that was indistinguishable from the positive control (p = 0.75). Similarly monoclonal anti-IL-10 totally blocked the immune suppressive effect (p = 0.46 vs. the positive control). Injecting isotype control rat antibody into JP-8-treated mice had no effect on JP-8-induced immune suppression. Nor did injecting anti-IL-10 or SC 236 into acetone-treated control mice affect the generation of CHS. These data indicate that blocking the induction of PGE2 and/or neutralizing the activity of IL-10 blocks JP-8-induced immune suppression. The results from this experiment, which was repeated 3 times, suggest that IL-10 and PGE2 are released by JP-8-treated epidermal cells and induce systemic immune suppression. Effect of recombinant IL-12 on JP-8-induced immune suppression. From the data presented above, suppression of cell-mediated immune reactions such as CHS (Table 2) and anti-CD-3-driven T-cell proliferation (Figs. 1 and 2) but no effect on antibody production (Fig. 3), it appears that JP-8 is selectively suppressing cellular immune reactions driven by T helper-1 cells. (Cher and Mosmann, 1987; Mosmann and Sad, 1996). Moreover, both IL-10 and PGE2 suppress T helper-1 cell function (Fiorentino et al., 1991; Hilkens et al., 1995). Because IL-12 is the primary cytokine driving the activation/maturation of T helper-1 cells (Hsieh et al., 1993; Magram et al., 1996; McKnight et al., 1994), we next tested the hypothesis that injecting exogenous IL-12 will prevent JP-8-induced immune suppression. Mice were injected with 0.01 to 0.1 μg of recombinant IL-12 at 2 h prior to the application of jet fuel or acetone. The mice were then sensitized with DNFB and the effect that rIL-12 had on JP-8-induced immune suppression was measured (Table 3). As shown previously, JP-8 exposure resulted in significant immune suppression (p < 0.0001 vs. the positive control). Compared to 100% immune suppression in JP-8-treated mice, mice treated with JP-8 and 0.1 μg of IL-12 showed 37% immune suppression. The restorative effect decreased as the doses of IL-12 used decreased, 64 and 77% immune suppression was noted in JP-8-treated mice injected with 0.05 and 0.01 μg of IL-12, respectively. Injecting rIL-12 into acetone-treated mice had no effect, as the Δ ear measurement post-injection observed in these mice was not significantly different from the positive control. These findings indicate that IL-12 administration will prevent the immunosuppressive effects of JP-8 and, in combination with the data presented in Figures 2 and 3, suggest that JP-8 exposure selectively targets cell-mediated immune reactions driven by T helper-1 type T cells. This experiment was repeated twice and similar results were noted in both experiments. DISCUSSION The focus of the experiments presented here was to begin to determine the mechanism involved in JP-8-induced immune suppression. Our findings suggest that one mechanism by which JP-8 induces system-wide immune suppression is through the production of immune regulatory cytokines and biological-response modifiers. This conclusion is based on the observation that injecting JP-8-treated mice with a selective COX-2 inhibitor, a non-steroidal anti-inflammatory drug that inhibits the enzymatic activity of cyclooxygenase-2 blocks JP-8-induced immune suppression. In addition, we find that neutralizing the activity of the anti-inflammatory cytokine, IL-10, with a highly specific monoclonal antibody restores immune function in JP-8-treated mice. We also examined and further characterized the timing and duration of the immune suppression induced by a single acute exposure to JP-8. The observation that 3 to 4 days must elapse between JP-8-treatment and the induction of immune suppression is consistent with cytokine release as the mechanism behind immune suppression. From our previous work we know that maximal serum IL-10 secretion occurs 24 to 48 h after JP-8 exposure (Ullrich 1999). We propose that it takes 2 to 3 days for immune suppressive cytokines, such as IL-10 and PGE2 to be produced, enter the circulation, and interact with the target cells, thus inducing immune suppression. The exact identity of the target cells is not clear at this time, but based on the fact that IL-10 and PGE2 down-regulate the action of antigen-presenting cells (Fiorentino et al., 1991; Kalinski et al., 1998), we suspect that antigen-presenting cells are targeted by JP-8-induced IL-10 and PGE2. Experiments are now in progress to test this hypothesis. The other major route of toxin exposure is via inhalation of aerosolized JP-8. It is of interest to note the similarities between the immune suppression induced by dermal exposure and inhalation of jet fuel. In both cases, T cell function is suppressed, immune suppression first appears 3 to 4 days after initial exposure and the effect lasts for 3 to four weeks (Harris et al., 1997a,b). These findings are alarming because, in the course of their duties on a typical Air Force base, fuel handlers, engine mechanics, and flight line personnel are in daily contact with JP-8 (Pleil et al., 2000). This suggests that the induced immune suppression may be of longer duration than demonstrated here. Alternatively, repeated JP-8 exposure may have a chronic dampening effect on the immune reaction of affected individuals. Whether this translates into increased susceptibility to infection remains to be seen. Our findings indicate, however, that the consequence of JP-8 exposure is not global immune suppression. Rather JP-8 has a selective effect on the immune response. We base this conclusion on the fact that exposure to JP-8 has no effect on antibody formation. When we exposed mice to doses of JP-8 (300 μl) that caused 95 to 100% suppression of cell-mediated immune reactions (Fig. 1, Table 1), we noted no suppression of antibody formation. These data, coupled with the observation that IL-12 blocks JP-8-induced immune suppression, indicate to us that JP-8 is selectively suppressing T helper-1-driven, cell-mediated immune reactions. This would suggest that immune reactions driven by T helper-1 cells, such as delayed-type hypersensitivity and immunity to intracellular microorganisms, might be more susceptible to the toxic effects of JP-8. In one of the classic papers of immunotoxicity, Luster and colleagues suggested that a multi-tiered approach was the best way to detect immunotoxic compounds. Further they presented data to indicate that one of the best tests for determining immunotoxicity was suppression of antibody formation (Luster et al., 1992). We have used a similar multi-tiered approach in our study of the immunotoxicity of JP-8. Although cell-mediated immune reactions, such as delayed and contact hypersensitivity and CD3-driven T cell proliferation, were suppressed by jet-fuel exposure, antibody formation was not. Does the failure to suppress antibody formation suggest that JP-8 is not immunotoxic? We think not, but rather we propose that some immune toxins may have a more subtle and selective effect on the immune response than those originally described by Luster and colleagues. During the past decade, immunologists identified different subclasses of T-helper cells and demonstrated that T helper-1 cells generally help cell-mediated immune reactions and T helper-2 cells help antibody formation (Mosmann and Sad 1996). Furthermore, distinct classes of antigen-presenting cells have been described: one that presents antigen to T helper-1 cells and one that presents to T helper-2 cells (Rissoan et al., 1999; Siegal et al., 1999). Our data strongly suggest that JP-8 exposure only targets T helper-1 cell function. In this regard, the immune regulation induced by JP-8 is similar to that found after exposure to the ubiquitous environmental immune toxin, ultraviolet radiation, which preferentially suppresses T helper-1 cell function (Brown et al., 1995). If, in either of these studies, we concentrated only on the ability of either toxin to suppress the direct plaque-forming cell function, which is a measure of IgM production, the immunotoxicity of JP-8 or ultraviolet radiation would have been missed. Thus, the findings presented here further reconfirm the need for a multi-tiered approach in determining immunotoxicity, as suggested by Luster et al. (1992). However, they further suggest that different classes of immunotoxins are present and that these toxins may not function via the same suppressive mechanism as those described by Luster and colleagues. Perhaps the most important finding reported here is the prevention of JP-8-induced immunotoxicity by IL-12, monoclonal anti-IL-10, and the selective COX-2 inhibitor. These findings are important because they provide insight into the mechanisms involved in JP-8-induced immune suppression but more importantly provide insight into preventing JP-8-induced immunotoxicity in the field. Of the 3 agents used, monoclonal anti-IL-10 is the most specific, and the specificity of antibody treatment in blocking JP-8-induced immune suppression is a compelling argument for a role of IL-10 in jet fuel induced immunotoxicity. Due to its specificity, antibody treatment would be ideal in preventing JP-8-induced immune suppression in individuals who come into contact with this toxin; however, the overall lack of humanized monoclonal antibodies makes this approach impractical. Blocking JP-8 immune suppression by IL-12 is also an important observation regarding the mechanisms involved. Because IL-12 is the primary cytokine involved in activating T helper-1 cells in vivo, the blocking of immune suppression, as demonstrated here, in combination with the type of immune reactions suppressed (cell-mediated immunity versus humoral immunity) argues that JP-8 is interfering with T helper-1-cell-driven immune reactions. In addition, we recently demonstrated that IL-12 blocks IL-10 secretion in vivo, primarily by interfering with the transcription of the gene (Schmitt et al., 2000). Therefore, it is possible that IL-12 is blocking IL-10 production in JP-8-treated mice, which probably contributes to the prevention of immune suppression. Unfortunately, the use of IL-12 to block JP-8-induced immune suppression in humans is risky because of the severe toxicity associated with its use (Car et al., 1999). Using the selective COX-2 inhibitor to prevent immune suppression in Air Force personnel who come in contact with JP-8 during the course of their duties holds the most promise. Selective COX-2 inhibitors are commercially available and in use clinically for the treatment of chronic aliments such as arthritis (Megeff and Strayer, 2000) and colon cancer (Rao et al., 1995). They are well tolerated with minimal side effects Kaplan-Machlis and Klostermeyer, 1999). Their use here provides compelling evidence to suggest the involvement of PGE2 in the immune suppression induced by JP-8. Furthermore, blocking the production of PGE2 may have the added benefit of inhibiting a cascade of events ultimately resulting in IL-10 production. In a study of the immune suppression induced by ultraviolet radiation, we documented that PGE2 induces a cytokine cascade involving IL-4 and IL-10 that ultimately suppresses cell-mediated immune reactions. Pertinent to the results presented here was the finding that blocking COX-2 activity in vivo with SC 236 prevented the secretion of IL-4 and IL-10. Moreover, injecting PGE2 into normal mice resulted in IL-4 and IL-10 production (Shreedhar et al., 1998). If the same result happens after JP-8 exposure and the suppression of JP-8-induced immune suppression by SC 236, coupled with the secretion of IL-10 into the serum following JP-8-treatment, suggests that it might, then using a selective COX-2 inhibitor will have the added benefit of blocking down stream events. Thus, the selective COX-2 inhibitor may prevent a cascade of events in vivo and may be, due to its limited toxicity and potent restorative effect, the ideal way to overcome the immunotoxicity of JP-8. In summary, the data presented here indicate that JP-8 exposure has a selective effect on immune function. T helper-1 cell-driven cell-mediated immune reactions, such as DTH, CHS, and T-cell proliferation are susceptible to the effects of JP-8, whereas antibody formation is not suppressed. The mechanism through which dermal JP-8 application induces systemic immune suppression appears to be via cytokine release, in particular PGE2 and IL-10. Furthermore, injecting JP-8-treated mice with a selective COX-2 inhibitor, which suppresses PGE2 production in vivo with minimal side effects, totally restores immune function. These findings suggest that interfering with cycloooxygenase-2 activity in vivo may provide a reasonable method of suppressing JP-8-induced immunotoxicity in exposed personnel. TABLE 1 Dermal Application of JP-8 Suppresses the Induction of CHS in Vivo JP-8/acetonea  DNFBb  Δ ear size (edema)c  Specific swellingd  % Suppressione  pf  aMice were treated with 300 μl of a mixture of JP-8 and acetone on the shaved dorsal skin.  bMice were sensitized with 50 μl of 0.3% DNFB on the shaved ventral skin 3 h after JP-8/acetone treatment.  cMean values from 5 mice (mm × 10–2) ±SD.  dThe Δ ear size found in the controls that were treated with JP-8/acetone but were not sensitized was subtracted from the Δ ear size found in the group treated with an equal amount of JP-8/acetone and sensitized with DNFB.  e% suppression = 1−[(specific swelling of JP-8/acetone-treated sensitized mice ÷ specific swelling of the positive control) × 100].  fp values determined by two-tailed Students t-test vs. the positive control (0/300 + DNFB).  0/300  –  3.0 ± 0.6  –      0/300  +  17.5 ± 0.3  14.5  0  –   300/0  –  3.4 ± 0.6  –  –    300/0  +  11.1 ± 1.1  7.7  47  0.0006   200/100  –  3.9 ± 1.1  –      200/100  +  12.8 ± 0.3  8.9  39  0.0001   100/200  –  2.7 ± 0.3  –      100/200  +  13.6 ± 1.0  10.9  25  0.007   50/250  –  2.4 ± 0.2  –      50/250  +  16.8 ± 0.8  14.4  1  0.432  JP-8/acetonea  DNFBb  Δ ear size (edema)c  Specific swellingd  % Suppressione  pf  aMice were treated with 300 μl of a mixture of JP-8 and acetone on the shaved dorsal skin.  bMice were sensitized with 50 μl of 0.3% DNFB on the shaved ventral skin 3 h after JP-8/acetone treatment.  cMean values from 5 mice (mm × 10–2) ±SD.  dThe Δ ear size found in the controls that were treated with JP-8/acetone but were not sensitized was subtracted from the Δ ear size found in the group treated with an equal amount of JP-8/acetone and sensitized with DNFB.  e% suppression = 1−[(specific swelling of JP-8/acetone-treated sensitized mice ÷ specific swelling of the positive control) × 100].  fp values determined by two-tailed Students t-test vs. the positive control (0/300 + DNFB).  0/300  –  3.0 ± 0.6  –      0/300  +  17.5 ± 0.3  14.5  0  –   300/0  –  3.4 ± 0.6  –  –    300/0  +  11.1 ± 1.1  7.7  47  0.0006   200/100  –  3.9 ± 1.1  –      200/100  +  12.8 ± 0.3  8.9  39  0.0001   100/200  –  2.7 ± 0.3  –      100/200  +  13.6 ± 1.0  10.9  25  0.007   50/250  –  2.4 ± 0.2  –      50/250  +  16.8 ± 0.8  14.4  1  0.432  View Large TABLE 2 Blocking PGE2 Synthesis and Neutralizing IL-10 Activity in Vivo Prevents JP-8-Induced Immune Suppression Groupa  Δ ear size (edema)b  Specific swellingc  Suppression (%)d  pe  aMice were treated with acetone (negative and positive controls) or JP-8 (neat 300 μl) on the shaved dorsal skin and 3 h later sensitized with 50 μl of 0.3% DNFB. Two h prior to JP-8 treatment, some mice were injected with 0.2 μg of the selective COX-2 inhibitor, SC 236 or 100 μg of monoclonal anti-IL-10. Negative controls were not sensitized but were challenged. Positive controls were sensitized and challenged.  bMean values from 5 mice (mm × 10–2) ± SD.  cThe Δ ear size found in the negative controls was subtracted from the Δ ear size found in the experimental groups.  d% suppression = 1−[(specific swelling of JP-8-treated mice ÷ specific swelling of the positive control) × 100].  ep values determined by two-tailed Students t-test vs. the positive control.  Negative control  3.6 ± 2.0  –      Positive control  16.1 ± 3.0  12.5  –     Anti-IL-10  15.8 ± 2.1  12.2  2  0.87   SC 236  16.8 ± 2.1  13.2  0  0.70   JP-8  9.2 ± 3.2  5.6  55  0.011   JP-8 + rat IgG  8.3 ± 1.0  4.7  62  0.002   JP-8 + anti-IL-10  14.6 ± 2.6  11  12  0.46   JP-8 + SC 236  15.4 ± 3.0  11.8  6  0.75  Groupa  Δ ear size (edema)b  Specific swellingc  Suppression (%)d  pe  aMice were treated with acetone (negative and positive controls) or JP-8 (neat 300 μl) on the shaved dorsal skin and 3 h later sensitized with 50 μl of 0.3% DNFB. Two h prior to JP-8 treatment, some mice were injected with 0.2 μg of the selective COX-2 inhibitor, SC 236 or 100 μg of monoclonal anti-IL-10. Negative controls were not sensitized but were challenged. Positive controls were sensitized and challenged.  bMean values from 5 mice (mm × 10–2) ± SD.  cThe Δ ear size found in the negative controls was subtracted from the Δ ear size found in the experimental groups.  d% suppression = 1−[(specific swelling of JP-8-treated mice ÷ specific swelling of the positive control) × 100].  ep values determined by two-tailed Students t-test vs. the positive control.  Negative control  3.6 ± 2.0  –      Positive control  16.1 ± 3.0  12.5  –     Anti-IL-10  15.8 ± 2.1  12.2  2  0.87   SC 236  16.8 ± 2.1  13.2  0  0.70   JP-8  9.2 ± 3.2  5.6  55  0.011   JP-8 + rat IgG  8.3 ± 1.0  4.7  62  0.002   JP-8 + anti-IL-10  14.6 ± 2.6  11  12  0.46   JP-8 + SC 236  15.4 ± 3.0  11.8  6  0.75  View Large TABLE 3 Reversal of JP-8-Induced Immune Suppression by rIL-12 Groupa  IL-12  Δ ear size (edema)b  Specific swellingc  Suppression (%)d  pc  aMice were treated with acetone (negative and positive controls) or JP-8 (neat 300 μl) on the shaved dorsal skin and 3 h later sensitized with 50 μl of 0.3% DNFB. Two h prior to JP-8 treatment, some mice were injected with rIL-12 diluted in PBS. Negative controls were not sensitized but were challenged. Positive controls were sensitized and challenged.  bMean values from 5 mice (mm × 10–2) ± SD.  cThe Δ ear size found in the negative controls was subtracted from the Δ ear size found in the experimental groups.  d% suppression = 1−[(specific swelling of JP-8-treated mice ÷ specific swelling of the positive control) × 100].  ep values determined by 2-tailed Students t-test vs. the positive control.  Negative control  –  1.2 ± 0.24  0  –    Positive control  –  20.8 ± 5.6  19.6  0    JP-8  –  0.7 ± 0.24  0  100  0.0001   JP-8  0.1 μg  13.5 ± 0.9  12.3  37  0.036   JP-8  0.05 μg  8.2 ± 1.2  7  64  0.0024   JP-8  0.01 μg  5.8 ± 2.5  4.6  77  0.0013   Acetone  0.1 μg  22.4 ± 1.7  21.2  0  0.699   Acetone  0.05 μg  25.5 ± 2.0  24.3  0  0.155   Acetone  0.01 μg  17.5 ± 3.2  16.3  17  0.299  Groupa  IL-12  Δ ear size (edema)b  Specific swellingc  Suppression (%)d  pc  aMice were treated with acetone (negative and positive controls) or JP-8 (neat 300 μl) on the shaved dorsal skin and 3 h later sensitized with 50 μl of 0.3% DNFB. Two h prior to JP-8 treatment, some mice were injected with rIL-12 diluted in PBS. Negative controls were not sensitized but were challenged. Positive controls were sensitized and challenged.  bMean values from 5 mice (mm × 10–2) ± SD.  cThe Δ ear size found in the negative controls was subtracted from the Δ ear size found in the experimental groups.  d% suppression = 1−[(specific swelling of JP-8-treated mice ÷ specific swelling of the positive control) × 100].  ep values determined by 2-tailed Students t-test vs. the positive control.  Negative control  –  1.2 ± 0.24  0  –    Positive control  –  20.8 ± 5.6  19.6  0    JP-8  –  0.7 ± 0.24  0  100  0.0001   JP-8  0.1 μg  13.5 ± 0.9  12.3  37  0.036   JP-8  0.05 μg  8.2 ± 1.2  7  64  0.0024   JP-8  0.01 μg  5.8 ± 2.5  4.6  77  0.0013   Acetone  0.1 μg  22.4 ± 1.7  21.2  0  0.699   Acetone  0.05 μg  25.5 ± 2.0  24.3  0  0.155   Acetone  0.01 μg  17.5 ± 3.2  16.3  17  0.299  View Large FIG. 1. View largeDownload slide Minimal time required for induction of immune suppression following a single exposure to JP-8. One to 4 days after dermal application of JP-8 (300 μl/mouse), spleen cells were removed, and T cells were prepared by nylon wool filtration and stimulated in vitro with plate-bound anti-CD3 antibody (10 μg/ml). Background responses were measured in wells containing tissue-culture medium. The positive control was T cells isolated from mice painted with acetone one day prior to harvest. T cell proliferation was determined by incorporation of 3H-Tdr. *p < 0.001 vs. the positive control. FIG. 1. View largeDownload slide Minimal time required for induction of immune suppression following a single exposure to JP-8. One to 4 days after dermal application of JP-8 (300 μl/mouse), spleen cells were removed, and T cells were prepared by nylon wool filtration and stimulated in vitro with plate-bound anti-CD3 antibody (10 μg/ml). Background responses were measured in wells containing tissue-culture medium. The positive control was T cells isolated from mice painted with acetone one day prior to harvest. T cell proliferation was determined by incorporation of 3H-Tdr. *p < 0.001 vs. the positive control. FIG. 2. View largeDownload slide Duration of JP-8-induced immune suppression. Mice were painted with JP-8 or acetone on day 0. Four to 21 days later, the mice were killed, spleen removed, and T cells isolated. The T cells were then stimulated with plate-bound anti-CD3. Background responses were determined by culturing the T cells in wells containing tissue-culture medium. T-cell proliferation was determined by incorporation of 3H-Tdr. *p < 0.05 vs. the positive control (acetone/anti-CD3). FIG. 2. View largeDownload slide Duration of JP-8-induced immune suppression. Mice were painted with JP-8 or acetone on day 0. Four to 21 days later, the mice were killed, spleen removed, and T cells isolated. The T cells were then stimulated with plate-bound anti-CD3. Background responses were determined by culturing the T cells in wells containing tissue-culture medium. T-cell proliferation was determined by incorporation of 3H-Tdr. *p < 0.05 vs. the positive control (acetone/anti-CD3). FIG. 3. View largeDownload slide Failure of JP-8 treatment to suppress antibody production. Mice were exposed to JP-8 (100 to 300 μl/mouse); 3 h later the mice were immunized with KLH (100 μg/mouse) emulsified in complete Freund's adjuvant. One week later the mice were re-treated with jet fuel and boosted with KLH in incomplete Freund's adjuvant. One week after that the mice were sacrificed/bled and KLH-specific IgM, IgG1, and IgG2b were measured by ELISA (n = 5 mice/group). The serum from each animal was assayed individually. The data represent the mean OD ± SD from the group of 5. Negative control mice were treated with acetone but not immunized. Positive control mice were treated with acetone and immunized with KLH. FIG. 3. View largeDownload slide Failure of JP-8 treatment to suppress antibody production. Mice were exposed to JP-8 (100 to 300 μl/mouse); 3 h later the mice were immunized with KLH (100 μg/mouse) emulsified in complete Freund's adjuvant. One week later the mice were re-treated with jet fuel and boosted with KLH in incomplete Freund's adjuvant. One week after that the mice were sacrificed/bled and KLH-specific IgM, IgG1, and IgG2b were measured by ELISA (n = 5 mice/group). The serum from each animal was assayed individually. The data represent the mean OD ± SD from the group of 5. Negative control mice were treated with acetone but not immunized. Positive control mice were treated with acetone and immunized with KLH. 1 To whom correspondence should be addressed. Fax: (713) 745-1633. E-mail: sullrich@notes.mdacc.tmc.edu. 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TI - Mechanisms Involved in the Immunotoxicity Induced by Dermal Application of JP-8 Jet Fuel
JF - Toxicological Sciences
DO - 10.1093/toxsci/58.2.290
DA - 2000-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mechanisms-involved-in-the-immunotoxicity-induced-by-dermal-dWHil6ZDaU
SP - 290
EP - 298
VL - 58
IS - 2
DP - DeepDyve
ER -