Dermal Application of JP-8 Jet Fuel Induces Immune Suppression

Transcription

1 TOXICOLOGICAL SCIENCES 52, (1999) Copyright 1999 by the Society of Toxicology Dermal Application of JP-8 Jet Fuel Induces Immune Suppression Stephen E. Ullrich 1 Department of Immunology-178, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas Received March 25, 1999; accepted March 30, 1999 Chronic exposure to JP-8 jet fuel induces lung toxicity, adverse neurological effects and some liver and kidney dysfunction. In addition, inhalation of JP-8 induces immune suppression. Besides the lung, the other major route of JP-8 exposure is via the skin. In this study we tested the hypothesis that dermal exposure to JP-8 is immune suppressive. JP-8 was applied to the skin of adult female C3H/HeN mice and various immune parameters were examined. Dermal exposure to JP-8, either multiple small exposures (50 l for 5 days) or a single large dose ( l) resulted in immune suppression. The induction of contact hypersensitivity was impaired in a dose-dependent manner regardless of whether the contact allergen was applied directly to the JP-8 treated skin or at a distant un-treated site. In addition, the generation of a classic delayed-type hypersensitivity reaction to a bacterial antigen (Borellia burgdorferi) injected into the subcutaneous space was suppressed by dermal application of JP-8 at a distant site. The ability of splenic T lymphocytes from JP-8 treated mice to proliferate in response to plate-bound monoclonal anti-cd3 was also significantly suppressed. Interleukin-10, a cytokine with potent immune suppressive activity, was found in the serum of JP-8 treated mice, suggesting that the mechanism of systemic immune suppression may involve the upregulation of cytokine release by JP-8. These findings confirm the immunosuppressive effects of JP-8 and demonstrate that dermal exposure to JP-8 is immunotoxic. Key words: jet fuel; JP-8; immunotoxicity; immune suppression; IL-10; contact hypersensitivity; delayed-type hypersensitivity. Since the early 1990s, the United States Air Force and the NATO allies have been converting to JP-8 as their main jet fuel. The higher flash point and lower vapor pressure of JP-8 yields significant advantages in comparison to previously used fuels in regard to minimizing evaporative losses during storage and handling and decreased susceptibility to crash and/or combat-induced fires and explosions. During the conversion to JP-8, complaints of fatigue, headache, blocked nasal passages and skin irritation among fuel handlers, mechanics, and flight line personnel prompting the initiation of systemic studies to determine the toxicity of JP-8. Studies with experimental animals indicated that JP-8 was not irritating to the eyes and 1 To whom correspondence should be addressed. Fax: (713) sullrich@notes.mdacc.tmc.edu dermal exposure resulted in, at best, very weak sensitization (Kinkead et al., 1992). The oral LD 50 for JP-8 is 16 g/kg body weight, and although chronic exposure to high doses of JP-8 by gastric gavage resulted in decreased body weight, elevated levels of liver enzymes, and gastric and perianal irritation, little to no morbidity or mortality was noted in the JP-8 treated rats, nor were any pathologic changes noted in the livers of the treated animals (Mattie et al., 1995). Similarly, ingestion of JP-8 ( mg/kg/day) by pregnant rats on days 6 through 15 of gestation did not induce fetal malformation (Cooper and Mattie, 1996). Other studies, however, suggest that JP-8 exposure is capable of inducing toxicity. Exposure to JP-8 fuel vapors altered the postural sway response of volunteers, indicating a subtle effect of this fuel on neurologic function (Smith et al., 1997). Inhalation of JP-8 also caused a reduction in pulmonary function and induced lung pathology. Pulmonary resistance was increased in rats treated with JP-8 for 7, 28, and 56 days. Microscopic examination of the treated lungs indicated thickening of the alveolar septa resulting from JP-8 induced endothelial damage (Hays et al., 1995; Pfaff et al., 1995). Additional evidence for pulmonary dysfunction included a JP-8 induced decrease in the concentration of neuropeptide substance P in the bronchoalveolar lavage fluid (Pfaff et al., 1996). Inhalation studies also demonstrated that exposure to JP-8 is immunotoxic. Short term exposure (once a day for 7 days) to low concentrations (100 mg/m 3 ) of aerosolized JP-8 decreased immune organ weights, decreased the number of viable immune cells recovered, and suppressed the ability of splenic T cells to respond to mitogenic stimulation (Harris et al., 1997a). The effect of JP-8 on immune organ weights was found 24 h after treatment, and the suppressed T-cell mitogenesis persisted for up to 4 weeks postexposure (Harris et al., 1997b). Besides the lung, the other prominent route of JP-8 exposure is the skin. Although previous studies have addressed the irritant effects of JP-8 (Kinkead et al., 1992), there is no data to indicate whether dermal exposure to JP-8 induces immunotoxicity. Dermal exposure to JP-8 is a common and often unavoidable consequence for fuel handlers, engine mechanics, and flight line personnel exposed to unburned fuel that passes through a jet engine during starts in very cold weather. In addition, the decreased vapor pressure of JP-8 results in slower 61

2 62 ULLRICH rates of evaporation and presents an opportunity for potential fuel absorption into the skin. The focus of the studies presented here is to test the hypothesis that dermal exposure to JP-8 induces immune suppression. MATERIALS AND METHODS Mice. Specific pathogen-free (SPF) 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. 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 Major Thomas E. Miller, 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, and nitrile rubber-based gloves (Touch N Tuff, Fisher Scientific Co.) were used in the place of normal latex gloves because of 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 were applied directly to the dorsal skin of the animals. The mice (3 5 per group) were held individually in the hood for 3 h after exposure to prevent cage mates from grooming and ingesting the fuel. In addition, 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 SPFbarrier facility. Delayed-type hypersensitivity reactions. The dorsal hair of the mice was removed with electric clippers, and a micropipete was used to apply the JP-8 directly to the dorsal skin as described above. Control mice were shaved and treated with acetone. Three hours later, the mice were immunized by injecting 2.35 g of Borrelia burgdorferi antigen emulsified in complete Freund s adjuvant (CFA) into each flank, as described previously (Brown et al., 1995). Ten days later, each hind footpad was measured with an engineer s micrometer (Swiss Precision Instruments, Los Angles, CA); the footpad thickness was recorded and the animals were challenged by injecting 0.5 g of Borrelia antigen into each hind footpad. Footpad thickness was measured again h later and the swelling was recorded. The specific footpad swelling was calculated by subtracting the response found in control mice that were not immunized, but were challenged, from the swelling seen in mice that were both immunized and challenged. There were five mice per group. The data is expressed as specific footpad swelling the standard deviation. Statistical differences between the controls and experimental groups were determined by use of a two-tailed Student s t-test, with a probability of less than 0.05 considered significant. Contact hypersensitivity. The dorsal hair of the mice was removed with electric clippers, and a micropipette was used to apply the JP-8 directly to the dorsal skin as described above. Control mice were shaved and treated with acetone. Three hours later, the mice were sensitized by painting 100 lofa3% (w/v) solution of oxazolone in acetone onto the shaved abdominal skin. Six days after sensitization, the thickness of each ear was measured and recorded, and the mice were challenged by applying 10 l of a 3% oxazolone solution to each ear. Ear thickness was measured again h later and recorded. The specific ear swelling was calculated as described above. There were five 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 two-tailed Student s t-test, with a probability of less than 0.05 considered significant. ELISA to detect serum interleukin-10 levels. The capture antibodies and biotinylated detecting antibodies were purchased from PharMingen (San Diego, CA) and used according to the manufacturer s instructions in a sandwich ELISA procedure, as reported previously (Shreedhar et al., 1998). Generally the limit of detection for interleukin-10 (IL-10) was pg/ml. There were three mice per group. Each animal was bled and the serum obtained and assayed in triplicate. The IL-10 concentration of each mouse was determined and data presented represents the mean and standard deviation of the serum IL-10 concentration of all three animals in the group. Serum samples were also obtained from normal control mice treated with acetone. Statistical differences between the experimental groups were determined by use of a two-tailed Student s t-test, with a probability of less than 0.05 considered significant. T-cell proliferation in vitro. Seven days 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; M 2-mercaptoethanol; 100 units/ml streptomycin; 2 mm L-glutamine; 10% sodium pyruvate; 10 mm HEPES buffer; 1X vitamins; and 1X nonessential amino acids (GIBCO-BRL, Grand Island, NY). The cells ( /well) were then cultured at 37 in an atmosphere of 5% CO 2, 95% air for 5 days in 96-well tissue culture plates coated with monoclonal anti-cd3 (30 l ofa10 g/ml solution overnight prior to culture; PharMingen, San Diego, CA). During the last 18 h of culture 1 Ci 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 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 two-tailed Student s t-test, with a probability of less than 0.05 considered significant. RESULTS Effect of JP-8 on Contact and Delayed-Type Hypersensitivity In the first series of experiments, we tested the hypothesis that dermal application of JP-8 induces immune suppression in vivo. Fifty microliters of undiluted JP-8 was applied to the shaved back skin of the mice. One group of mice received 50 l of JP-8 Monday through Friday and was sensitized with the contact allergen 3 h following the last application of JP-8 (50 l 5). A second group received 50 l of JP-8 Tuesday through Friday (50 l 4) and was sensitized with the contact allergen 3 h after the last application of JP-8. This procedure was repeated until we had five groups of mice, each treated with JP-8 on 1 (50 l 1) through 5 (50 l 5) days. The negative and positive control mice were painted with 50 l of acetone on 5 successive days. We chose acetone as our control because we knew from previous studies that the application of

3 DERMAL EXPOSURE TO JP-8 IS IMMUNOTOXIC 63 acetone did not interfere with the generation of contact hypersensitivity (CHS). The positive control mice were sensitized with the contact allergen 3 h after the final acetone treatment; the negative control mice were not sensitized. Six days later all the mice were challenged on the ears with the contact allergen; the resulting ear swelling was measured h later. Data from a representative experiment is found in Figure 1A. At the two highest doses tested (50 l 5 and 50 l 4), JP-8 application caused a significant impairment (p 0.05; Student s t-test compared to the positive control) of the immune response. No impairment was found at the lower doses; the slight decrease in the response found in the 50 l 1 group is not statistically different from the positive control. These data indicate that JP-8 can induce immune suppression in a dosedependent fashion. What was not clear from these observations was whether multiple applications of JP-8 was required to induce immune suppression or whether a single application would be sufficient. Because the data presented in Figure 1A suggest that application of approximately 250 l of JP-8 is needed for immune suppression, we repeated this experiment, but this time the mice were treated with 300 l of JP-8 instead of 50 l. These data, presented in Figure 1B, indicate that at all the application schedules used, 300 l of JP-8 induced significant immune suppression. From these data we conclude that one application of JP-8 above a threshold level of l/mouse will induce significant immune suppression. In the generation of CHS, the contact allergen must be carried from the skin to the draining lymph nodes by the epidermal antigen-presenting cell, the Langerhans cell (Kripke et al., 1990). Once the Langerhans cell arrives at the draining lymph node, the antigen is presented to T cells and the reaction proceeds. One possible mechanism by which JP-8 could cause immune impairment may be to alter the ability of Langerhans cells to migrate from the site of sensitization to the draining lymph node. In order to test this hypothesis, the following experiment was done. Mice were treated with JP-8 on the shaved dorsal skin as described above. The mice received l JP-8 on each day of the week for a total of five treatments. Three hours after the last treatment, the mice were sensitized by applying the contact allergen to the shaved abdominal skin, a site not previously treated with JP-8. The data from a representative experiment (Fig. 2) clearly indicate that JP-8 application can have a systemic effect on the immune response. Immune suppression was observed in a dose-dependent fashion when the JP-8 and the contact allergen were applied at different body sites. It also appears that significant suppression (p 0.05; Student s t-test compared to the positive control) occurs when the mice are treated with a cumulative amount of JP-8 in excess of 250 l. This was tested directly in the next experiment. The mice were treated with 100, 200, and 300 l JP-8 on the shaved dorsal skin; 3 h later, Borellia burgdorferi protein antigen was injected into the subcutaneous tissue underlying the abdominal skin. Data from such an experiment is found in Table 1. Compared to the FIG. 1. Suppression of CHS by dermal application of JP-8. Fifty microliters (panel A) or 300 l (panel B) of JP-8 was applied to the shaved dorsal skin of mice once or multiple times over the course of 5 days. Three hours after the last treatment a contact allergen was applied directly to the JP-8-treated skin. CHS was measured 7 days later. The data is expressed as ear swelling SD. * indicates a significant difference (p 0.05) from the positive control. Representative experiments are shown; the experiment in panel A was repeated three times and the experiment in panel B repeated twice with identical results. positive control, significant immune suppression was found only in mice treated with 300 l JP-8. The slight depression in the response found in mice treated with 100 and 200 l JP-8 was not significantly different from the positive control. These data confirm that JP-8 can induce systemic immune suppression by demonstrating that JP-8 will suppress the induction of a classic delayed type hypersensitivity (DTH) reaction to a

4 64 ULLRICH FIG. 2. Systemic immune suppression by dermal application of JP-8. Twenty five to 300 l of JP-8 was applied to the shaved dorsal skin of mice once a day for 5 consecutive days. Three hours after the last treatment a contact allergen was applied at a distant (abdominal skin) site. CHS was measured 7 days later. The data is expressed as ear swelling SD. *indicates a significant difference (p 0.05) from the positive control. A representative experiment is shown; this experiment was repeated three times with identical results. TABLE 1 Effect of JP-8 on Delayed-Type Hypersensitivity to Borrelia burgdorferi Group a Footpad swelling b Specific swelling c % Suppression d p* Negative control 8 3 Positive control l NS 200 l NS 300 l a Mice were treated with acetone (negative and positive control) or JP-8 (neat; l) on the shaved dorsal skin and 3 h later immunized with B. burgdorferi (2.35 g in CFA) into the flank. Negative control refers to mice that were not immunized but were challenged; positive control refers to mice that were immunized and challenged. b Mean values from five mice (mm 10 2 ) SD. c The footpad swelling found in the negative control was subtracted from the footpad swelling observed in the experimental groups. d % suppression 1-[specific swelling of JP-8 treated mice specific swelling of the positive control] 100 *p values determined by two-tailed Student s t-test comparing the experimental groups to the positive control; NS p FIG. 3. Dose-response curves for JP-8 induced immune suppression. The ear swelling data was converted to percent immune suppression (see legend for Table 1) and plotted versus the dose of JP-8 applied to the skin. Local (}) refers to application of the contact allergen directly to the JP-8 treated skin; systemic ( ) refers to the situation where the contact allergen was applied to a distant site. bacterial antigen. Moreover, they provide additional evidence that a single exposure to JP-8 is sufficient to suppress the immune response. For the sake of comparison, the dose-response data generated in the various experiments was converted to percentage immune suppression and plotted versus the dose of JP-8 applied. These curves are presented in Figure 3. Both doseresponse curves are shown: immunization on the JP-8 treated site (local) and immunization at a distant site (systemic). Because the curves are identical, these data suggest that a similar mechanism is involved in the induction of immune suppression whether the antigen is applied directly to the JP-8 treated skin or at a distant site. Effect of JP-8 Treatment on Body Weight One indication of a systemic effect of a toxic chemical is a decrease in body weight after treatment. Although relatively short exposures were used in these experiments, a systemic effect of JP-8 on the immune response was noted, so changes in body weights were monitored. No major changes in body weight were observed during the course of the JP-8 treatment (Table 2). Although a slight but significant depression was observed following 300 l of JP-8 4 treatments (.01 p.05), a similar depression was found in the acetone-treated negative control group, suggesting this change in body weight is not biologically relevant. Effect of JP-8 on Anti-CD3 Driven T-cell Proliferation Spleens were removed from the mice at the termination of the CHS experiments described above (i.e., 7 days after sensitization with the contact allergen) and the T cells isolated. The T cells were stimulated in vitro with plate-bound anti-cd3 monoclonal antibody. A dose-dependent suppression of T-cell

5 DERMAL EXPOSURE TO JP-8 IS IMMUNOTOXIC 65 Treatment b TABLE 2 Effect of JP-8 on Body Weight Before Weight (g) a After Negative control p.05 Positive control NS 300 l NS 300 l NS 300 l NS 300 l p l NS a Mice were weighed immediately prior to the first JP-8 treatment and when the experiment was terminated 7 11 days later. Mean values are from five mice SD. b Mice were treated with acetone (negative and positive control) or JP-8 (neat; 300 l on 1 to 5 successive days) on the shaved dorsal. Negative control refers to mice that were not immunized but were challenged; positive control refers to mice that were immunized and challenged. * p values determined by two-tailed Student s t-test comparing the body weights before the start of the experiment and after termination. proliferation was noted (Table 3). Significant immune suppression (p compared to the positive control) was found when T cells from JP-8 treated mice (300 l 1) were stimulated with anti-cd3. No suppression was seen following treatment with 100 or 200 l JP-8, confirming that suppression occurs after threshold levels greater than 250 l are reached. Induction of Interleukin-10 by JP-8 Other environmental immunotoxins that interact with the skin, most notably ultraviolet radiation, activate immune suppressive pathways by upregulating the production of immunoregulatory cytokines such as IL-10 (Rivas and Ullrich, 1992; Rivas and Ullrich, 1994; Shreedhar et al., 1998; Ullrich, 1994). To determine whether JP-8 induces IL-10, the following experiment was done. Three hundred microliters of JP-8 or p* acetone was applied to the shaved dorsal skin of mice. Twentyfour, 48, 72, and 96 h later, the mice were bled and the presence and/or absence of IL-10 in the serum was determined by ELISA. Twenty-four hours after exposure, no IL-10 was found in the serum of the acetone-treated or JP-8 treated mice. At 48 h, IL-10 was found in the JP-8 treated mice, whereas no IL-10 was found in the control serum (p 0.01; JP-8 versus acetone treated; Student s t-test). At 72 h, the amount of IL-10 in the serum of the JP-8 treated mice decreased; however, the difference between the IL-10 levels in the JP-8-treated mice and the acetone-treated controls remained statistically significant (p 0.02). The concentration of IL-10 in the JP-8 treated mice gradually decreased with time, and by 96 h the difference between the IL-10 concentration in the serum of the jet fueltreated mice and the acetone-treated controls was not significantly different (Fig. 4). These data indicate that IL-10, a potent inhibitor of immune reactions such as CHS, DTH, and T-cell proliferation, is found in the serum of JP-8 treated mice and further indicate that the effects of dermal application of JP-8 are apparent as early as 48 h after exposure. DISCUSSION The purpose of the experiments reported here was to test the hypothesis that application of JP-8 jet fuel to the skin induces immune suppression. We used a multi-tiered immunologic approach, as suggested by Luster et al. (1992), to test this hypothesis. Our findings indicate that dermal application of JP-8 will suppress both antigen-driven (DTH and CHS) as well as polyclonal activation (anti-cd3 induced T-cell proliferation) of the immune response. This occurs whether the antigen (DTH) or contact allergen (CHS) is applied directly onto the JP-8 treated skin or is introduced at a distant untreated site. In addition, we find IL-10 in the serum of JP-8 treated mice. This observation not only indicates that JP-8 can modulate the production of immune regulatory cytokines, but because IL-10 is a potent inhibitor of inflammatory immune reactions such as TABLE 3 Effect of JP-8 Treatment on T-cell Proliferation Treatment a CPM SD Background response b Anti-CD3 c CPM d p* Acetone l NS 250 l NS 300 l a Mice were treated with 300 l acetone or JP-8 (neat; l, single exposure) on the shaved dorsal skin. b Background response was measured in wells without plate-bound anti-cd3. c 30 l anti-cd3 (10 g/ml; clone 145 2C11) was added to the wells and incubated overnight. d CPM of wells receiving anti-cd3 minus the background response. * p values determined by two-tailed Student s t-test comparing experimental to the acetone control.

6 66 ULLRICH FIG. 4. Dermal JP-8 application induces serum IL-10 production. Mice were treated with 300 l of acetone ( ) or JP-8 (F) and at various times later serum IL-10 was measured by ELISA. * indicates a significant difference (p 0.05) between the JP-8 and the acetone-treated mice. DTH and CHS and can inhibit T cell proliferation in vitro (reviewed by Moore et al., 1993), we suggest that the induction of IL-10 by JP-8 may be involved in its mechanism of action. Thus, the data reported here clearly support the hypothesis that dermal exposure to JP-8 is immunotoxic. It appears that once l of JP-8 is applied to the skin of a mouse, a threshold level for the activation of immune suppression is achieved. This may occur in a single dose or may be fractionated and delivered in smaller doses applied over a prolonged period of time. This observation may have an important practical application to those encountering JP-8 during occupational exposure. If the relationship mentioned above (i.e., 300 l per mouse; surface area m 2 ) applies to humans, exposing a 200-lb technician (surface area 2.15 m 2 )to approximately 100 ml of JP-8 would be enough to provide the amount needed to induce immune suppression. It is conceivable that fuel handlers, mechanics, and flight line personnel may in the course of a day be exposed to this level of JP-8 and activate the immune suppressive pathways described here. Harris et al. (1997b) suggest that immune function is most sensitive to JP-8 induced damage because immunotoxicity is noted before toxic effects are found in any other system. Our findings support this idea. First, we see IL-10 in the serum of JP-8 treated mice 48 h following a single dermal exposure. Second, although a decrease in body weight is often used as a measure of systemic toxicity, no effect on body weight was noted in our studies at doses that yielded greater than 70% systemic immune suppression (compare Table 2 with Figure 2). Many unanswered questions remain concerning the immune suppression induced following dermal exposure to JP-8. What is not clear is how long immune suppression persists following JP-8 treatment. Nor is it entirely clear when immune suppression is first noted after dermal exposure. The IL-10 data suggest immune deviation as early as 48 h after JP-8 exposure. Experiments are currently in progress to confirm this observation by asking whether T-cell proliferation is suppressed 1, 2, or 3 days following JP-8 exposure. Furthermore, experiments are also in progress to determine the duration of JP-8 induced immune suppression. Perhaps the most important unanswered question involves the mechanism of action of immune suppression. As mentioned above, antigens encountered in the skin must be taken up, processed, and transported to the draining lymph node by epidermal Langerhans cells. Once at the draining lymph node, the antigen-bearing Langerhans cell interacts with the T cell. Proliferation, differentiation, and cytokine production occur, and the net result is expansion of a clone of antigen-specific effector T cells. Although not the focus of these initial studies, our data can shed some light on the potential mechanism(s) involved. JP-8 does not appear to be altering the permeability of the skin to prevent antigen uptake or migration of the epidermal Langerhans cell to the draining lymph nodes. This conclusion is based on the data demonstrating that application of the contact allergen directly to the JP-8 treated skin or at a distant site is equally effective at inducing immune suppression. In addition, the identical dose-response curves generated when the hapten is applied to treated skin or at a distant site support the conclusion that an effect on Langerhans cell migration is not involved (Figure 3). We suggest that modulation of cytokine production is the most reasonable explanation for the effects reported here. JP-8 could be working at multiple levels. Because volatile organic chemicals are known to induce keratinocytes (Ullrich, 1997) to produce and secrete immune modulatory cytokines such as IL-10, JP-8 may be activating cytokine release by cells within the skin. This could start a cascade of events, as recently described following ultraviolet radiation (Shreedhar et al., 1998), and the cytokines released by epidermal cells may cause downstream targets such as T cells to release immunosuppressive cytokines such as IL-10. Alternatively, JP-8 may be penetrating through the skin and into the circulation where it directly activates circulating leukocytes to secrete immune suppressive cytokines. It is also equally possible that JP-8 in the circulation may be directly affecting T-cell function and that the cytokine release we note is not involved in the induction of immune suppression. These questions can be addressed by neutralizing the function of IL-10 in vivo and asking what effect this procedure has on JP-8 induced immune suppression. In addition, we can inject JP-8 treated mice with IL-12, a cytokine that counteracts the suppressive activity of IL-10 (Schmitt et al., 1995) and determine whether this treatment reverses immune suppression. On the other hand, if JP-8 is directly affecting the T cells, then we would expect to see alterations in T-cell numbers in the lym-

7 DERMAL EXPOSURE TO JP-8 IS IMMUNOTOXIC 67 phoid organs of the treated mice. Experiments are currently in progress to test these different hypotheses. In summary, data is provided to support the hypothesis that dermal exposure to JP-8 is immune suppressive. The data indicate that a threshold dose of l/mouse given either in a single exposure or over 5 successive days will induce immune suppression. Although the mechanism is not entirely clear, the appearance of the immune regulatory cytokine IL-10 in the serum of JP-8 treated animals suggests that the modulation of cytokine production may be involved. Whether commercial jet fuel (Jet-A), which is similar in composition to JP-8 is also a dermal immunotoxin remains to be seen. However, the findings reported here suggest that care should be taken to minimize dermal exposure to jet fuel to avoid immunotoxicity. ACKNOWLEDGMENTS The author thanks Todd Giese and Heather Lyons for their technical assistance with this project and Dr. Eric Brown, Texas A& M University Institute of Biotechnology, Houston, Texas, for the gift of the B. burgdorferi antigen. This study was supported by a grant from the United States Air Force Office of Scientific Research (F ). The animal facilities at the M. D. Anderson Cancer Center are supported in part by a core grant from the National Cancer Institute (CA 16672). REFERENCES Brown, E. L., Rivas, J. M., Ullrich, S. E., Young, C. R., Norris, S. J., and Kripke, M. L. (1995). Modulation of immunity to Borrelia burgdorferi by ultraviolet irradiation: Differential effect on Th1 and Th2 immune responses. Eur. J. Immunol. 25, Cooper, J. R., and Mattie, D. R. (1996). Developmental toxicity of JP-8 jet fuel in the rat. J. Appl. Toxicol. 16, Harris, D. T., Sakiestewa, D., Robledo, R. F., and Witten, M. (1997a). Immunotoxicological effects of JP-8 jet fuel exposure. Toxicol. 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