www.elsevier.com/locate/ytaap

Toxicology and Applied Pharmacology 195 (2004) 331–338

Platelet activating factor receptor binding plays a critical

role in jet fuel-induced immune suppression

Gerardo Ramos,a,b Nasser Kazimi,a Dat X. Nghiem,a,b

Jeffrey P. Walterscheid,a,b and Stephen E. Ullricha,b,*

aDepartment of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USAbThe Graduate School of Biomedical Sciences, Houston, TX 77030, USA

Received 3 June 2003; accepted 29 July 2003

Abstract

Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-

dependant manner. The release of biological response modifiers, particularly prostaglandin E2 (PGE2), is a critical step in activating

immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks

immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2

production and PGE2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced

immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE2 secretion. Jet fuel-induced

PGE2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating

the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet

fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative

stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with

immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function.

Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic

environmental agents that target the skin.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Jet fuel; Immune suppression; Platelet-activating factor; Prostaglandins

Introduction rather the product is refined to meet a series of perfor-

During the past decade, the United States Air Force and

the Air Forces of the NATO allies began a gradual

conversion to a new jet fuel, JP-8. Commercial and

military jet fuels are complex mixtures of hydrocarbons

whose exact compositions are not rigorously defined, but

0041-008X/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.taap.2003.07.014

Abbreviations: BHT, beta-hydroxy toluene; c-PAF, carbamyl platelet-

activating factor; DTH, delayed-type hypersensitivity; JP-8, jet propulsion-

8 military jet fuel; Jet-A, commercial jet fuel; Oct Br., octylonium bromide;

PAF, platelet-activating factor.

* Corresponding author. Department of Immunology, The University of

Texas MD Anderson Cancer Center, PO Box 301402, Houston, TX 77030-

1903. Fax: +1-713-563-3357.

E-mail address: sullrich@mdanderson.org (S.E. Ullrich).

mance specifications (White, 1999). The new generation of

military jet fuel is refined to have a higher flash point, a

lower vapor pressure, and a lower freezing point than

previous fuels. The net result is a safer fuel, one that is

less combustible and more resistant to exploding than

previous fuels, and also, one that performs better at high

altitudes. The lower vapor pressure also minimizes evap-

orative loses during storage, a serious financial and logis-

tical concern.

Initial toxicological studies of JP-8 suggested that the

new fuel presented minimal adverse effects (Kinkead et al.,

1992; Mattie et al., 1991, 1995). However, as the conver-

sion to JP-8 progressed, reports of health problems by

personnel exposed to the fuel, especially engine mechanics,

fuel handlers, and flight line personnel, prompted further

examinations of JP-8 toxicity. It is now apparent that JP-

G. Ramos et al. / Toxicology and Applied Pharmacology 195 (2004) 331–338332

8 has subtle effects on the nervous system (Smith et al.,

1997), more serious effects on pulmonary function (Hays et

al., 1995; Pfaff et al., 1995; Robledo et al., 2000) and

induces dermal toxicity (McDougal et al., 2000; Monteiro-

Riviere et al., 2001). Furthermore, treating cell lines with

JP-8 induces DNA damage (Grant et al., 2001) and

oxidative stress (Boulares et al., 2002). Immune function

appears to be particularly sensitive to JP-8-induced toxicity.

The two major routes of human jet fuel exposure are

inhalation of aerosols and vapors and absorption through

the skin. Pulmonary (Harris et al., 1997a,b,c, 2000) and

dermal (Ullrich, 1999) exposure to JP-8 induced immune

suppression. Moreover, immune toxicity is noted much

before toxic effects are found in other organ systems

(Harris et al., 1997c).

One mechanism responsible for immune suppression

following dermal JP-8 exposure is the induction of immune

regulatory cytokines and biological response modifiers.

Interluekin-10 is found in the serum of JP-8-treated mice,

and neutralizing IL-10 activity with anti-IL-10 monoclonal

antibody blocked JP-8-induced immune suppression (Ull-

rich and Lyons, 2000). In addition, administering a selec-

tive cyclooxygenase-2 inhibitor to JP-8-treated mice

(Ullrich and Lyons, 2000), or mice treated with commercial

jet fuel (Jet-A), reversed jet fuel-induced immune suppres-

sion (Ramos et al., 2002). These findings imply that

prostaglandin E2 (PGE2) production is an essential early

step in the sequence of events that leads to immune

suppression.

The essential role that PGE2 plays in jet fuel-induced

immune suppression caused us to focus our attention on

platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glyc-

ero-3-phosphocholine). PAF is a phospholipid inflammato-

ry mediator with a wide variety of biological effects. In

addition to platelets, PAF activates monocytes, mast cells,

and polymorphonuclear leukocytes. It also induces the

migration of granulocytes to sites of inflammation and is

mitototic for fibroblasts and lymphocytes (Prescott et al.,

2000). PAF plays a role in cell-to-cell communication in a

variety of different organ systems, including the vascular

system, the central nervous system, the endocrine system,

and the gastrointestinal tract. Cells responsive to PAF

express a seven transmembrane spanning G-coupled pro-

tein. Binding of PAF to its receptor induces a variety of

intracellular events, including the activation of the mito-

gen-activated protein kinase pathway, the activation of

phospholipases, and the biosynthesis of a variety of

cytokines and prostaglandins (Ishii and Shimizu, 2000).

Of particular interest are the findings that PAF-receptor

binding results in the up-regulation of cyclooxygenase-2

expression (Pei et al., 1998) and increased transcription of

cyclooxygenase-2 and IL-10 genes (Walterscheid et al.,

2002). These observations, coupled with the fact that

blocking PAF-receptor binding prevents the activation of

immune suppression by another dermal immunotoxin (UV

radiation) (Walterscheid et al., 2002), prompted us to

question whether PAF-receptor binding is an important

step in jet fuel-induced immune suppression.

Materials and methods

Animals. Specific pathogen-free female C3H/HeNCr

(MTV�) mice were obtained from the National Cancer

Institute Frederick Cancer Research Facility Animal Pro-

duction Area (Frederick, MD). The animals were main-

tained 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

matched. The mice were 8–10 weeks old at the start of

each experiment.

Reagents and cell lines. The metabolically stable analogue

of PAF, carbamyl-PAF (c-PAF) and the specific PAF-recep-

tor antagonists, PCA-4248, CV-3988; trans-2,5-bis (3,4,5-

trimethoxyphenyl)-1,3-dioxolone (hereafter referred to as

dioxolone), and octylonium bromide (Oct Br) were pur-

chased from Biomol Research Labs, Plymouth Meeting, PA.

Stock solutions were prepared in 50% DMSO/PBS and

diluted further in PBS or tissue culture medium immediately

before use. Vitamin C, Vitamin E, and beta-hydroxy toluene

(BHT) were purchased from Sigma Chemical Co (St. Louis,

MO). The transformed murine keratinocyte cell, PAM 212,

was acquired from Dr. Stuart Yuspa, NCI. Tissue culture

medium was obtained from Gibco BRL (Grand Island, NY);

bovine calf serum was purchased from Hyclone Laborato-

ries (Logan, UT).

Application of jet fuels. Military jet fuel, JP-8 (lot #

3509) and commercial jet fuel (Jet-A) were acquired from

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

the fuels. The undiluted fuel (800 Ag/ml) was applied

directly to the shaved dorsal skin of the animals, as

described previously (Ullrich, 1999). 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. 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.

Fig. 1. PGE2 secretion by jet fuel-treated keratinocytes. Semi-confluent

keratinocyte cultures were treated with 0.1 Ag/ml of JP-8 or Jet-A for 1 h

at 37jC, in the presence or absence of PCA-4248, a specific PAF receptor

antagonist. The cells were then washed and re-plated in cRPMI medium.

Eighteen to 24 h later, supernatants were removed and PGE2 levels were

determined by ELISA. Results are expressed as pg/ml F SD. * indicates

a statistically significant difference ( P < 0.05) versus the Jet-A only-

treated control. # indicates a statistically significant difference ( P < 0.05)

versus the JP-8 only-treated control. A representative experiment is

shown, this experiment was repeated three times and similar results were

obtained each time.

plied Pharmacology 195 (2004) 331–338 333

Suppression of the elicitation of delayed-type hypersensi-

tivity by jet fuel exposure. On day 0, mice were immu-

nized by subcutaneous injection of 107 formalin-fixed

Candida albicans into each flank. Ten days later, each

hind footpad was measured with an engineer’s micrometer

(Mitutoyo, Tokyo, Japan) and then challenged by intra-

footpad injection of 50 Al of Candida antigen (Alerchek

Inc., Portland, ME). Eighteen to 24 h after challenge, the

thickness of each foot was re-measured, and the mean

footpad swelling for each mouse was calculated (left foot +

right foot H 2). The background footpad swelling (neg-

ative control) was determined in a group of mice that were

not immunized but were challenged. The specific footpad

swelling response was calculated by subtracting the back-

ground response observed in the negative controls from the

mean footpad swelling found in mice that were immunized

and challenged. There were five mice per group; the mean

footpad thickness for the group F the standard error of the

mean was calculated. Statistical differences between the

control and experimental groups was determined using a

one-way analysis of variance (ANOVA) followed by the

Dunnett’s multiple comparison test (GraphPad Software,

San Diego, CA). Percentage immune suppression was

determined by the following formula: % immune suppres-

sion = 1 � [specific footpad swelling of the jet fuel-

treated mice H specific footpad swelling of the positive

control] � 100. For the most part, the jet fuel was applied

to the dorsal skin of the mice 1 day before challenge.

Previous studies have indicated that applying the jet fuel

on days 6 through 9 post-immunization will suppress the

elicitation of delayed-type hypersensitivity (DTH) (Ramos

et al., 2002).

PGE2 analysis. Five million Pam 212 cells were sus-

pended in 5 ml of RPMI 1640 medium containing 10%

bovine calf serum and added to 100-mm tissue culture

dishes. Eighteen to 24 h later, the medium was removed;

the sub-confluent monolayer was washed with PBS and

treated with jet fuel, in the presence or absence of PCA-

4248. In initial experiments, keratinocyte monolayers were

treated with different concentrations of the jet fuel (2.0–

0.02 Ag/ml), and the cell viability was determined by the

MTT assay. In all subsequent experiments, we used the

highest dose of jet fuel (0.1 Ag/ml) that did not induce

overt cellular toxicity (data not shown). As positive con-

trols, the keratinocytes were exposed to 100 pmol of c-

PAF as described previously (Walterscheid et al., 2002).

One hour later, the medium was removed, the cells were

washed and resuspended in complete RPMI 1640. Back-

ground responses were measured in supernatants from cells

that were not treated with jet fuel, but were otherwise

treated in an identical manner. Eighteen to 24 h later, the

supernatants were collected and PGE2 levels were deter-

mined by a PGE2-specific ELISA according to the manu-

facturer’s instructions (Cayman Chemical, Ann Arbor MI).

The limit of detection is approximately 30 pg/ml. Each

G. Ramos et al. / Toxicology and Ap

sample was measured in triplicate; the data are presented

as the mean F the standard deviation of the samples.

Statistical differences between the jet fuel-treated controls

and the PAF-receptor antagonist-treated groups were de-

termined by the use of the two-tailed Student’s t test

(GraphPad Software).

Results

Keratinocytes produce PGE2 following the application of jet

fuel

Previously, we reported that injecting jet fuel-treated

mice with a selective cyclooxygenase-2 inhibitor blocked

immune suppression (Ramos et al., 2002; Ullrich and

Lyons, 2000). These data imply that dermal jet fuel

treatment activates keratinocytes to secrete PGE2. To

directly test this hypothesis, keratinocyte cultures were

treated with jet fuel and PGE2 secretion was measured

(Fig. 1). Prostaglandin E2 was found in supernatants

isolated from keratinocytes cultures treated with JP-8 and

Jet-A. Because PAF-receptor binding up-regulates eicosa-

noid secretion by keratinocytes (Pei et al., 1998), we also

tested the role of PAF-receptor binding in jet fuel-induced

PGE2 secretion. To do this, keratinocytes were pretreated

with a specific PAF-receptor antagonist (PCA-4248; 1 h at

G. Ramos et al. / Toxicology and Applied Pharmacology 195 (2004) 331–338334

37jC), washed with PBS, and then treated with JP-8, Jet

A, or c-PAF. Induction of PGE2 release by JP-8, Jet A,

and/or PAF, coupled with the inhibition of jet fuel-induced

PGE2 secretion by the PAF-receptor antagonist (Fig. 1),

suggests that jet fuel treatment activates PGE2 secretion by

a PAF-receptor binding-dependent pathway.

PAF suppresses DTH in vivo

Next we wanted to determine if PAF suppresses the

elicitation of DTH in vivo (Fig. 2). Mice were injected with

c-PAF 1 day before challenge. As positive controls for

suppressor activity in this experiment, two other groups of

mice were treated with immunosuppressive doses of Jet-A

and JP-8 (Ramos et al., 2002). Injecting c-PAF suppressed

DTH in a dose-dependent fashion. Significant immune

suppression was observed in mice injected with 1 and 10

nmol of c-PAF. At the higher dose, the immune suppression

observed was indistinguishable from the suppression ob-

served in jet fuel-treated mice. These data indicate that PAF

can suppress the elicitation of DTH.

Platelet-activating receptor antagonists block jet

fuel-induced immune suppression

In the next series of experiments, we wanted to determine

whether blocking PAF-receptor binding would interfere

with jet fuel-induced immune suppression. Mice were

immunized with C. albicans on day 0, and treated with an

Fig. 2. PAF suppresses the elicitation of DTH. Mice were injected with c-

PAF or treated with JP-8 or Jet-A 1 day before challenge. The background

response (negative control) was measured in mice that were not immunized

but were challenged. The positive control was measured in mice that were

immunized and challenged. Results are expressed as means F SEM. The

asterisk (*) indicates a statistically significant difference ( P < 0.005) from

the positive control. A representative experiment is shown, this experiment

was repeated twice and similar results were obtained each time.

Fig. 3. A specific PAF receptor antagonist blocks JP-8-induced immune

suppression. Mice were treated with an immunosuppressive dose of JP-8

(A) or Jet-A (B) 1 day before challenge. Two hours before jet fuel

treatment, 10–200 nmol of PCA-4248 was injected into the peritoneal

cavity. Results are expressed as means F SEM. The asterisk (*) indicates a

statistically significant difference ( P < 0.01) from the positive control. A

representative experiment is shown, this experiment was repeated three

times and similar results were obtained each time.

immunosuppressive dose of JP-8 (Fig. 3A) or Jet-A (Fig.

3B) on day 9. Groups of mice were injected (intraperitoneal)

with a specific PAF-receptor antagonist, PCA-4248, 2

h before jet fuel treatment. As expected, treating the mice

with JP-8 or Jet-A caused significant immune suppression

(P < 0.01 vs. the positive control). Injecting 10 nmol of

PCA-4248 into the jet fuel-treated mice had no effect on

G. Ramos et al. / Toxicology and Applied Pharmacology 195 (2004) 331–338 335

immune suppression (P < 0.05 vs. the positive control).

When the mice received 100 nmol of PCA-4248, the DTH

reaction was reversed to a degree; however, it was still

significantly different from the positive control (P < 0.01).

Complete restoration of DTH was noted when the mice

were injected with 200 nmol of PCA-4248, in that the

response generated was not significantly different from that

found in the positive control.

To confirm these results, and to ensure that the effects we

see are not unique to PCA-4248, we repeated this experi-

ment using three other structurally diverse PAF-receptor

antagonists, CV-3988, dioxolone, and Oct Br (Corey et al.,

1988; Fernandez-Gallardo et al., 1990; Terashita et al.,

1985). Two hours before jet fuel exposure, the mice were

injected with 200 nmol of the PAF-receptor antagonist and

their effect on the suppression of DTH was measured. In

Fig. 4, panel A, the PAF-receptor antagonists were injected

into mice that were immunized, but not treated with jet fuel.

Injecting the PAF-receptor antagonists into non-jet fuel-

Fig. 4. Structurally diverse PAF receptor antagonists block jet fuel-induced

immune suppression. Mice received intraperitoneal injections of 200 nmol

of PCA-4248, CV-3988, dioxolane or Oct Br, 24 h before challenge. In

panel A, the mice received no further treatment. In panel B, the mice were

treated with Jet-A, 2 h after injection of the PAF receptor antagonists. In

panel C, the mice were treated with JP-8, 2 h after injection of the PAF

receptor antagonists. Results are expressed as means F SEM. The asterisk

(*) indicates a statistically significant difference ( P < 0.002) from the

positive control. A representative experiment is shown, this experiment was

repeated three times and similar results were obtained each time.

Fig. 5. Vitamin C reverses JP-8-induced immune suppression. Mice were

treated with JP-8 1 day before challenge. Two hours before JP-8 treatment,

the mice received intraperitoneal injections of 10–1000 nmol of Vitamin C.

Results are expressed as means F SEM. The asterisk (*) indicates a

statistically significant difference ( P < 0.002) from the positive control. A

representative experiment is shown, this experiment was repeated three

times and similar results were obtained each time.

treated mice had no effect on the immune response (P >

0.05 vs. the positive control). The effects of the structurally

diverse PAF-receptor antagonists on jet fuel-induced im-

mune suppression are found in Fig. 4, panels B and C. As

before, treating the mice with Jet-A (Fig. 4B) or JP-8 (Fig.

4C) suppressed DTH. No immune suppression was noted,

however, in mice treated with jet fuel and injected with any

of the PAF-receptor antagonists. These data indicate that

pharmacological blockade of the PAF receptor with any of

four structurally diverse receptor antagonists prevents jet

fuel-induced immune suppression.

Reversal of jet fuel-induced immune suppression with

anti-oxidants

Because dermal exposure to jet fuel induces oxidative

stress (Rogers et al., 2001), we wanted to determine whether

anti-oxidant treatment would reverse jet fuel-induced im-

mune suppression. Mice were immunized with C. albicans

on day 0 and treated with JP-8 on day 9. Two hours before

JP-8 treatment, the mice were injected i.p. with different

doses of Vitamin C. As shown in Fig. 5, we noted a dose-

dependent inhibition of JP-8-induced immune suppression.

At the lowest dose of Vitamin C injected (10 nmol), no

effect was noted (P < 0.002 vs. the positive control).

When, however, the mice were injected with 10- or 100-

fold more Vitamin C, no immune suppression was observed.

The DTH reaction in these mice was indistinguishable from

the positive control.

G. Ramos et al. / Toxicology and Applied Pharmacology 195 (2004) 331–338336

We extended these findings in the next experiment by

adding two additional anti-oxidants, Vitamin E and BHT,

and asking whether anti-oxidant treatment interferes with

Jet-A-induced immune suppression (Fig. 6). The data pre-

sented in panel A confirm that injecting the anti-oxidants

into non-jet fuel-treated mice has no suppressive effect. In

panel B, we examined the effect that Vitamin C, Vitamin E,

and BHT had on Jet-A-induced immune suppression. When

the mice were injected with 100 nmol of any one of the

three anti-oxidants, 2 h before Jet-A treatment, no immune

suppression was observed. The response found in these

mice was statistically similar to that found in the positive

control (P > 0.05). Similar results were obtained when 100

nmol of Vitamin C, Vitamin E, and/or BHT was injected

Fig. 6. Treatment with anti-oxidants blocks jet fuel-induced immune

suppression. Mice received intraperitoneal injections of 100 nmol of

Vitamin C, Vitamin E or BHT, 24 h before challenge. In panel A, the

mice received no further treatment. In panel B, the mice were treated with

Jet-A, 2 h after receiving the anti-oxidants. In panel C, the mice were

treated with JP-8, 2 h after receiving the anti-oxidants. Results are

expressed as means F SEM. The asterisk (*) indicates a statistically

significant difference ( P < 0.001) from the positive control. A

representative experiment is shown, this experiment was repeated three

times and similar results were obtained each time.

into JP-8-treated mice. All three anti-oxidants blocked JP-8-

induced immune suppression. Because oxidative stress also

stimulates the production of PAF and PAF-like oxidized

phospholipids (Lewis et al., 1988; Marathe et al., 1999) that

can signal through the PAF receptor (Davies et al., 2001;

McIntyre et al., 1999), these findings are consistent with the

data presented above suggesting a role for PAF-receptor

binding in jet fuel-induced immune suppression.

Discussion

The focus of the studies presented here was to deter-

mine the mechanisms underlying the activation of immune

suppression following the dermal application of military

and commercial jet fuels. Results from previous studies

imply that PGE2 production, presumably by jet fuel-treated

keratinocytes, is a critical early step in jet fuel-induced

immune suppression (Ramos et al., 2002; Ullrich and

Lyons, 2000). Here, we provide direct evidence that

PGE2 is secreted by jet fuel-treated keratinocytes. More-

over, we demonstrate that injecting jet fuel-treated mice

with PAF-receptor antagonists blocks jet fuel-induced

immune suppression. Furthermore, we show that treating

mice with anti-oxidants will block jet fuel-induced immune

suppression. These data indicate that PAF-receptor binding

is an essential early step in the cascade of events that leads

to jet fuel-induced immune suppression.

As mentioned above, jet fuels are complex chemical

mixtures, containing more than 200 aliphatic and aromatic

hydrocarbons. The precise composition of a jet fuel will

vary from batch to batch, as the fuel is refined to meet

performance specifications (White, 1999). Military jet fuel,

JP-8, is essentially commercial jet fuel (Jet-A) to which

three components are added, an anti-static agent, an anti-

corrosive agent, and an anti-freeze. Although the immune

suppressive effects of JP-8 are well documented (Harris et

al., 1997a,c, 2000; Ullrich, 1999; Ullrich and Lyons,

2000), less is known about the effect of Jet-A on the

immune response (Ramos et al., 2002). This is somewhat

surprising in view of the fact that commercial airlines in

the United States are estimated to use in excess of 68

million gallons of Jet-A a day (White, 1999). Our data

clearly demonstrate that commercial Jet-A induces immune

suppression. In addition to our previous results, showing

almost identical dose-response curves for JP-8 and Jet-A-

induced immune suppression (Ramos et al., 2002), the data

presented here, showing that both JP-8 and Jet-A activate

keratinocytes to secrete PGE2, coupled with the fact that

anti-oxidants and PAF-receptor antagonists interfere with

both JP-8 and Jet-A induced immune suppression, support

our previous conclusion that jet fuel-induced immune

suppression is a function of the base kerosene fuel, Jet-

A. What remains unresolved is the identity of the exact

hydrocarbon, or mixture of hydrocarbons, which activates

immune suppression.

G. Ramos et al. / Toxicology and Applied Pharmacology 195 (2004) 331–338 337

Synthesis of PGE2 is a critical step in wound healing

and scar formation (Reno et al., 2001; Rys-Sikora et al.,

2000), the immune suppression associated with burns

(Schwacha et al., 2002), the immune suppression induced

by some chemical carcinogens (Andrews et al., 1991), and

the sunburn and immune suppression caused by UV

radiation (Rhodes et al., 2001; Shreedhar et al., 1998).

We suggest that a common mechanism underlying PGE2

synthesis in all these examples of dermal trauma is

activation via the PAF receptor. Cells that secrete PAF,

such as keratinocytes, do not contain preformed stores of

the molecule, but rapidly synthesize PAF in response to

cellular stress. Secreted PAF then binds to PAF receptors

on target cells, inducing downstream effects such as

calcium flux, activation of the MAP kinase cascade, and

activation of phospholipase-A2 to cleave arachidonic acid

from the cell membrane. An acetyl residue is then trans-

ferred from Acetyl-CoA to the free hydroxyl of arachi-

donic acid to form biologically active PAF. In addition,

arachidonic acid servers as a substrate for PAF-induced

cyclooxygenase-2. This series of events ultimately results

in the secretion of PGE2 and a variety of inflammatory and

immune regulatory cytokines (Pei et al., 1998). Manipu-

lating this pathway may have practical implications. For

example, the systemic immune suppression associated with

burns may be blocked by PAF-receptor antagonists, in a

manner similar to the inhibition of immune suppression we

describe here.

Perhaps the most successful public health campaign of

the 20th century was the widespread use of vaccination to

prevent the morbidity and mortality due to infectious dis-

eases. Previously, we demonstrated that dermal jet fuel

exposure suppresses secondary immune reactions (Ramos

et al., 2002). We believe that this is a very relevant model to

study jet fuel-induced immune toxicity. Jet fuel-induced

immune suppression of the elicitation of DTH suggests that

jet fuel exposure has the potential to weaken resistance to

infectious disease afforded by previous immunization. This

may be particularly relevant in situations such as combat,

where stress and fatigue, coupled with jet fuel-induced

immune suppression, may contribute to a weakened im-

mune response.

Unfortunately, minimizing jet fuel exposure may not be

easy. As mentioned above, the active moieties within jet

fuel (both JP-8 and Jet-A) that induce immunotoxicity

remain unknown. So removing them from the blend of

hydrocarbons that constitute jet fuel is impossible at the

present time. Spills during refueling, contact with residual

fuel during fuel tank and/or engine maintenance, fuel leaks,

and exposure of flight line personnel to un-burnt fuel

during ‘‘cold’’ engine starts are common ways in which

Air Force and commercial airline employees encounter jet

fuel. Because risk avoidance is probably impossible, it may

be more useful to attempt to block the induction of immune

suppression. Previously, we demonstrated that blocking

PGE2 production, with selective cyclooxygenase-2 inhib-

itors, very effectively blocked jet fuel-induced immune

suppression (Ramos et al., 2002; Ullrich and Lyons,

2000). In this study, we targeted an event up-stream of

PGE2 production, PAF-receptor binding, and saw similar

results. Although the safety of PAF-receptor antagonists in

humans is unknown, blocking jet fuel-induced immune

suppression with anti-oxidants, such as Vitamins C and

E, has the potential to be useful in protecting humans

against jet fuel-induced immunotoxicity.

Acknowledgments

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, The Department of Defense,

or the US Government. This work was supported by grants

from the United States Air Force Office of Scientific

Research (F49620-02-0121) and the National Cancer

Institute (CA 075575). Gerardo Ramos was supported by

the USAF Institute of Technology. The animal facilities at

the MD Anderson Cancer Center are supported in part by

a core grant from the NCI (CA 16672).

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