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Published ahead of print on May 11, 2006, doi:10.1165/rcmb.2006-0059OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 479-487, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2006-0059OC

Expression of JP-8–Induced Inflammatory Genes in AEII Cells Is Mediated by NF-{kappa}B and PARP-1

Luis A. Espinoza, Fnu Tenzin, Andrea O. Cecchi*, Zun Chen, Mark L. Witten and Mark E. Smulson

Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC; and Joan B. and Donald R. Diamond Lung Injury Laboratory, Department of Pediatrics, The University of Arizona Health Sciences Center, Tucson, Arizona

Correspondence and requests for reprints should be addressed to Dr. Mark E. Smulson, Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, 3900 Reservoir Road NW, Washington, DC 20057. E-mail: smulson{at}georgetown.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Lung epithelial cells are critical in the regulation of airway inflammation in response to environmental pollutants. Altered activation of NF-{kappa}B is associated with expression of several proinflammatory factors in respiratory epithelial cells in response to an insult. Here we show that a low threshold dose (8 µg/ml) of the jet fuel JP-8 induces in a rat alveolar epithelial cell line (RLE-6TN) a prolonged activation of NF-{kappa}B as well as the increased expression of the proinflammatory cytokines TNF-{alpha} and IL-8, which are regulated by NF-{kappa}B. The up-regulation of IL-6 mRNA in cells exposed to JP-8 appears to be a reaction of RLE-6TN cells to reduce the enhancement of proinflammatory mediators in response to the fuel. Moreover, lung tissues from rats exposed to occupational levels of JP-8 by nasal aerosol also showed dysregulated expression of TNF-{alpha}, IL-8, and IL-6, confirming the in vitro data. The poly(ADP-ribosyl)ation of PARP-1, a coactivator of NF-{kappa}B, was coincident with the prolonged activation of NF-{kappa}B during JP-8 treatment. These results evidenced that a persistent exposure of the airway epithelium to aromatic hydrocarbons may have deleterious effects on pulmonary function.

Key Words: epithelial cells • inflammation • JP-8 jet fuel • NF-{kappa}B • PARP-1


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Persistent exposure of the airway epithelium to environmental pollutants, such as aromatic hydrocarbons, may have deleterious effects on pulmonary function (1) and pathologic changes to lung tissue (2, 3). Previous studies in our laboratory have shown that JP-8 jet fuel (mixture of aliphatic and aromatic hydrocarbons) at 80 µg/ml induces in alveolar epithelial type II (AEII) cells the generation of high levels of reactive oxygen species (ROS) and the depletion of intracellular glutathione (GSH) (4), followed by the induction of biochemical and morphologic markers of apoptotic cell death. These included caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, cytochrome c release from mitochondria, and genomic DNA cleavage into both oligonucleosomal (DNA ladder) and high-molecular-weight (HMW) fragments (5). Furthermore, nasal exposure of mice lung to low concentrations of aerosolized JP-8 had adverse effects on pulmonary function, increasing pulmonary resistance and permeability, as well the accumulation of inflammatory cells (6, 7).

We have recently described that a short-term exposure of rat pulmonary airways with JP-8, under conditions that mimic the occupational exposure of Air Force personnel to this fuel, evidenced marked changes specifically in the expression of various genes whose functions are related to defense against oxidative and toxicant-induced stress in lung tissue (3). Among the most affected alveolar cells after chronic exposure by inhalation to JP-8 jet fuel aerosol/vapor were lung AEII cells and pulmonary alveolar macrophages (PAM) (6, 8), which are characterized by a fuel-induced lung inflammatory state. In support, roles for PARP in the inflammatory process have also recently been described (9). In addition, the immunomodulatory role of AEII cells in the secretion of cytokines is a critical inflammatory mechanism of these cells in response to stressors (10, 11). The prolonged production of inflammatory mediators by epithelial cells, together with the proteases produced by activated macrophages and neutrophils, are capable of producing a sustained immune response with increased risk of lung damage.

PARP-1 is an enzyme that binds nonspecifically to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of various nuclear proteins using NAD+ as a substrate, with PARP-1 itself being one of the major targets of this modification (12, 13). PARP-1 plays pleiotropic roles in various nuclear processes, including DNA repair, DNA replication, and apoptosis (13, 14). PARP-1 has also been reported to be a coactivator of NF-{kappa}B, a transcription factor that controls the expression of a variety of genes involved in inflammatory responses, cell division, and apoptosis (15). Acetylated PARP-1, NF-{kappa}B, and p300 form a complex, which affects both NF-{kappa}B transactivation and binding to proinflammatory promoters (16).

Recently, our laboratory showed that prevention of PARP-1 activation, using either PARP-1 knockout mice or the PARP-1 inhibitor, 3-aminobenzamide (3-AB), protects against both ROS-induced airway epithelial cell injury in vitro and in an airway asthma inflammation model in vivo (17). The inhibition of NF-{kappa}B induction and expression of IL-8 in this study support the notion that PARP-1 contributes to the NF-{kappa}B signaling pathway operative in lung inflammation.

The NF-{kappa}B transcriptional activity is under control by an inducible feedback pathway regulated by I{kappa}B proteins in the cytoplasm (18). A variety of stress signals induce phosphorylation with the subsequent ubiquitination and degradation of I{kappa}B by proteasomes, which has been reported to promote the nuclear translocation and binding of NF-{kappa}B to specific {kappa}B consensus sequences. This leads to the activation of specific subsets of genes, such as those that encode cytokines, including TNF-{alpha}, IL-1, IL-6, and IL-8 (15).

In the present work we investigated the hypothesis that at a lower threshold dose of JP-8 (8 µg/ml), compared with that which induces apoptosis (80 µg/ml) (4, 5), induces the expression of proinflammatory mediators regulated by NF-{kappa}B in respiratory epithelial cells. Here, we demonstrate that with prolonged exposure of pulmonary epithelial cells, but at a low concentration of JP-8, there is a sustained NF-{kappa}B activation. The observed activation of PARP-1 in cells exposed to the fuel indicates that long exposure to the fuel induces DNA damage in lung epithelial cells. In this context, we hypothesize that a persistent PARP-1 and NF-{kappa}B activation by a continuous exposure of pulmonary epithelial cells to JP-8 jet fuel can lead to chronic inflammatory responses and result in lung damage.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
Primary antibodies used were: anti-I{kappa}B-{alpha}, anti–phospho-I{kappa}B-{alpha} (phosphorylated on Ser32), anti–Lamin A, anti-p50, anti-p65 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); anti-IKKbeta (phosphorylated on Ser181; New England Biolabs, Beverly, MA); anti-iNOS, anti-PARP (BD Transduction Laboratories, San Diego, CA); anti-GADPH (Ambion, Austin, TX), and anti-GST-P (Stressgen Bioreagents, Victoria, BC, Canada).

Cell Culture and Treatment
The rat lung epithelial cell line (RLE-6TN) supplied by the ATCC was grown in DMEM and Ham's F12 (1:1; Invitrogen, Carlsbad, CA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum and 100 U/ml penicillin–streptomycin in a humidified 5% CO2. Cells growing logarithmically were treated with JP-8 solution (prepared in absolute ethanol) in fresh media. The 8 µg/ml concentration of JP-8 (> 98% purity; density = 0.8 kg/liter) diluted in ethanol was used in all our experiments. Control cells were exposed with equivalent volumes of absolute ethanol.

JP-8 Aerosol Generation and Animal Exposure
An aerosol of JP-8 (Wright-Patterson Air Force Base, Dayton, OH) was generated with an Ultra-Neb 99 nebulizer (DeVilbiss, Somerset, PA) and was allowed to mix with ambient air. The fuel–air mixture was drawn through a 12-port nose-only exposure chamber (IN-TOX, Albuquerque, NM) with the use of a constant vacuum. The JP-8 aerosol was characterized as described previously (19). We employed four animals per group (treated and control), which represents four separate tissue samples per group. Consequently, the number of samples we used in our experiments consistently result in > 2 degrees of freedom, thus providing valid information. Fischer 344 rats were exposed for 1 h/d for 7 d to JP-8 at a mean concentration of 352 mg/m3 that became 44 mg/m3/h. This treatment corresponds to a typical human exposure over a 7-d work period at an air base (20, 21). Occupational personnel can be exposed to even higher levels of JP-8, such as 4,000 mg/m3 for 1 h, which is an 11.36-fold higher exposure level than the rats that were exposed to a 1-h for 7 d. Accordingly, Air Force personnel exposed to 4,000 mg/m3 JP-8 for 1 h could be exposed to 32,000 mg/m3 total exposures in an 8-h period. In our study, the current permissible exposure level for JP-8 was 350 mg/m3. The animals were subjected to nose-only exposure to minimize oral ingestion of JP-8 during grooming and to simulate more closely occupational exposure in humans. A group of control rats was exposed in a similar manner to air only. At the end of the 7-d exposure period, the lungs of each rat were perfused with ice-cold PBS, removed, and rapidly placed in separate tubes containing RNAlater (Ambion) at room temperature. The rats were killed by CO2 asphyxiation in a CO2 chamber fed by a pressurized tank. All rats were housed in an American Association for Accreditation of Laboratory Animal Care–approved animal facility of the Department of Animal Resources at the University of Arizona Health Sciences Center.

Viability Assay
Cell viability was determined by the calcein method. Cells were treated with JP-8 for different time intervals. Calcein-AM (Molecular Probes, Eugene, OR) was then added directly to the media in each well, without washing, to a final concentration of 2.5 µM and then incubated for 30 min at 37°C. Fluorescence resulting from the deesterification of calcein-AM was monitored with a CytoFluor 4000 fluorometer (PerSeptive Biosystems, Framingham, MA) at excitation and emission wavelengths of 488 and 520 nm, respectively. Cell viability was presented as percentages relative to control samples.

Assay for GSH
Lysates of cells treated and respective untreated controls were depleted of protein with 10% sulfosalicylic. We used supernatants for spectrophotometric determination of total glutathione (GSH+GSSG) based on the enzymatic recycling of the DTNB (5,5'-dithio-bis-[2-nitrobenzoic acid]) in the presence of glutathione reductase at 412 nm. In each experiment, standard curves were prepared using known amounts of GSH as described (4).

Measurement of ROS Production
To monitor the generation of ROS, cells exposed to JP-8 were incubated with 10 µM of the fluorogenic probe dichlorodihydro-fluorescein diacetate H2DCF for 15 min in the dark. In the presence of ROS, the nonfluorescent H2DCF (diacetate form) is oxidized to the highly fluorescent DCF. Fluorescence was measured at excitation and emission wavelengths of 485 and 530 nm, respectively.

Western Blot Analysis
Cells were either left treated or untreated with JP-8 at the specific indicated time points. Cells were lysed in ice-cold buffer as described (22). Forty micrograms of protein per lane was subjected to SDS-PAGE analysis, followed by transfer to nitrocellulose membrane. Thereafter, membranes were probed with the respective primary antibody. Proteins were visualized with horseradish peroxidase–conjugated secondary antibodies (Amersham, Piscataway, NJ) followed by use of ECL chemilomuniscence (Pierce, Rockford, IL). Gel loadings were normalized with GADPH or Lamin-A antibody.

Isolation of RNA
Total RNA was extracted from cells and both lungs of each animal with Trizol reagent (Invitrogen) and was purified with the use of an RNeasy Mini Kit (Qiagen, Valencia, CA). The concentration of the isolated RNA were determined by measurement of absorbance at 260 nm, and its high quality was verified by electrophoresis on a 1% agarose gel and ethidium bromide staining.

Semiquantitative RT-PCR Analysis
cDNAs were synthesized from 0.5 µg of total RNA using the OneStep RT-PCR kit (Qiagen). Efficient cDNA synthesis was achieved with 30 min incubation at 50°C. The Primer3 Input software package was used to design specific primers. PCR amplifications were started with an initial heating step at 95°C for 15 min to activate HotStart Taq DNA polymerase and to simultaneously inactivate the reverse transcription, followed by 30 cycles. Each cycle comprised denaturation at 94°C for 15 s, annealing at 60–65°C (depending on the primer set) for 30 s, and the extension at 70°C for 1 min, with a 10-min final extension at 72°C. PCR products were separated in 1.5% agarose gel electrophoresis, visualized by ethidium bromide staining, and the images were digitized on a DC120 Camera (Kodak, Rochester, NY). Reproducibility of the method was confirmed by repeating the RT-PCR three times, and the results have shown to be consistent.

Electrophoretic Mobility Shift Assay
The electrophoretic mobility shift assay (EMSA) analysis of DNA-binding activity was performed as described. The oligonucleotide containing the NF-{kappa}B consensus sequence (Promega, Madison, WI) was end-labeled with T4 polynucleotide kinase (New England Biolabs) using [{gamma}32P]ATP and purified by G-25 spin column (Boehringer Mannheim, Indianapolis, IN). Nuclear extracts were prepared using the NE-PER nuclear and cytoplasmic extraction reagent (Pierce). For binding assays, 5 µg of nuclei extract was added to binding buffer (10 mM Tris, pH 7.5, 0.5 mM EDTA, 1 mM MgCl2, 4% glycerol, 0.05 mg/ml of poly[dI-dC]·poly[dI-dC], and 0.5 mM DTT), and incubated with 32P-labeled DNA probe (100,000 cpm) in a final volume of 20 µl for 30 min at 37°C. The DNA-protein complexes were then analyzed by electrophoresis on 4% DNA retardation gel (Invitrogen) in 0.5X TBE buffer, transferred to Biodyne nylon membrane (Pierce), and subjected to autoradiography overnight at -80°C for visualization of NF-{kappa}B signals. To confirm the specificity of NF-{kappa}B signals, a competition binding assay for NF-{kappa}B consensus binding sites with unlabeled (cold) probe was performed. To identify the subunits of NF-{kappa}B responsible for DNA-binding activity, supershift experiments were performed by pre-incubating 2 µL of antibodies targeted to p50 or p65 (Santa Cruz Biotechnology Inc.) with the nuclear extracts for 2 h at 4°C before addition of the radiolabeled probe.

PARP-1 Activity Assay
At indicated time intervals after exposure to JP-8, treated and untreated control cells were harvested and washed with ice-cold PBS. Then cells were incubated in a cold buffer containing 50 mM Tris-Hcl, pH 8.0, 28 mM KCl, 10 mM MgCl2, digitonin 0.01%, 1 mM DTT, and {alpha}32P-NAD (0.5 µCi/ml) for 30 min at room temperature and spined down by centrifugation. Thereafter, cells were lysed in cold-buffer containing 0.1 M NaCl, 0.01 M Tris-HCl pH 7.6, 0.001 M EDTA, aprotinin (10 µg/ml), PMSF (100 g/ml) and clarified by centrifugation. Protein extracts were subjected to SDS-PAGE and transferred to nitrocellulose membrane. PARP-1 activity was visualized by autoradiography.

Statistical Analysis
All the grouped data are expressed as mean ± SD and were evaluated by one-way ANOVA. Statistical significance was determined using the Student's t test. A difference of P >= 0.05 was considered to indicate statistical significance. All the results were expressed as means for three different experiments in each group. Otherwise, representative data are shown where appropriate.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effects of JP-8 on Cell Viability of Respiratory Epithelial Cells
The lung is one of the primary routes of JP-8 exposure; we therefore established an in vitro model allowing the effects of JP-8 on rat epithelial lung cells, as an alternative method to animal studies. Previously, we have studied the molecular mechanisms involved in the JP-8 toxicity at higher doses (80 µg/ml) (4, 5). In contrast, in the current study, we used a nonlethal dose of JP-8 to assess how this much lower dose (8 µg/ml) alters the response of pulmonary epithelial cells. Because the respiratory tract is constantly exposed to environmental pollutants, such as aromatic hydrocarbons, we used in this study the lung epithelial cell line RLE-6TN, which was originated from rat AEII cells and preserves many of the original phenotypic characteristics (23). The calcein-AM staining revealed that incubation of RLE-6TN cells for 1–12 h with JP-8 (8 µg/ml) did not affect cell viability (Figure 1). To determine whether longer exposures times with 8 µg/ml of JP-8 might induce cell death in pulmonary cells, RLE-6TN cells were incubated for 24, 36, and 48 h with the fuel. As shown in Figure 1, longer incubations with 8 µg/ml of JP-8 had no impact on the cell viability of pulmonary epithelial cells. These values were not different to those observed in control cells exposed with the equivalent dilution of ethanol for each time course (Figure 1).


Figure 1
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  		Figure 1. RLE-6TN cells were incubated for the indicated times with 8 µg/ml of JP-8 (open squares), after which cell viability was assessed by measurement of calcein-AM fluorescence. Control cells (filled circles) were incubated in media containing 0.05% of ethanol. Values are expressed as percentage of ethanol-treated cells (control). Data represent means ± SD of three independent experiments performed in duplicate.

 
Effects of JP-8 on Phosphorylation and Degradation of I{kappa}B-{alpha} in Epithelial Cells
To gain insight of the role of pulmonary epithelial cells in the regulation of lung inflammation, we wanted to determine whether NF-{kappa}B is activated in RLE-6TN cultures exposed to JP-8 (8 µg/ml) for 24, 36, and 48 h, respectively. NF-{kappa}B activation in several models is preceded by the phosphorylation of the inhibitory I{kappa}B-{alpha} protein (18). Treatment of RLE-6TN cells with the fuel resulted in a modest increase of I{kappa}B-{alpha} phosphorylation after a 24-, 36-, and 48-h treatment with JP-8 (Figure 2A). We used an antibody that is specific for I{kappa}B-{alpha} phosphorylated at Ser32. Untreated control cells harvested at 48 h had negligible or very low levels of phospho-I{kappa}B-{alpha}. Since the nuclear localization of p65 and p50 is preceded by the phosphorylation and degradation of I{kappa}B-{alpha}, the slight reduction of I{kappa}B-{alpha} levels in a time-dependent manner observed in the total protein extract (Figure 2B) are indicative of the degradation of this cytoplasmic protein by the 26S proteosome (15). These results suggested that JP-8 promotes the phosphorylation and degradation of I{kappa}B-{alpha}, which is a critical step to the activation of NF{kappa}B in RLE-6TN cells treated with JP-8.


Figure 2
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  		Figure 2. Effect of JP-8 on I{kappa}B-{alpha} phosphorylation and degradation in AEII cells. Cells were exposed for 24, 36, and 48 h in the presence or absence of JP-8 (8 µg/ml). Control cells were harvested at 48 h after incubation with ethanol only (MATERIALS AND METHODS). Total protein extracts were analyzed by Western blot using specific antibodies for p-I{kappa}B-{alpha} (A), I{kappa}B-{alpha} (B), and p-IKKbeta (C). To confirm equal protein loading, the membranes were again stripped and reblotted using an antibody against GADPH. The blots are representative of three independent experiments.

 
Because the phoshorylation of I{kappa}B-{alpha} by IKKbeta, an I{kappa}B kinase, is essential for the activation and regulation of NF-{kappa}B, we next examined whether IKKbeta phosphorylates the inhibitory I{kappa}B-{alpha} in RLE-6TN cells treated with JP-8. Using a phospho-specific antibody, we showed that JP-8 stimulates in lung epithelial cells a progressive phosphorylation of IKKbeta, which is considered an indicator of its activated form, during all the different time intervals compared with untreated control cells. IKKbeta181 phosphorylation increases in a time-dependent manner after JP-8 exposure in RLE-6TN cells (Figure 2C), which appears to be directly related with the nuclear entry of NF-{kappa}B in lung epithelial cells treated with JP-8.

JP-8 Prolongs NF-{kappa}B Activation in RLE-6TN Cells
To demonstrate that a prolonged, nontoxic concentration of JP-8 directly induces a marked DNA-binding activity of NF-{kappa}B after stimulation of rat pulmonary epithelial cells with the fuel, gel shift assay were performed. Nuclear extracts were obtained from RLE-6TN cells treated with the fuel for 24, 36, and 48 h and from unstimulated control cells. RLE-6TN cells exposed to JP-8 showed significant NF-{kappa}B nuclear translocation (Figure 3A, lanes 2, 3, and 4) compared with 8 µg/ml untreated control cells (Figure 3A, lane 1). These data were confirmed by densitometry (Figure 3B). The specificity of the complex formation was confirmed by incubation with a 100-fold molar excess of unlabeled probe during DNA–protein binding, and no shift was observed (data not shown). To identify the specificity of NF-{kappa}B binding activity, antibodies against p65 (Figure 3A, lanes 5–8) or p50 (Figure 3C, lanes 5–8) subunits of NF-{kappa}B that associate with specific NF-{kappa}B promoter sequence were used in supershift analysis. The results show that the mobility of the NF-{kappa}B–DNA complex was only supershifted by anti-p65 antibody (Figure 3C, lanes 2, 3, and 4) but not by the anti-p50 antibody (Figure 3C, lanes 6, 7, and 8), which suggests that only the p65 subunit of NF-{kappa}B is involved in gene activation in RLE-6TN cells treated with JP-8. The translocation of p65 and p50 NF-{kappa}B subunits into the nucleus was confirmed by Western blot analysis. As shown in Figure 3D, nuclear expression of p65 and p50 proteins are increased upon JP-8 exposure compared with untreated control cells.


Figure 3
Figure 3
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  		Figure 3. NF-{kappa}B activation in AEII cells exposed to JP-8. (A) Involvement of p65. AEII cells were incubated in the absence or presence of JP-8 for 24, 36, and 48 h (lanes 2–4). Nuclear protein extracts were prepared and assayed for the DNA-binding activity of NF-{kappa}B by EMSA. Control cells (lane 1) were harvested at 48 h after incubation with ethanol only (MATERIALS AND METHODS). Nucelar protein extracts were pre-incubated with antibodies reactive Supershifts experiments using antibodies to p65 are shown in lanes 5–8. (B) Quantitative data (intensity of each band was quantified using a Phosphor Imager) indicating the time course of NF-{kappa}B activation in RLE-6TN cells (lanes 1–4). Results are expressed as mean ± SD of three experiments. *P < 0.05 compared with group 1 (control), group 2 (24 h), group 3 (36 h), and group 4 (48 h). (C) Involvement of p50. The experiment was performed identically to (A) with the exception that antibodies reactive to p50 proteins were utilized. Representative results from at least three independent experiments are shown. (D) For the identification of translocation of cytoplasmic p65 and p50 subunits of NF-{kappa}B into the nuclei of AEII cells, nuclei were isolated (MATERIALS AND METHODS) and equal volumes of nuclear protein lysates from untreated control or treated RLE-6TN cells at time points (24, 36, and 48 h) were resolved by SDS-PAGE electrophoresis and blotted onto nitrocellulose membranes. The blots were probed with monoclonal anti-p50, polyclonal anti-p65 antibodies, and monoclonal anti-Lamin A as a control for sample loading. The results were identical in three independent experiments.

 
Although I{kappa}B-{alpha} and IKKbeta were phosphorylated in a time-dependent manner, it is interesting to note that the degradation of I{kappa}B-{alpha} was not exacerbated. It was found, however, that treatment of RLE-6T cells with 8 µg/ml of JP-8 did not affect cell viability (Figure 1). Thus, it is perhaps not surprising that NF-{kappa}B activity was found to have a maximal DNA activity as early as at 24 h after JP-8 exposure.

Oxidative Stress Induced by JP-8 Exposure
We previously showed that JP-8 toxicity is mediated by the generation of ROS and depletion of intracellular GSH (4). Therefore, to elucidate the effects of numerous oxidants present in a nonlethal dose of JP-8 (8 µg/ml), we monitored changes in the GSH content and Glutathione S-transferases Pi (GST-P) protein expression in RLE-6TN cells exposed to the fuel. GST-P is part of a superfamily of enzymes, responsible for the detoxification of a wide range of xenobiotics. This enzyme is involved in the detoxification of electrophiles by conjugation with the nucleophilic thiol reduced GSH. As shown in Figure 4A, the immunoblot analysis showed that GST-P expression is increased in a time-dependent manner on cells exposed to JP-8 (Figure 4A). Similarly, we found a significant increase in the intracellular amount of GSH during the 24–48 h of treatment (Figure 4B). The increase in GSH and GST-P levels indicates that both antioxidants may act to directly limit the effects of ROS generation (Figure 4C). The increase on GSH amount and GST-P expression at the three treatment periods with JP-8 elicit a protective effect to regulate enhanced ROS-induced NF-{kappa}B activation.


Figure 4
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  		Figure 4. Effect of JP-8 on antioxidants levels and ROS generation. RLE-6TN cells were exposed for 24, 36, and 48 h in the presence or absence of JP-8 (8 µg/ml). Control cells were harvested at 48 h after incubation with ethanol only (MATERIALS AND METHODS). (A) Proteins were subjected to immunoblot analysis using antibodies against GST-P. To confirm equal protein loading, the membranes were stripped and reblotted using an antibody against GADPH. The blots are representative of three independent experiments. (B) Cells extracts were assayed for GSH content as described in MATERIALS AND METHODS. (C) After cells were incubated in the absence or presence of JP-8, cultures were loaded with H2DCF for 15 min, after which the oxidation of H2DCF was assessed by fluorometer. Data in B and C are means ± SD of three experiments. Significant differences from control on values normalized to the control mean for each treatment (*P < 0.05).

 
Effect of JP-8 on the Expression of Proinflammatory Mediators
The IL-8 gene, whose expression is regulated by NF-{kappa}B, plays an important role in several types of inflammatory responses, such as in chronic obstructive pulmonary disease (COPD)-associated lung inflammation, by inducing the chemotaxis of polymorphonuclear leukocytes (24). Total RNA isolated from untreated and treated RL-6TN cells with JP-8 (8 µg/ml) was subjected to semiquantitative RT-PCR analysis. Figure 4A shows that JP-8 increased the expression of IL-8 gene transcripts at 24 h, with a stable expression of IL-8 mRNA through the 36- and 48-h exposure period. Because TNF-{alpha}–inducing signal transduction in respiratory cells is regulated through NF-{kappa}B activation, changes in the expression levels of this proinflammatory mediator were also determined by semiquantitative RT-PCR. We observed a time-dependent increase in TNF-{alpha} transcripts (Figures 5A and 5B) with a pronounced induction at 48 h of treatment.


Figure 5
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  		Figure 5. Effects of JP-8 on IL-8 and TNF-{alpha} up-regulation in lung cells and tissues. (A) RLE-6TN cells were incubated in the absence or presence of 8 µg/ml of JP-8 for 24, 36, and 48 h. Control cells were harvested at 48 h after incubation with ethanol only (MATERIALS AND METHODS). Total RNA was then isolated from the cells and subjected to semiquantitative RT-PCR analysis with specific primers to IL-8 and TNF-{alpha} as described in MATERIALS AND METHODS. (B) Histogram showing the arbitrary densitometric readings of the IL-8 (shaded bars) and TNF-{alpha} (open bars) normalized for GADPH. Bars represent the mean ± SD of three independent experiments. *P < 0.05 and #P < 0.05, difference from untreated cells. (C) In vivo verification. Semiquantitative RT-PCR analysis of IL-8 and TNF-{alpha} in lung of four different rats (L1, L2, L3, and L4) exposed to JP-8 by nasal-aerosol for 1 h during seven consecutive days (MATERIALS AND METHODS). A representative sample from one of four rat lung controls was used to compare the altered expression levels of these cytokines.

 
To determine whether JP-8 also upregulates the expression of IL-8 and TNF-{alpha} transcripts in vivo, we examined the expression of these inflammation mediators in lung tissues of rats previously exposed for 7 d at doses that mimic the level of occupational exposure in humans, as described in detail in MATERIALS AND METHODS and in another study of ours recently published (3). As shown in Figure 5C, exposure to JP-8 induces in quadruplicate rat lung tissues (L1, L2, L3, L4) the upregulation of both IL-8 and TNF-{alpha} transcripts, which appear to be stable during the entire course of rat lung exposure.

Dysregulated Expression of the Anti-Inflammatory Cytokine IL-6 in RLE-6TN Cells Treated with JP-8
Because IL-6 is induced together with the alarm cytokine TNF-{alpha} in acute phase reactions, we examined the effects of JP-8 treatment on the levels of this anti-inflammatory regulator in RLE-6TN cells. Figure 6A shows the representative results of the gene-specific PCR product of IL-6 by gel electrophoresis. IL-6 mRNA expression was almost undetectable in the untreated control cells but it was markedly stimulated at the 24-, 36-, and 48-h time points in epithelial cells treated with the fuel (Figures 6A and 6B). The levels of IL-6 became more pronounced in epithelial cells 48 h after exposure to the fuel. At this specific time exposure, RLE-6TN cells also exhibited a prominent induction of TNF-{alpha}. These data suggest that the induction of IL-6 in RLE-6TN cells may be an alternative mechanism to regulate the production of proinflammatory mediators induced by JP-8 fuel.


Figure 6
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  		Figure 6. Effects of JP-8 on IL-6 induction in lung cells and tissues. (A) RLE-6TN cells were incubated in the absence or presence of JP-8 for 24, 36, and 48 h. Control cells were harvested at 48 h after incubation with ethanol only (see MATERIALS AND METHODS). Total RNA was then isolated from the cells and was employed in mRNA expression analysis of IL-6 and TNF-{alpha} using RT-PCR analysis as described in MATERIALS AND METHODS. (B) Histogram showing the arbitrary densitometric readings of the IL-6 (shaded bars) and TNF-{alpha} (open bars) normalized for GADPH. Bars represent the mean ± SD of three independent experiments. *P < 0.05 and #P < 0.05 when compared with untreated cells. (C) In vivo verification. Semiquantitative RT-PCR analysis of IL-6 in lung of four different rats (L1, L2, L3, and L4) exposed to JP-8 by nasal-aerosol for 1 h during seven consecutive days (MATERIALS AND METHODS). A representative sample from one of four rat lung controls was used to compare the altered expression levels of this cytokine.

 
Furthermore, as with IL-8, the RT-PCR analysis for IL-6 mRNA in quadruplicate lung tissue extracts of rats exposed to JP-8 (L1, L2, L3, L4) by nose-only aerosol administration (as described in MATERIALS AND METHODS), revealed that IL-6 levels were also substantially increased after a prolonged treatment with JP-8 (Figure 6C). Thus, overall, the RT-PCR data obtained from animals lung tissues exposed to JP-8 treatment for 7 d are consistent with those observed in the cell culture system.

JP-8 Induces Activation of PARP-1 in Lung Epithelial Cells
Because PARP-1 has been indicated to act as a co-activator of NF-{kappa}B in inflammatory disorders (25), we investigated whether activation of PARP-1 is concomitant with the prolonged activation of NF-{kappa}B in RLE-6TN cells exposed to JP-8–mediated oxidative stress. The time course treatment revealed that JP-8 induces poly(ADP-ribosylation) automodification of PARP-1 (Figure 7A). The progressive increase in PARP-1 activity was noted using 32P NAD. This prolonged poly(ADP-ribosyl)ation of PARP-1 is in agreement with the increased and prolonged NF-{kappa}B activation after JP-8 exposure, compared with early time points (data not shown) and in control cells. While several proteins, including histones, topoisomerase 1, and p53, are modified in Figure 6A, by far the major acceptor was PARP-1 (116 kD) itself. It was of significance that the maximal PARP-1 activity was observed at 24 h, which corresponds to the maximal JP-8–mediated activation of NF-{kappa}B (Figure 3A, lane 2). At 36 h there was less PARP-1 activity, and at 48 h no poly(ADP-ribosyl) ation was observed, in spite of the fact that PARP-1 protein was constant and nondegraded by caspase-3–like activity through the experiment (Figure 7B). In addition, no cell death was observed at this time point (Figure 1) as was evident by the constant presence of intact pro–caspase-3 (Figure 7B) and no caspase-3 activation (data not shown). A number of possibilities for the lack of PARP activity at 48 h are consistent with the literature in this field. High activity at this extended period in the presence of threshold JP-8 would consume significant NAD (and ATP), which would be required for cell viability. In addition, considerable evidence indicates that both PARP-1 activation and poly (ADP-ribose) (PAR) accumulation is proliferation- and cell cycle–dependent. We showed earlier that in synchronized HeLa and WI-38 cells (26) that PARP-1 mRNA is highest mainly in mid S and significantly higher in G2 phase. This was confirmed in nuclear runoff transcription studies. Kidwell and Mage (27) also showed in synchronized intact cells, using PAR antibodies, that there are two major points of PAR accumulation in cells, one in mid S and a second at G2 phase, due to synthesis and probably also due to less PAR turnover. Accordingly, the repeated neglible activity at 48 h could be caused by a combination of these aspects of PARP-1 cell biology. However, the high activity of both NF-{kappa}B and PARP-1 at 24 h was the key observation of this experiment, as noted above. Poly(ADP-ribosyl)ation is a phenomenon that contributes to various physiologic and pathophysiologic events associated with DNA strand breakage, repair of DNA damage, gene expression, and apoptosis (12, 13). Hence, either apoptosis or necrosis associated with persistent PARP-1 activation and NAD/ATP depletion is indicative of DNA strand breakage. This conclusion is supported by our previous studies using apoptotic doses (80 µg/ml) of JP-8 exposure and the generation of ROS, inducing DNA damage in lung epithelial cells (4). However, this damage to the DNA appears to be insufficient to induce cell death using threshold levels of JP-8 exposure (Figure 1).


Figure 7
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  		Figure 7. Effects of JP-8 on PARP-1 activity. (A) JP-8 induces PARP-1 catalytic activity in RLE-6TN cells in a time-dependent manner. RLE-6TN cells were cultured in the presence or absence of JP-8. Control cells were harvested at 48 h after incubation with ethanol only (MATERIALS AND METHODS). At the indicated time intervals after exposure to 8 µg/ml JP-8, exposed and control untreated cells were harvested, then washed in ice-cold PBS and incubated with the assay buffer (MATERIALS AND METHODS). Total protein extracts were analyzed by SDS-PAGE autoradiography. (B) Cells exposed to JP-8 (8 µg/ml) at different times were subjected to immunoblot analysis with antibodies to PARP-1, pro– caspase-3, and GADPH (loading control). Control cells were harvested at 48 h after incubation with ethanol only (MATERIALS AND METHODS). The results are representative of at least three experiments performed at different times.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
AEII cells secrete a variety of proinflammatory chemokines, which are important for the recruitment of monocytes, macrophages, and T cells into the lung interstitium and alveolar space in response to stress stimuli (28, 29). Recent studies performed both in vitro and in vivo have shown that JP-8 exposure induces pathologic injury in both lung cell lines and tissue (4, 30). Personnel exposed to this fuel exhibit levels of JP-8 components in their breath that are 40 times greater than ambient levels (31). In vitro assays have also shown that JP-8 toxicity (elevated doses) is associated with its capacity to cause genomic DNA damage, alter mitochondrial structure and function, as well as induce the generation of ROS in RLE-6TN cells (4, 5). ROS also play a pivotal role in mediating the activation and expression of NF-{kappa}B, a transcription factor that is implicated in the pathogenesis of several respiratory diseases and lung epithelial cell injury (15). Therefore, the prolonged activation of NF-{kappa}B in RLE-6TN cells during the extensive treatment with JP-8 (low dose) may play a critical role in stimulating the production and expression of proinflammatory mediators. Many of the genes that code for these proinflammatory factors, including several chemokines activated in lung epithelial cells in response to oxidative stress, are target genes of NF-{kappa}B (32). Both IL-1beta and TNF-{alpha} induce specific binding of p50/p65 heterodimer and p65/p65 homodimer to the NF-{kappa}B consensus sequence. In our study, only the p65 subunit, not the p50 subunit, appears to regulate the transcriptional of the basal transcription machinery in RLE-6TN cells treated with JP-8.

The significant phosphorylation of I{kappa}B-{alpha} observed at 24 h treatment supports the augmented activation of NF-{kappa}B at this time point. Because of the enhanced phosphorylation of I{kappa}B-{alpha} and IKKbeta after 36 and 48 h of treatment, we expected an enhanced activation of NF-{kappa}B. Nevertheless, the increased levels of the detoxyfing enzymes, GSH and GST-P, compared with the control untreated cells represent a defense mechanism displayed by respiratory epithelial cells, against the numerous oxidants present in the JP-8. We hypothesize that changes in GSH content and GST-P levels regulate ROS-mediated enhanced activation of NF-{kappa}B, and protect cells from injury during the prolonged exposure to JP-8. This supports the observations that NF-{kappa}B activation was not increased in a time-dependent manner at the 36- and 48-h time points. In addition, the augmented expression of GST-P may also be correlated with the absence of cell death in RLE-6TN cells exposed to JP-8. Recent studies have revealed that GST-P limits the degree of JNK signaling in response to increased ROS generation treatments (33).

We found that the expression level of TNF-{alpha} was increased in RLE-6TN cells exposed to JP-8. TNF-{alpha} together with IL-1beta is considered an alarm cytokine required for the induction of subsequent cytokines in lung in response to stressors. However, we did not observe an augmented expression of IL-1beta transcript by JP-8, under our experimental conditions (data not shown), which suggests that in lung epithelial cells exposed to the jet fuel, only TNF-{alpha} may have a prominent effect on the expression of other genes such as IL-8. Nevertheless, the reduced levels of TNF-{alpha} at early time treatments (24 and 36 h) indicate that NF-{kappa}B activation coordinates the expression of IL-8 in RLE-6TN cells exposed to 8 µg/ml JP-8. IL-8 has been indicated as the major chemotactic factor for macrophages and polymorphonuclear leukocytes to sites upon inflammation occurring in both asthma and COPD. The augmented expression of IL-8 observed in our cultured airway epithelial cells is also in agreement with previous reports, indicating that this chemokine is increased in epithelial cells by oxidative stress (34). We have previously shown that JP-8 induces in RLE-6TN cells the generation of ROS and depletion of intracellular GSH in respiratory epithelial cells, events that are critical for the toxicity of this fuel (4). Repeated inhalation of toxicants might be expected to generate augmented levels of ROS by macrophages and AEII cells. Therefore, the increased and constant induction of IL-8 in RLE-6TN during the 3-d treatment with JP-8 probably represents the primary response of these cells to the oxidative stress imposed during the insult. The continuous production of free radicals by airway cells may affect the homeostasis in lung tissue, leading to the outcome of pulmonary disease. For example, radical species play a pivotal role in mediating the activation of NF-{kappa}B in several models of lung inflammation (35). In addition, the up-regulation of IL-8 in RLE-6TN cells showing increased activation of NF-{kappa}B is consistent with the notion that proteolysis of I{kappa}B-{alpha}, a critical event for NF-{kappa}B activation, is the regulator of IL-8 expression in AEII cells (36). Furthermore, evidence showing that cigarette smoking induces augmented IL-8 expression in human bronchial epithelial cells (37), an effect that is enhanced by TNF-{alpha} treatment in airway smooth muscle cells (38), suggests that JP-8 may induce in a AEII cells inflammatory conditions, with some cytokine responses similar to those induced by cigarette smoking.

IL-6 has been indicated as an important inhibitor for TNF-{alpha} production in several experimental conditions (39). Accordingly, IL-6 induction appears to have an inhibitory effect on the synthesis and secretion of TNF-{alpha}. The levels of this cytokine usually is elevated in mice treated with antibody against IL-6 and in mice deficient in IL-6 gene, compared with the corresponding untreated or wild-type (IL-6+/+) control animals (40). Consistent with these observations, we found that IL-6 expression is increased in a time-dependent manner. The abundance of IL-6 appears to inhibit the expression of TNF-{alpha} transcript, which is slightly induced at 24 and 36 h in RLE-6TN cells exposed to JP-8. However, the TNF-{alpha} mRNA level was markedly up-regulated in these cells at the 48-h time period. It is conceivable that the amount of IL-6 produced by the RLE-6TN cells was insufficient to inhibit the up-regulation of TNF-{alpha} transcript in a persistent stress environment induced by the fuel, which permanently can alter the responsiveness of AEII cells to modulate immune responses.

Recently it was indicated that PARP-1 activation in response to oxidative stress regulates the inflammatory process through a signal transduction mechanism mediated by AP-1 (41) and NF-{kappa}B (9) in fibroblasts. A massive poly(ADP-ribosyl)ation of PARP-1 has been associated with the increased catalytic activity of this enzyme in several lung-related studies (17, 42). Therefore, the pronounced activation of PARP-1 in respiratory epithelial cells with long exposure to JP-8 suggests that the persistent oxidative stress induced by the fuel induces substantial DNA damage in these cells. In our findings, the activation of PARP-1, concomitant with the prolonged activation of NF-{kappa}B activation, is in agreement with the role of this enzyme (in its automodified state), as a co-activator of NF-{kappa}B activation in inflammatory responses in the lung. Consequently, a continuous PARP-1 activation can sustain an extended NF-{kappa}B activation provoking a chronic expression of proinflammatory factors in respiratory epithelial cells exposed to JP-8.

Repetitive exposure to the JP-8 hydrocarbon mix induces long-term effects on the immune system (43, 44), which may increase the susceptibility of personnel exposed to JP-8 to infectious agents. This sensitization has been indicated as responsible in the exacerbated induction of proinflammatory cytokines in stressed animals exposed to a stressor compared with nonstressed controls (45). Repeated inhalation of toxicants might be expected to generate augmented levels of ROS by macrophages and AEII cells. The chronic oxidative damage, largely due to the continuous production of free radicals, can permanently alter the responsiveness of AEII cells in the lung to modulate immune responses and may also affect the homeostasis in pulmonary tissue, leading to improperly controlled responses by respiratory epithelial cells. This in turn, may induce morphologic and functional changes, increasing the risk of development of diseases in the lung. Therefore, the induction of proinflammatory cytokines through increased transcription on AEII cells exposed to JP-8 may have critical consequences for the outcome of disease in the lung.

In conclusion, the present study clearly demonstrates that JP-8 at a high dilution (8 µg/ml) increases the expression of proinflammatory mediators in pulmonary epithelial cells during in vitro exposure. Similarly, in vivo experiments supporting the in vitro effects are showing the targeting of the expression of these factors, as a primary response to JP-8 exposure. It is clearly difficult to extrapolate data from cell lines to animal or human exposure. However, our results suggest several potential mechanisms underlying JP-8–induced toxicity on the lung, and implicate jet fuels, in part, as potential environmental risk factors leading to the development of pulmonary disease.


   Footnotes
 
This work was supported by the grants: F49620-94-1-0297 to M.W. and FA9550- 04-1-0395 to M.E.S., from the US Air Force Office of Scientific Research.

* Present affiliation: Universidade de Franca, Franca, Brazil Back

Originally Published in Press as DOI: 10.1165/rcmb.2006-0059OC on May 11, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 6, 2006

Accepted in final form May 2, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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