Roles of Reactive Oxygen Species and Heme Oxygenase-1 in Modulation of Alveolar Macrophage-Mediated Pulmonary Immune Responses to Listeria monocytogenes by Diesel Exhaust Particles

Xuejun J. Yin*, Jane Y. C. Ma{dagger}, James M. Antonini{dagger}, Vincent Castranova{dagger} and Joseph K. H. Ma*,1

* School of Pharmacy, West Virginia University, Morgantown, West Virginia 26506; and {dagger} Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505

Received March 26, 2004; accepted August 9, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diesel exhaust particles (DEP) have been shown to suppress alveolar macrophage (AM)-mediated pulmonary immune responses to Listeria monocytogenes in vivo. In this study, effects of DEP-derived reactive oxygen species (ROS) and heme oxygenase (HO)-1 on AM-mediated immune responses to L. monocytogenes were investigated. Brown Norway rats were intratracheally inoculated with 100,000 L. monocytogenes, and AM were isolated at 7 days post-infection. Exposure to DEP or their organic extract (eDEP), but not the washed DEP (wDEP) or carbon black, increased intracellular ROS and HO-1 expression in AM. Induction of ROS and HO-1 by eDEP was partially reversed by {alpha}-naphthoflavone, a cytochrome P450 1A1 inhibitor, and totally blocked by N-acetylcysteine. In addition, exposure to eDEP, but not wDEP, inhibited lipopolysacchride-stimulated secretion of tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-12 (IL-12), but augmented production of IL-10 by AM. Kinetic studies showed that modulation of cytokines by eDEP was preceded by ROS and HO-1 induction. Furthermore, pretreatment of AM with superoxide dismutase (SOD) or zinc protoporphrin IX (Znpp), which attenuated eDEP-induced HO-1 expression/activity, substantially inhibited eDEP effect on IL-10. Finally, direct stimulation with pyrogallol (PYR), a superoxide donor, upregulated HO-1 and IL-10 but decreased secretion of IL-12 in L. monocytogenes-infected AM. These results show that DEP, through eDEP-mediated ROS, induce HO-1 expression and IL-10 production and at the same time inhibit AM production of TNF-{alpha} and IL-12 to dampen the host immune responses. The results also suggest that HO-1 may play an important role in regulating production of IL-10 by DEP-exposed and L. monocytogenes-infected AM.

Key Words: diesel exhaust particles; Listeria monocytogenes; heme oxygenase-1; reactive oxygen species; cytokines.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidemiologic studies have shown a consistent association between elevated levels of particulate matter in ambient air and increased incidence of pulmonary infections, or respiratory mortality and morbidity in high-risk groups (Dockery et al., 1993Go; Pope et al., 1991Go; Schwartz et al., 1996Go). Effect of diesel exhaust particles (DEP), a major component of environmental particulate pollutants in most industrialized urban areas, on pulmonary infections is of great environmental and occupational concern. DEP are carbon-based particles that contain adsorbed organic compounds including polycyclic aromatic hydrocarbons (PAHs), quinones, and nitro-PAHs. Both the organic and particulate components play a role in DEP-induced pulmonary toxicity. Studies from our laboratory showed that the particulate component induces macrophage respiratory burst, augments ovalbumin-mediated allergic sensitization, and suppresses cytochrome P450 (CYP) 2B1 and glutathione-S-transferase activities in rat lungs (Al-Humadi et al., 2002Go; Castranova et al., 2001Go; Rengasamy et al., 2003Go). The organic component, on the other hand, induces CYP 1A1 and suppresses the response of alveolar macrophages (AM) to lipopolysacchride (LPS) in the production of nitric oxide (NO), interleukin-1ß (IL-1ß), and tumor necrosis factor-{alpha} (TNF-{alpha}) (Rengasamy et al., 2003Go; Yang et al., 1997Go, 1999Go). This dual effect of DEP on the pulmonary system has been the subject of a recent review (Ma and Ma, 2002Go).

Listeria monocytogenes (L. monocytogenes), a ubiquitous species of gram-positive, facultative intracellular bacteria, has been used in our laboratory as a pulmonary infection model for studies of host immune responses (Yang et al., 2001Go; Yin et al., 2002Go, 2003Go, 2004Go). Unlike most typical extracellular pathogens, L. monocytogenes induces both innate (nonspecific) and cell-mediated (antigen-specific) immune responses upon infection. This distinguishing feature of L. monocytogenes infection makes it feasible to serve as an experimental probe to assess how an immunotoxic xenobiotic affects both innate and cell-mediated host immunity. Among the various cell types involved in the innate immune system, AM are responsible for the clearance of inhaled particles and/or microorganisms from the distal airways and alveolar spaces. Activated AM release reactive oxygen species (ROS), cytokines, and a variety of mediators that are capable of killing microorganisms (Laskin and Pendino, 1995Go; Sibille and Reynolds, 1990Go). A number of AM-derived cytokines are known to be necessary for the generation of a protective immune response against L. monocytogenes (Bancroft et al., 1989Go; Czuprynski et al., 1992Go). It has been well documented that AM-derived pro-inflammatory cytokines, such as IL-1ß and TNF-{alpha}, provide innate resistance to bacterial infection, promote the inflammatory process by recruiting neutrophils into the air spaces, and stimulate these phagocytes to release ROS and enzymes (Laskin and Pendino, 1995Go; Le and Vilcek, 1987Go). A successful pulmonary host defense, on the other hand, also needs specific cell-mediated immunity (Kaufmann, 1993Go). In this aspect, studies have already shown that AM, through their secretion of cytokines in response to specific antigen exposure, provide a critical link between these two systems. For example, AM produce IL-12 in response to L. monocytogenes to elicit the development of interferon-{gamma} (IFN-{gamma})-secreting T helper (Th)1 CD4+ lymphocytes (Hsieh et al., 1993Go). This cytokine, which is produced rapidly after infection, initiates the development of the appropriate CD4+ Th subset and plays a role in maintaining the Th1 response (Park and Scott, 2001Go) and increased T cell production of IFN-{gamma} (Trinchieri, 1995Go, 1998Go). In addition, both IL-1ß and TNF-{alpha} activate NK cells to release IFN-{gamma}, which activates macrophages to kill the bacteria. These cytokines are also T cell activators (Akira et al., 1990Go; Hsieh et al., 1993Go). IL-10, on the other hand, is a potent immunosuppressive factor that downregulates macrophage bactericidal activity (Fleming et al., 1999Go). The effect of DEP exposure on the production of IL-10 by AM is of interest because some intracellular pathogens, including L. monocytogenes, specifically target macrophages for infection and use IL-10 to dampen the host immune response and thus prolong their survival (Redpath et al., 2001Go).

We have shown previously that DEP exposure markedly retarded the ability of L. monocytogenes-infected Brown Norway rats to clear the bacteria (Yin et al., 2002Go, 2004Go). DEP suppressed AM phagocytotic function and their secretion of pro-inflammatory cytokines including IL-1ß, TNF-{alpha}, and IL-12, but increased AM production of IL-10. The mechanism through which DEP alter cytokine production by AM and suppress the pulmonary immunity to bacterial infection is not yet clear, but may involve certain responses to DEP-induced oxidative stress. Studies have shown that DEP induce the generation of ROS in AM and promote apoptosis via mitochondrial release of cytochrome c and the activation of downstream caspase activities (Hiura et al., 1999Go, 2000Go). These studies also showed that the induction of ROS by DEP was due to the organic compounds of DEP, but not the washed particles. This is significant, since many PAHs and redox-active quinones are known to induce ROS (Ng et al., 1998Go; Park et al., 1996Go) and apoptosis (Lei et al., 1998Go). DEP-derived chemicals, in fact, have been shown to produce superoxide and hydroxyl radicals when incubated with lung microsomal enzymes (Kumagai et al., 1997Go).

Demple and colleagues (Bouton and Demple, 2000Go; Demple, 1999Go) have shown that the threat of ROS damage is countered by coordinated cellular responses that modulate the expression of sets of gene products, one of which is heme oxygenase-1 (HO-1), which, along with other inducible enzymes, constitutes a cellular adaptive resistance pathway for defense against various oxidants. The organic extract of DEP has been shown to induce HO-1 gene expression in AM (Koike et al., 2002Go) and macrophage cell lines (Li et al., 2000Go, 2002Go) as an early response to oxidative stress. Li et al. (2000)Go demonstrated that DEP extract, its aromatic and polar fractions, and a benzo[a]pyrene quinone all induce HO-1 expression in macrophages. HO-1 has been shown to modulate cellular production of pro- and anti-inflammatory cytokines. Lee and Chau (2002)Go showed that HO-1 mediated the anti-inflammatory effect of IL-10 in mice. Here, IL-10 induced the expression of HO-1 in macrophages via p38 mitogen-activated protein (MAP) kinase pathway. Inhibition of HO-1 protein synthesis or activity significantly reversed the inhibitory effect of IL-10 on LPS induction of TNF-{alpha}. On the other hand, Inoue et al. (2001)Go have shown that the production of IL-10 by AM in mice was upregulated by either hemin-induced HO-1 expression or an intratracheal inoculated adenovirus that encodes HO-1.

The present study was carried out to test the hypothesis that DEP, through their organic component, induce ROS generation in AM, resulting in over-expression of HO-1 and altered cytokine production, and lead to depressed host immune responses to bacterial infections. To achieve this objective, we studied effects of DEP and their organic and carbonaceous components on ROS generation, HO-1 expression and activation, and production of TNF-{alpha}, IL-10, and IL-12 by AM in response to L. monocytogenes infection. Because cellular enzymes and signaling cascades are involved in the regulation of ROS and cytokine production and HO-1 expression, we evaluated in the present study the contribution of DEP-derived ROS, HO-1, and protein kinases in the regulation of AM-mediated cytokine release by the use of specific inhibitors/scavengers and ROS as a direct cellular stimulant.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. 2',7'-Dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probes (Eugene, OR), and used at 10 µM as a ROS probe. Pyrogallol (PYR) was purchased from Sigma Chemical Co. (St. Louis, MO) and used at 300 µM as a superoxide donor. Superoxide dismutase (SOD, Sigma) at 2000 U/ml was used as superoxide scavenger. N-acetylcysteine (NAC, Sigma) at 10 mM, was used as an antioxidant. {alpha}-Naphthoflavone (ANF, Sigma) at 10 µM was used as a CYP 1A1 inhibitor. SB203580 (Calbiochem, San Diego, CA), at 10 µM, SP600125 (Biomol, Plymouth Meeting, PA), at 100 µM, and PD98059 (Calbiochem), at 20 µM, were used to inhibit the p38 MAP kinase, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) pathways, respectively. Zinc protoporphrin IX (Znpp, Porphrin products, Logan, UT) at 10 µM, was used as an inhibitor of HO-1, and hemin (Sigma), a known HO-1 inducer at 100 µg/ml, was used as a positive control. LPS (1 µg/ml, Sigma) was used to enhance AM cytokine production. Dimethyl sulfoxide (DMSO, Sigma) at 0.25% was used as a solvent control for eDEP.

Preparation of DEP, carbon black (CB), and DEP components. The diesel particulate matter SRM 2975 was purchased from the National Institute of Standards and Technology (Gaithersburg, MD). CB (particle size: 0.1–0.6 µm) was obtained from Cabot Co. (Boston, MA). DEP or CB was suspended in sterile phosphate-buffered solution (PBS, pH 7.4) by sonication using an ultrasonic processor (Heat System-Ultrasonics, Plainview, NY). The organic extract of DEP (eDEP) and the washed DEP (wDEP) were prepared as follows: 100 mg of DEP were suspended in 50 ml of CH2Cl2, vortexed and sonicated for 10 min on ice, and centrifuged for 10 min at 500 x g. After solvent removal, DEP were further extracted with 50 ml of a 1:1 (v/v) mixture of acetone and methanol. The two extracts were combined, evaporated to dryness, and weighed. This procedure yielded 41 mg of the organic content from 100 mg DEP. The eDEP and wDEP were dissolved or suspended in DMSO or sterile PBS to yield a concentration corresponding to 20 mg/ml of the original DEP and stored at –20°C. When used in experiments, samples were diluted with sterile saline. All concentrations expressed for eDEP or wDEP refer to the extract or particles, respectively, from the same concentration of DEP.

Animals and bacterial infection. Male Brown-Norway rats (BN/CrlBR), weighing 225–250 g, were purchased from Charles River Laboratories (Wilmington, MA). The animals were housed in a clean-air and viral-free room with restricted access, given a conventional laboratory diet and tap water ad libitum, and allowed to acclimate for one week in an animal facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). L. monocytogenes (strain 10403s and serotype 1) was grown overnight in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, MI) with aeration at 37°C in a shaking incubator. One milliliter was then transferred into 50 ml of BHI broth and incubated at 37°C with aeration until the culture was in log growth (3 h). The bacterial concentration was determined spectrophotometrically at 600 nm and diluted for animal infection. Rats were lightly anesthetized with methohexital sodium (35 mg/kg body weight; Eli Lilly Co., Indianapolis, IN) and intratracheally inoculated with ~100,000 cells of L. monocytogenes in 500 µl of sterile saline or the vehicle alone according to the method previously described (Antonini et al., 2000Go).

Isolation of AM and cell culture. At 7 days after intratracheal instillation, the L. monocytogenes-infected and uninfected rats were deeply anesthetized with sodium pentobarbital (50 mg/kg; Butler, Columbus, OH) and then exsanguinated by cutting the abdominal aorta. The trachea was cannulated and the lungs lavaged with 8-ml aliquots of ice-cold Ca2+/Mg2+-free PBS (pH 7.4) containing 145 mM NaCl, 5 mM KCl, 1.9 mM NaH2PO4, 9.35 mM Na2HPO4, and 5.5 mM glucose. A total of 80 ml of the bronchoalveolar lavage (BAL) fluid was collected from each rat and centrifuged at 500 x g for 10 min at 4°C. Cell pellets from individual rats were combined, washed, and resuspended in 1 ml of PBS. AM in the BAL cell suspension were enumerated according to their unique cell diameter (Kang et al., 1992Go) using an electronic cell counter equipped with a cell-sizing unit (Coulter Electronics, Hialeah, FL). The BAL cells were then suspended in RPMI-1640 medium (Gibco, BRL) supplemented with 2 mM glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, and 10% fetal bovine serum (FBS). Aliquots of 1 ml cell suspensions, containing 2 x 106 AM, were added to each well of 24-well culture plates (Costar, Cambridge, MA) and incubated in a humidified incubator (37°C and 5% CO2) for 2 h to allow cell attachment to plastic plate. The nonadherent BAL cells were removed by rinsing the monolayer three times with RPMI-1640 medium. The AM-enriched cells were then directly used for primary cell culture to determine various cellular parameters as described later.

Determination of ROS generation. AM were incubated ex vivo with 50 µg/ml of DEP, eDEP, wDEP or CB at 37°C and 5% CO2 for up to 24 h. DMSO (0.25%) was used as a solvent control. Inhibitors/scavengers were added 2 h before eDEP treatment. After the treatment, AM were gently scraped from the plates and washed three times with a washing buffer (PBS with 2% FBS and 0.02% NaN3, pH 7.4). The cells were then resuspended in culture medium containing 10 µM of DCF-DA (Molecular Probes, Eugene, OR), and incubated in dark for 30 min. DCF-DA is a nonfluorescent compound that can enter the cells and is trapped by removal of the diacetate group. Upon interaction with intracellular ROS, DCF is converted into a fluorescent product. After staining, DCF-DA was removed by washing the cells with the washing buffer. Propidium iodide (3 µg/ml) was added to the samples, which were then immediately subjected to flow cytometric analysis using a FACScan (Becton-Dickinson, Mountain View, CA) at excitation 488 nm and emission 575 nm.

Western blot analysis for HO-1 expression. AM were incubated ex vivo with 50 µg/ml of DEP, eDEP, wDEP, CB, or PYR (300 µM) at 37°C and 5% CO2 for 24 h. Inhibitors/scavengers were added 2 h before eDEP or PYR treatment. DMSO (0.25%) and hemin (100 µg/ml) were used as a solvent and positive control, respectively. After the treatment, the cells were washed three times with PBS and suspended in 100–200 µl of a lysis buffer (50 mM Tris-HCl, 1% NP40, 2 mM EDTA, 100 mM NaCl, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride; pH 7.5) for 30 min on ice. Cytoplasmic extracts were separated from the nuclei by centrifugation at 14,000 rpm for 10 min at 4°C and determined for protein content using a BCA Protein Assay Kit (Pierce, Rockford, IL). An equal amount of protein (30 µg/well) for each sample was boiled for 5 min, loaded, and run for electrophoresis in a 4–20% Tris-Glycine gel (Invitrogen, Carsbad, CA) at 125 V. The gel was transferred electrophoretically (Bio-Rad Laboratories, Hercules, CA) to nitrocellulose membranes (Schleicher & Schuell, Keene, NH), and the blots were blocked with 5% milk in TBST buffer (20 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20; pH 7.5) for 1 h at room temperature. Membranes were then probed with a polyclonal rabbit antibody against HO-1 (Stressgen, Victoria, Canada) and with a horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz, Santa Cruz, CA). Blots were developed using an enhanced chemiluminescence reagent commercially developed (Amersham Biosciences, Piscataway, NJ). For quantification, bands in photographs were scanned by a densitometer linked to a computer system (Personal Densitometer SI, Amersham Biosciences, Piscataway, NJ).

Assay for heme oxygenase activity. AM were incubated ex vivo with 50 µg/ml of eDEP or wDEP at 37°C and 5% CO2 for up to 24 h. DMSO (0.25%) and hemin (100 µg/ml) were used as a solvent and positive control, respectively. Inhibitors/scavengers were added 2 h before eDEP treatment. The total HO activity was determined by measuring the generation of bilirubin from heme, as described by Motterlini et al. (1996)Go. Briefly, microsomes of a known quantity from harvested cells were added to 1 ml of a reaction mixture containing NADPH (0.8 mM), glucose-6-phosphate (2 mM), glucose-6-phosphate dehydrogenase (0.2 unit), hemin (10 µM), and 2 mg rat liver cytosol protein as a source of biliverdin reductase in a potassium phosphate buffer (100 mM, pH 7.4). The reaction was carried out in dark at 37°C for 1 h and terminated by adding 1 ml of chloroform to the reaction mixture. The extracted bilirubin concentration was determined spectrophotometically based on the difference in absorbance between 464 and 530 nm ({varepsilon} = 40 mM–1 cm–1). The HO activity was expressed as picomoles of bilirubin per milligram of protein per h. The protein content of microsomes was determined using a BCA Protein Assay Kit (Pierce).

Cytokine assays. AM were incubated ex vivo with 50 µg/ml of eDEP, wDEP, or 300 µM of PYR in the presence of 1 µg/ml LPS at 37°C and 5% CO2 for up to 24 h. DMSO (0.25%) was used as a solvent control, and hemin (100 µg/ml) was used as a positive control for HO-1. Inhibitors/scavengers were added 2 h before eDEP or PYR treatment. After the treatment, the AM-conditioned media were collected, centrifuged (1200 x g for 4 min), and aliquots of the supernatants were stored at –70°C until assayed.

The concentration of IL-10 and TNF-{alpha} in the culture supernatants collected under various exposure conditions was quantified by the enzyme linked immunosorbent assay (ELISA) using the OptEIA ELISA sets according to the manufacturer's instructions (BD PharMingen, San Diego). The levels of IL-12 in the culture media were quantified by ELISA using an ELISA kit from BioSource International, Inc. (Camarillo, CA). Absorbance of samples was read at 450 nm with a SpectraMax 250 plate spectrophotometer and analyzed using Softmax Pro 2.6 software (Molecular Devices Co., Sunnyvale, CA). All cytokine levels were determined from the linear portion of the standard curves generated using recombinant cytokines. The range of detection was: 31.3–1000 pg/ml for TNF-{alpha}, 15.6–500 pg/ml for IL-10, and 7.8–500 pg/ml for IL-12.

Statistical analysis. All data are expressed as means ± standard error (SE) of multiple measurements. Statistical analyses were carried out with the JMP IN statistical program (SAS, Inc., Cary, NC). The significance of the interaction among different treatment groups for the different parameters at each time point was assessed using an analysis of variance (ANOVA). The significance of difference between individual groups was analyzed using the Tukey-Kramer's Honestly Significant Different (HSD) Test. For all analyses, the criterion of significance was set at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Role of Organic Compounds in DEP-Induced ROS Production
To determine the respective role of the organic compounds and the carbonaceous core, we compared the effects of DEP, eDEP, wDEP, and CB particles on intracellular ROS generation using DCF-DA probe, which yields a fluorescent product by interaction with ROS. Exposure to DEP or eDEP, but not wDEP or CB, increased ROS generation in L. monocytogenes-infected AM (Fig. 1A). The induction of ROS by eDEP was time-dependent, with the maximum oxidant production occurring within 2 h of exposure (Fig. 1B). This rapid production of ROS was partially blocked by preincubation of cells with ANF, a CYP 1A1 inhibitor, and strongly attenuated by NAC (Fig. 1A). These results highlight a role of the organic compounds in DEP-derived ROS production and suggest that the eDEP-induced ROS involves the action of the NADPH cytochrome P450 enzymes.



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FIG. 1. DEP-induced ROS generation (A) and kinetics of ROS induction by eDEP (B) in AM. (A) AM were obtained from L. monocytogenes-infected rats at 7 days post-infection and incubated with DMSO (0.25%), 50 µg/ml of DEP, eDEP, wDEP, or CB for 2 h. Some of the eDEP-containing cultures were pretreated with ANF (10 µM) or NAC (10 mM) for 2 h prior to the addition of eDEP. (B) AM from L. monocytogenes-infected rats at 7 days post-infection were exposed ex vivo to DMSO (0.25%,{circ}) or 50 µg/ml of eDEP (•) for varying time periods. Intracellular ROS generation was determined by flow cytomitric analysis using DCF-DA as a probe and expressed as fold increase in DCF fluorescence compared to that in DMSO-treated (A) and untreated (B) controls. Data represent the mean ± SE of at least four experiments. *Significantly different from that of the corresponding controls, p < 0.05; #Significantly different from that of the eDEP-exposed AM, p < 0.05.

 
Roles of Organic Compounds and ROS in DEP-Induced HO-1 Expression and Activation
To clarify the effects of the organic compounds and ROS on HO-1 expression in L. monocytogenes-infected AM, the cells were treated with various DEP components, and hemin was used as a positive control. Ex vivo exposure to DEP or eDEP markedly increased expression of HO-1 in the AM (Fig. 2A). In contrast, wDEP or CB, which failed to induce ROS generation (Fig. 1A), was shown to have no effect on HO-1 expression. The eDEP-induced HO-1 expression was markedly inhibited by ANF, and completely blocked by NAC (Fig. 2B). These findings suggest a role of organic compounds in DEP-induced HO-1 expression and the effect of ROS in this response. The results also show that both the MEK and JNK inhibitors, PD98059 and SP600125, failed to alter HO-1 expression. On the other hand, SB203580, which inhibits p38 MAP kinase, markedly inhibited the eDEP-induced HO-1 expression (Fig. 2B). This suggests that induction of HO-1 by DEP involves activation of p38 MAP kinase, but not JNK, ERK, or the upstream MEK kinase pathway. In the initial experiments, we also treated AM from uninfected rats in the same manner to determine whether these DEP effects are specific to macrophages infected with L. monocytogenes. The baseline of HO-1 expression in uninfected AM was about three times lower than that in the infected controls. Ex vivo exposure to DEP or eDEP, but not wDEP or CB, resulted in a 2.5- to 3-fold induction of HO-1 expression in uninfected AM (data not shown). These results suggest that the DEP effect on HO-1 induction may be common to all macrophages, but to a greater extent in L. monocytogenes-infected cells.



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FIG. 2. Western blot analysis of HO-1 expression in AM exposed ex vivo to DEP, eDEP, wDEP, CB, or hemin (A) and the effects of various inhibitory agents on HO-1 expression (B). (A) AM were obtained from L. monocytogenes-infected rats at 7 days post-infection and incubated with DMSO (0.25%), 50 µg/ml of DEP, eDEP, wDEP, CB, or 100 µg/ml hemin for 24 h. B. AM were obtained from L. monocytogenes-infected rats at 7 days post-infection and incubated with DMSO (0.25%) or 50 µg/ml of eDEP for 24 h. Some of the eDEP-containing cultures were preincubated with ANF (10 µM), NAC (10 mM), PD98059 (20 µM), SB203580 (10 µM), or SP600125 (100 µM) for 2 h prior to the addition of eDEP. The top panels show representative results of the Western blot analysis of HO-1 expression in AM, and the bottom panels show respective fold increase in band density, which represent the means ± SE of at least four experiments. *Significantly different from that of the corresponding controls, p < 0.05; #Significantly different from that of the eDEP-exposed AM, p < 0.05.

 
To investigate the role of organic compound-derived ROS on activation of HO-1 in L. monocytogenes-infected AM and the possible link between these two cellular responses, a kinetic study was carried out to show the effects of organic compounds and ROS on HO-1 activation. The eDEP, but not wDEP, induced the total HO activity (Fig. 3). This induction, occurring significantly at 4 h and longer exposure times, was partially blocked by ANF and totally inhibited by NAC. These results indicate that the measured changes in total HO activity, which correlated with the induction of HO-1 by eDEP and hemin or inhibition of HO-1 expression by ANF and NAC, give an accurate indication of changes in HO-1 activity. The inhibitive effects of ANF and NAC on eDEP-induced HO-1 expression and activation, together with the fact that the time-dependent induction of HO activity was preceded by eDEP-induced ROS generation, strongly suggest that the DEP organic compound-derived ROS play a major role in the induction of HO-1 in L. monocytogenes-infected AM.



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FIG. 3. The kinetics of eDEP-induced total HO activity and the effect of ANF or NAC on HO activity in AM. AM were obtained from L. monocytogenes-infected rats at 7 days post-infection and incubated with DMSO (0.25%,{circ}), 50 µg/ml of eDEP (•), wDEP ({triangledown}), or 100 µg/ml hemin ({blacktriangledown}) for varying time periods. Some of the eDEP-containing cultures were pretreated with ANF (10 µM, {square}) or NAC (10 mM, {blacksquare}) for 2 h prior to the addition of eDEP. Data represent the means ± SE of at least four experiments. *Significantly different from that of the corresponding controls, p < 0.05; #Significantly different from that of the eDEP-exposed AM, p < 0.05.

 
Roles of ROS and HO-1 in eDEP-Modulated Cytokine Production by AM
To study the potential roles of eDEP-derived ROS and HO-1 in AM-mediated immune responses to L. monocytogenes infection, the effect of eDEP on LPS-stimulated production of IL-10 and TNF-{alpha} by L. monocytogenes-infected AM in the absence and presence of various scavengers/inhibitors was kinetically monitored. Ex vivo exposure to eDEP resulted in a time-dependent increase in LPS-stimulated IL-10 production by L. monocytogenes-infected AM compared to nonexposed controls (Fig. 4A). The wDEP, on the other hand, did not enhance AM production of this cytokine. The eDEP-induced IL-10 production was partially inhibited by ANF and completely abolished by NAC. The time curves for the eDEP-induced IL-10 production showed a significant elevation at 8 h or longer incubation times, indicating that this event was preceded by DEP-induced ROS generation (Fig. 1) and HO-1 expression (Fig. 3). These results suggest that DEP may enhance LPS-stimulated IL-10 production by L. monocytogenes-infected AM through increased production of ROS and expression of the stress response protein HO-1. Interestingly, hemin, a known HO-1 inducer, induced a moderate but significant increase in LPS-stimulated IL-10 by L. monocytogenes-infected AM.



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FIG. 4. The kinetics of eDEP-induced IL-10 (A) and TNF-{alpha} (B) and effect of ANF or NAC on cytokine production by AM. AM were obtained from L. monocytogenes-infected rats at 7 days post-infection and incubated with DMSO (0.25%, {circ}), 50 µg/ml of eDEP (•), wDEP ({triangledown}), or 100 µg/ml hemin ({blacktriangledown}) for varying time periods in the presence of 1 µg/ml LPS. Some of the eDEP-containing cultures were pretreated with ANF (10 µM, {square}) or NAC (10 mM, {blacksquare}) for 2 h prior to the addition of eDEP. Cytokine levels in culture supernatants were determined by ELISA and expressed as the means ± SE of at least four experiments. *Significantly different from that of the corresponding controls, p < 0.05; #Significantly different from that of the eDEP-exposed AM, p < 0.05.

 
In contrast, eDEP inhibited LPS-stimulated TNF-{alpha} production by L. monocytogenes-infected AM, with significant effect occurring at 16 and 24 h postexposure (Fig. 4B). This effect was not observed by using wDEP, but was duplicated in cells incubated with hemin. The eDEP effect on TNF-{alpha} production was partially reversed by ANF, which is consistent with ANF's ability to partially block the eDEP effect on AM production of ROS, HO-1, and IL-10. Similar to its effect on IL-10, NAC completely blocked the stimulation of AM production of TNF-{alpha} by LPS.

To further assess the roles of eDEP-derived ROS and HO-1 in AM-mediated immune responses to infection, LPS-stimulated production of IL-10 and IL-12 by the infected AM was determined, and respective levels of cytokines were compared to that of HO-1 expression (Fig. 5). Here, SOD was used as a superoxide scavenger, hemin as a HO-1 inducer, and Znpp as a selective HO-1 activity inhibitor. The Western blot analysis showed that the eDEP-upregulated HO-1 expression was attenuated by pretreatment of cells with SOD, but not Znpp (Fig. 5A). Exposure to eDEP significantly elevated the LPS-stimulated production of IL-10, as shown above (Fig. 4A), but decreased that of IL-12. Treatment with SOD, which decreased HO-1 expression, substantially inhibited the eDEP effect on IL-10 induction. Znpp, a HO-1 activity inhibitor, also markedly decreased the eDEP-induced IL-10 production (Fig. 5B). The inhibition of IL-12 by eDEP was found to be greatly reversed by the pretreatment of SOD, but not by Znpp (Fig. 5C). Hemin, a known HO-1 inducer, induced a significant increase in the LPS-stimulated IL-10 but decreased IL-12 by the L. monocytogenes-infected AM. Together with the corresponding alterations of HO-1 expression in each treatment, these results suggest that superoxide plays a role in eDEP-mediated HO-1 expression and production of IL-10 and IL-12 in L. monocytogenes-infected AM. The results also suggest that HO-1 may be involved in regulating AM production of IL-10.



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FIG. 5. Effects of SOD and Znpp on eDEP-modulated of HO-1 expression (A) and production of IL-10 (B) and IL-12 (C) in AM. AM were obtained from L. monocytogenes-infected rats at 7 days post-infection and incubated with DMSO (0.25%), 50 µg/ml of eDEP, or 100 µg/ml hemin for 24 h in the presence (B and C) or absence (A) of 1 µg/ml LPS. Some of the eDEP-containing cultures were pretreated with SOD (2000 U/ml) or Znpp (10 µM) for 2 h prior to the addition of eDEP. (A) The top panel shows representative result of the Western blot analysis of HO-1 expression in AM and the bottom panel (A) shows fold increase in band density which represents the means ± SE of at least three experiments. (B and C) Cytokine levels in culture supernatants were determined by ELISA and expressed as the mean ± SE of at least four experiments. *Significantly different from that of the corresponding controls, p < 0.05; #Significantly different from that of eDEP-exposed AM, p < 0.05.

 
Direct Effect of ROS on HO-1 Expression and Cytokine Production in AM
To illustrate the effect of ROS on cellular responses to L. monocytogenes, AM from L. monocytogenes-infected rats were directly treated with PYR, a superoxide anion donor, and measured for HO-1 expression and cytokine secretion (Fig. 6). The results showed that PYR strongly induced HO-1 expression (Fig. 6A). PYR was also found to induce the LPS-stimulated production of IL-10, but downregulated IL-12 by L-monocytogenes-infected AM. When the PYR-induced HO-1 activity was inhibited by pretreatment with Znpp, its effect on IL-10, but not IL-12, was markedly decreased. Znpp alone had no effect on HO-1 expression (Fig. 6A) and production of both cytokines (Figs. 6B and 6C). These results suggest that superoxide can directly induce HO-1 and IL-10 expression but decrease production of IL-12 by L-monocytogenes-infected AM. The fact that Znpp inhibited PYR-induced IL-10 secretion further suggests that HO-1 may play an important role in ROS-induced AM production of this cytokine. Again, the HO-1 inducer hemin induced a significant increase in the LPS-stimulated IL-10 but decreased IL-12 by the L. monocytogenes-infected AM.



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FIG. 6. Effect of PYR on HO-1 expression (A) and production of IL-10 (B) and IL-12 (C) in AM. AM were obtained from L. monocytogenes-infected rats at 7 days post-infection and incubated with DMSO (0.25%), 10 µM Znpp, 300 µM PYR, or 100 µg/ml hemin for 24 h in the presence (B and C) or absence (A) of 1 µg/ml LPS. Some of the PYR-containing cultures were pretreated with Znpp (10 µM) for 2 h prior to the addition of PYR. (A) The top panel shows representative result of the Western blot analysis of HO-1 expression in AM and the bottom panel shows fold increase in band density which represents the mean ± SE of at least three experiments. (B and C) Cytokine levels in culture supernatants were determined by ELISA and expressed as the means ± SE of at least four experiments. *Significantly different from that of the corresponding controls, p < 0.05; #Significantly different from that of eDEP-exposed AM, p < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although L. monocytogenes is not considered a major respiratory pathogen, several reports have shown that the L. monocytogenes infection model is also applicable to the respiratory system for assessing the pulmonary host defense mechanisms (Antonini et al., 2000Go; Jakob, 1993Go; Reasor et al., 1996Go; Van Loveren et al., 1988Go; Yang et al., 2001Go; Yin et al., 2002Go, 2003Go, 2004Go) and the importance of cytokines in resistance to bacterial infection (Bancroft et al., 1989Go; Pfeffer et al., 1993Go). The current study shows that DEP exhibited a significant effect on AM production of TNF-{alpha}, IL-12, and IL-10. These three cytokines are crucial for host to mount an effective immune response against L. monocytogenes. The ability of AM to secrete TNF-{alpha} is of major importance in innate immunity against bacteria. Mice that are deficient to TNF-{alpha} receptors have been shown to succumb to L. monocytogenes infection (Pfeffer et al., 1993Go; Rothe et al., 1993Go). IL-12, on the other hand, plays an important role in initiating and maintaining Th1 immunity (Hsieh et al., 1993Go; Park and Scott, 2001Go). Intracellular pathogens such as L. monocytogenes are also known to dampen host defense by inducing cellular production of IL-10 to delay their elimination from the infected cells (Fleming et al., 1999Go; Redpath et al., 2001Go). DEP markedly enhanced AM production of IL-10 and, at the same time, suppressed the production of pro-inflammatory cytokines TNF-{alpha} and IL-12. Previously, we have demonstrated that DEP can alter T cell proliferation and inhibit the production of IFN-{gamma} by L. monocytogenes-infected lymphocytes (Yin et al., 2003Go, 2004Go). Together, these results show that DEP inhibit innate immunity, interrupt the development of bacterial-specific T cell responses, and suppress T cell functions.

The effect of DEP on AM secretion of cytokines stems from the cellular action of the organic component. CB or wDEP, which are largely devoid of the organic content, had only moderate or no effect on cellular production of cytokines. DEP extract, but not wDEP, was also shown to induce ROS and HO-1 in AM. In fact, there was a sequential occurrence of these events in the order of ROS generation, HO-1 expression, and cytokine production. The kinetic studies showed that the induction of ROS by eDEP reached a peak value within 2 h of exposure (Fig. 1B), whereas the measured HO activity showed a delayed time course with significant activity occurring at 4–24 h after DEP exposure (Fig. 3). In comparison to the early induction of HO-1, the effects of DEP on AM production of IL-10 or TNF-{alpha} became significant only after 8 or 16 h of exposure. These results suggest that the induction of ROS by DEP elicited HO-1 expression and that HO-1 may indeed modulate the production of these cytokines. This observation was supported by the actions of various scavengers/inhibitors including ANF, NAC, SOD, and Znpp, and by results obtained through direct stimulation of AM with superoxide anion (Fig. 6). The literature has already shown that HO-1 mediates macrophage secretion of pro- and anti-inflammatory cytokines. On one hand, IL-10 induces HO-1 via p38 MAP kinase pathway and inhibits LPS-induced TNF-{alpha} production by macrophages, and on the other hand, activation of HO-1 by hemin induces AM production of IL-10 (Fleming et al., 1999Go). Our results show the involvement of p38 MAP kinase in eDEP-induced HO-1 expression and imply that this kinase may play a role in eDEP-modulated cytokine production by AM.

The ability of DEP to induce intracellular ROS has been noted in several studies. DEP are known to induce pulmonary expression of CYP 1A1 in AM, Clara cells, and type II pneumocytes (Rengasamy et al., 2003Go). The induction of CYP 1A1 by PAHs from DEP is associated with the generation of superoxide anion and hydroxyl radicals (Bonvallot et al., 2001Go; Takano et al., 2002Go). Our measurement of ROS via DCF fluorescence was consistent with the formation of these radicals, and ANF, a CYP 1A1 inhibitor, partially inhibited the induction of ROS by eDEP. ANF also inhibited eDEP-induced HO-1 expression and activity and partially reversed the DEP effects on AM production of IL-10 and TNF-{alpha} These results indicate that CYP 1A1 may play an important role in metabolizing DEP chemicals to produce ROS, upregulation of HO-1 expression, and modulation of IL-10 and TNF-{alpha} in AM by eDEP.

We have further shown that eDEP treatment or direct stimulation of AM by superoxide anion (via PYR) induced HO-1 expression and IL-10 production but decreased secretion of IL-12. Pretreatment of AM with SOD, which inhibited the effect of eDEP on HO-1 expression, decreased IL-10 secretion. Znpp, a known HO-1 activity inhibitor, also decreased eDEP- or PYR-induced IL-10 production. These results suggest that ROS, via increased HO-1 expression, upregulated the production of IL-10 by L. monocytogenes-infected AM. The effects of SOD and Znpp on eDEP- and PYR-induced HO-1 expression and/or IL-10 production also support the hypothesis that eDEP, through the induction of ROS and HO-1, modulate cytokine production by L. monocytogenes-infected AM. In addition, induction of IL-10 by hemin, a HO-1 inducer, further stresses the role of HO-1 in modulation of this cytokine. It should be mentioned, however, hemin at the concentration used resulted in a higher induction of HO-1 protein (Figs. 2A, 5A, and 6A) and HO activity (Fig. 3) but a lesser stimulatory effect on IL-10 secretion than eDEP (Figs. 4A and 5B), suggesting that factors other than HO-1 may also be involved in eDEP-induced IL-10 production. It was also observed that induction of HO-1 following hemin treatment significantly decreased IL-12 production by L. monocytogenes-infected AM. However, inhibition of HO-1 activity with Znpp did not increase the bacteria-induced IL-12 production when compared to control levels (Fig. 6C). Furthermore, the inhibitory effect of eDEP on IL-12 was reversed only by SOD, but not by Znpp, while both of them either downregulated the eDEP-induced HO-1 expression (Fig. 5) or acted as a HO-1 activity inhibitor. Similar results were also observed in the PYR-treated cells, where Znpp exhibited different effect on PYR-altered secretion of IL-10 and IL-12 (Fig. 6). These results suggest that inhibition of IL-12 by PYR or DEP-derived ROS may be through cellular mechanisms other than HO-1 expression. In this aspect, it has been reported that the intracellular thiol redox status may play a role in regulating the cytokine production by macrophages. Studies have shown that macrophages with reduced intracellular glutathione (GSH) content showed elevated production of IL-6 and IL-10, but decreased production of NO and IL-12, in responses to IFN-{gamma} and LPS stimulation. In contrast, macrophages with increased intracellular GSH content showed a reciprocal response (i.e., elevated production of NO and IL-12), but decreased production of IL-6 and IL-10 (Murata et al., 2001Go), suggesting a crucial role of intracellular GSH levels in determining which of Th1 or Th2 cytokine responses predominate in immune responses in macrophages. It has been demonstrated that, in the dose range 10–100 µg/ml, organic DEP extracts induce a progressive decline in the cellular GSH/GSSG ratio in the RAW 264.7 macrophage cell line (Xiao et al., 2003Go). Theses results provided a hint that the downregulation of IL-12 by PYR or DEP-derived ROS may be associated with the altered intracellular thiol redox status in AM.

In summary, the current study offers a plausible mechanism on how DEP dampen the pulmonary immune responses toward L. monocytogenes. DEP inhibit AM production of TNF-{alpha} and IL-12 that are crucial for the innate and T cell-mediated immunity, but enhance AM production of IL-10 that prolongs the survival of the bacteria. These effects were all attributable to the organic component of DEP through a mechanism that involves the induction of intracellular ROS and increased expression of HO-1.


    ACKNOWLEDGMENTS
 
This research was supported in part by grant NIH RO1 HL 62630 from the National Heart, Lung, and Blood Institute.


    NOTES
 

1 To whom correspondence should be addressed at School of Pharmacy, West Virginia University, 1 Medical Center Drive, Morgantown, WV 26506-9530. Fax: (304) 293-2576. E-mail: jma{at}hsc.wvu.edu.


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