* School of Pharmacy, West Virginia University, Morgantown, West Virginia 26506; and Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505
Received March 26, 2004; accepted August 9, 2004
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ABSTRACT |
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Key Words: diesel exhaust particles; Listeria monocytogenes; heme oxygenase-1; reactive oxygen species; cytokines.
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INTRODUCTION |
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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., 2001; Yin et al., 2002
, 2003
, 2004
). 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, 1995
; Sibille and Reynolds, 1990
). 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., 1989
; Czuprynski et al., 1992
). It has been well documented that AM-derived pro-inflammatory cytokines, such as IL-1ß and TNF-
, 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, 1995
; Le and Vilcek, 1987
). A successful pulmonary host defense, on the other hand, also needs specific cell-mediated immunity (Kaufmann, 1993
). 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-
(IFN-
)-secreting T helper (Th)1 CD4+ lymphocytes (Hsieh et al., 1993
). 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, 2001
) and increased T cell production of IFN-
(Trinchieri, 1995
, 1998
). In addition, both IL-1ß and TNF-
activate NK cells to release IFN-
, which activates macrophages to kill the bacteria. These cytokines are also T cell activators (Akira et al., 1990
; Hsieh et al., 1993
). IL-10, on the other hand, is a potent immunosuppressive factor that downregulates macrophage bactericidal activity (Fleming et al., 1999
). 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., 2001
).
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., 2002, 2004
). DEP suppressed AM phagocytotic function and their secretion of pro-inflammatory cytokines including IL-1ß, TNF-
, 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., 1999
, 2000
). 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., 1998
; Park et al., 1996
) and apoptosis (Lei et al., 1998
). DEP-derived chemicals, in fact, have been shown to produce superoxide and hydroxyl radicals when incubated with lung microsomal enzymes (Kumagai et al., 1997
).
Demple and colleagues (Bouton and Demple, 2000; Demple, 1999
) 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., 2002
) and macrophage cell lines (Li et al., 2000
, 2002
) as an early response to oxidative stress. Li et al. (2000)
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)
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-
. On the other hand, Inoue et al. (2001)
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-, 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.
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MATERIALS AND METHODS |
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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: 0.10.6 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 225250 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., 2000
).
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., 1992) 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 100200 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 420% 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). 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 (
= 40 mM1 cm1). 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- 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: 31.31000 pg/ml for TNF-
, 15.6500 pg/ml for IL-10, and 7.8500 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.
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RESULTS |
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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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DISCUSSION |
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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 424 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- 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-
production by macrophages, and on the other hand, activation of HO-1 by hemin induces AM production of IL-10 (Fleming et al., 1999
). 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., 2003). The induction of CYP 1A1 by PAHs from DEP is associated with the generation of superoxide anion and hydroxyl radicals (Bonvallot et al., 2001
; Takano et al., 2002
). 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-
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-
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- 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., 2001
), 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 10100 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., 2003
). 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- 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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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