Induction of apoptosis by particulate matter: role of TNF-alpha and MAPK

Beek Yoke Chin1,5, Mary E. Choi2,3, Marie D. Burdick4, Robert M. Strieter4, Terence H. Risby5, and Augustine M. K. Choi1,3,6

6 Section of Pulmonary and Critical Care Medicine and 2 Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven 06250; 3 Connecticut Veterans Affairs HealthCare System, West Haven, Connecticut 06516; 1 Division of Pulmonary and Critical Care Medicine and 5 Department of Environmental Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205; and 4 Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, Michigan 48109

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Particulate matter (PM) is a major by-product from the combustion of fossil fuels. The biological target of inhaled PM is the pulmonary epithelium and resident macrophages. In this study, we demonstrate that cultured macrophages (RAW 264.7 cells) exposed continously to a well-defined model of PM [benzo[a]pyrene adsorbed on carbon black (CB+BaP)] exhibit a time-dependent expression and release of the cytokine tumor necrosis factor-alpha (TNF-alpha ). CB+BaP also evoked programmed cell death or apoptosis in cultured macrophages as assessed by genomic DNA-laddering assays. The CB+BaP-induced apoptosis was inhibited when macrophages were treated with CB+BaP in the presence of a neutralizing antibody to TNF-alpha , suggesting that TNF-alpha plays an important role in mediating CB+BaP-induced apoptosis in macrophages. Interestingly, neither untreated carbon black nor benzo[a]pyrene alone induced apoptosis or caused the release of TNF-alpha in RAW 264.7 cells. Moreover, we observed that TNF-alpha activates mitogen-activated protein kinase (MAPK) activity, the extracellular signal-regulated kinases p42/p44, in a time-dependent manner. RAW 264.7 cells treated with PD-098059, a selective inhibitor of MAPK kinase activity, did not exhibit CB+BaP-induced apoptosis and TNF-alpha secretion. Furthermore, cells treated with the MAPK kinase inhibitor did not undergo TNF-alpha -induced apoptosis. Taken together, our data suggest that TNF-alpha mediates PM-induced apoptosis and that the MAPK pathway may play an important role in regulating this pathway.

tumor necrosis factor-alpha ; mitogen-activated protein kinase; programmed cell death; cytokines; carbon; signal transduction; macrophages

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

EPIDEMIOLOGICAL STUDIES conducted in urban centers have shown correlations between increases in morbidity and mortality in various respiratory ailments and exposure to environmental air pollution. Much of this increased morbidity and mortality has been demonstrated in susceptible populations, including people suffering from diseases such as asthma (14) or pulmonary fibrosis (32) as well as individuals who are immunocompromised (30). Among the multitude of agents contributing to environmental air pollution in our society, particulate matter (PM) generated from the combustion of fossil fuels represents a major culprit. PM derived from the combustion of fossil fuels consists of an inert carbonaceous core and multiple layers of adsorbed pollutant molecules. After inhalation and deposition in the lung, the majority of the adsorbed pollutants are released into the pulmonary surfactant and will eventually reach the pulmonary epithelial cells (3, 25). After this initial release, the particles with their residual burden of adsorbed pollutants will remain on the surface of the pulmonary surfactant until cleared by the mucociliary escalator or phagocytosed by the resident macrophages (18, 23) where the residual pollutants may be eventually released.

Our current understanding of how PM affects lung function and how it affects the inflammatory process in the lung is poor. Limited data exist supporting the notion that PM can modulate the inflammatory response in the lung. For example, an in vivo study (10) demonstrated that PM can induce an influx of inflammatory cells such as polymorphonuclear cells and macrophages into the airways. Cultured macrophages exhibit increased secretion of cytokines such as interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha ) when exposed to other types of particles such as fungus Micropolyspora faeni, mineral dusts, crystalline silica, titanium dioxide, and asbestos (8, 11, 12, 17, 20, 29).

In this study, we have chosen a well-defined model for PM, benzo[a]pyrene (BaP) adsorbed onto a carbon black (CB; CB+BaP) at a defined surface coverage, to examine the mechanism(s) by which PM modulates the inflammatory response in cultured macrophages. This particle complex has been shown previously to be a good model for the carbonaceous particles produced by the combustion of fossil fuels (28). We show in our in vitro model that PM (CB+BaP) evoked a time-dependent expression and release of the cytokine TNF-alpha . Additionally, we demonstrate that PM-induced TNF-alpha plays an important role in mediating PM-induced apoptosis and provide evidence that the mitogen-activated protein kinase (MAPK) pathway plays an important role in regulating PM- and TNF-alpha -induced apoptosis in this cell culture model.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. Murine peritoneal macrophage cell line RAW 264.7 cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM supplemented with 10% fetal bovine serum and gentamicin (50 µg/ml) at 37°C in a humidified atmosphere of 5% CO2-95% air. All experiments were conducted in subconfluent cells.

Model carbon particles. The CB (N339) selected for study was manufactured under well-defined conditions specified by the American Society for Testing Materials. This CB was selected because it has been shown to have similar surface properties to the carbonaceous particles produced by the combustion of fossil fuels (27). Moreover, because it has been well established that combustion of fossil fuels produces gaseous- and particle-phase pollutants, a defined amount (three-fourths of a unimolecular surface coverage) of a typical pollutant, BaP, was adsorbed onto the surface of the CB. This surface coverage was selected because Risby et al. (26) determined that particles have similar surface coverages after the initial release of adsorbed molecules into pulmonary surfactant.

Exposure of cultured macrophages to model carbon particles. The model particles (CB+BaP) were deaggregated to form stable particles, with a mean diameter of 0.1 µm by homogenization in DMEM at 3,000 g for 1 h. After this deaggregation, the particles remained suspended for at least 48 h. The cells were exposed to known concentrations of this suspension (2 µg/ml) for up to 24 h. Suspensions of deaggregated untreated CB or free BaP (2 µg/ml) were used as controls for this study.

Western blot analysis. Total cellular protein extracts were obtained for the Western analyses as previously described (29). Briefly, cells were lysed in buffer containing 1% Nonidet P-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of aprotinin. Protein concentrations of the cell lysates were determined by Coomassie blue dye-binding assay (Bio-Rad, Hercules, CA). An equal volume of 2× SDS loading buffer (0.125 mM Tris · HCl, pH 7.4, 4% SDS, and 20% glycerol) was added, and the samples were boiled for 5 min. Protein samples (100 µg) were resolved by 12% SDS-PAGE, then electroblotted onto polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were incubated with TNF-alpha rabbit polyclonal antibody (1:500; Biosource International, Camarillo, CA) for 1.5 h, followed by incubation with horseradish peroxidase-conjugated anti-rabbit antibody for 1.5 h. Signal development was carried out with an enhanced chemiluminescence detection kit (Amersham).

ELISA assay for TNF-alpha . Cell culture medium was collected at various time points after exposure to CB+BaP and stored at -80°C until assayed. TNF-alpha protein was determined by ELISA kits (Biosource International) and quantified with a plate reader (model EL340, Bio-Tek Instruments, Winooski, VT). Values are reported as nanograms per milliliter of medium. Neutralizing antibody to TNF-alpha was obtained from Biosource International.

Genomic DNA isolation and DNA laddering analysis. Genomic DNA isolation was performed as specified by the manufacturer's protocol (Puregene kit, Gentra Systems, Minneapolis, MN). Briefly, cells were lysed directly on the plate after medium removal with lysis buffer, followed by a 1-h incubation with RNase A. The cell lysates were precipitated for proteins and spun at 2,000 g for 15 min. Supernatant was precipitated with isopropanol for isolation of DNA. After an alcohol wash, DNA was hydrated and quantified, and 20 µg were analyzed in 1.5% agarose gel electrophoresis fractionation.

MAPK activity assays. MAPK activity assays were performed according to the manuufacturer's instructions (New England Biolabs, Beverly, MA) with minor modifications. Briefly, cells were lysed in a buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerophosphate, 1 mM Na3VO4, 1 µg/ml of leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and sheared by passage through a 25-gauge needle. Protein concentrations were determined as described in Western blot analysis. Total protein samples (200 µg) were incubated with phospho-specific p44/42 MAPK rabbit polyclonal antibody (1:50) overnight on a rocker at 4°C. The p44/42 MAPK antibodies detect only the 204-tyrosine-phosphorylated forms of extracellular signal-regulated kinase (ERK) 1 and ERK2 and thus select for the activated (phosphorylated) MAPK. As a positive control, 20 ng of active MAPK (ERK2) were incubated with a control cell extract. Protein A-Sepharose beads (Pharmacia Biotech, Piscataway, NJ) were then added to immunoprecipitate the activated MAPK complex. The immunoprecipitated pellets were incubated with 1 µg of Elk1 fusion protein in the presence of 100 µM ATP and a kinase buffer (25 mM Tris, pH 7.5, 5 mM beta -glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2). The reaction was terminated with SDS loading buffer [62.5 mM Tris · HCl, pH 6.8, 2% (wt/vol) SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% (wt/vol) bromophenol blue]. The samples were analyzed by 12% SDS-PAGE and electroblotted as described in Western blot analysis. ERK activity was assayed by detection of phosphorylated Elk1 with a phospho-specific Elk1 rabbit polyclonal antibody (1:1,000). After overnight incubation with the primary antibody at 4°C, the membrane was incubated for 1 h with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000) at room temperature with gentle rocking. The proteins were subsequently detected with LumiGLO (New England Biolabs) and exposed to X-ray film. Because ERK2 is known to be capable of phosphorylating Elk1, MAPK activity was determined by a phospho-specific Elk1 antibody that detects only the 383-serine-phosphorylated Elk1.

Statistical analysis. Data are expressed as means ± SE. Differences in measured variables between experimental and control groups were assessed with Student's t-test. Statistical calculations were performed on a Macintosh personal computer with the Statview II statistical package (Abacus Concepts, Berkeley, CA). Significant difference was accepted at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CB+BaP-induced TNF-alpha expression and secretion. RAW 264.7 cells were treated with CB+BaP (2 µg/ml), and the cells and medium were collected for Western blot and enzyme-linked immunosorbent assay (ELISA) analyses, respectively. Figure 1A shows rapid induction of TNF-alpha protein expression by Western blot analysis, with increased TNF-alpha expression by 1 h and sustained elevated TNF-alpha expression up to 8 h of CB+BaP treatment. The increase in TNF-alpha protein expression was associated with increased TNF-alpha protein secretion in RAW 264.7 cells in response to CB+BaP as assessed by ELISA. Figure 1B illustrates a time-dependent induction of TNF-alpha secretion in RAW 264.7 cells by CB+BaP.


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Fig. 1.   Macrophages (RAW 264.7 cells) exposed to carbon black (CB) and benzo[a]pyrene (BaP; CB+BaP) induce expression and secretion of tumor necrosis factor (TNF)-alpha . A: total cellular protein was extracted after CB+BaP (2 µg/ml) treatment and then analyzed for TNF-alpha protein expression by Western blot analyses as described in MATERIALS AND METHODS. Lane 1, untreated control; lane 2, 1-h CB+BaP; lane 3, 2-h CB+BaP; lane 4, 4-h CB+BaP; lane 5, 8-h CB+BaP; lane 6, 24-h CB+BaP. Data are representative of 3 independent experiments. B: supernatants were collected from cells at indicated times of CB+BaP (2 µg/ml) exposure, and ELISAs were performed as described in MATERIALS AND METHODS. CTL, untreated control cells. Data are means ± SE of measurements from 3 independent experiments. P < 0.03 for each CB+BaP-treated sample compared with CTL.

TNF-alpha mediates CB+BaP-induced apoptosis. In view of our observations that CB+BaP induces induction of TNF-alpha expression and secretion (Fig. 1) and that TNF-alpha has been strongly implicated in the induction of apoptosis in various in vitro models (1, 9, 15), we investigated whether TNF-alpha induction by CB+BaP may mediate CB+BaP-induced apoptosis in RAW 264.7 cells. Initially, we established whether CB+BaP alone can induce cell death via programmed cell death or apoptosis. Genomic DNA was isolated from RAW 264.7 cells after CB+BaP treatment and analyzed for nucleosomal fragmentation or DNA laddering. Figure 2A shows DNA laddering at 24 h, with pronounced DNA fragmentation at 48 h of CB+BaP treatment. We then demonstrated that TNF-alpha can also induce apoptosis in RAW 264.7 cells. Genomic DNA was isolated from RAW 264.7 cells after treatment with TNF-alpha (1 ng/ml) and then analyzed for DNA fragmentation by gel electrophoresis. Figure 2B shows marked DNA laddering in cells after treatment with TNF-alpha (1 ng/ml). To determine whether TNF-alpha played a role in the CB+BaP-induced apoptosis in RAW 264.7 cells, we exposed cells to CB+BaP in the absence and presence of a neutralizing antibody against TNF-alpha . As shown in Fig. 3A, DNA laddering is evident in RAW 264.7 cells exposed to CB+BaP (lane 2). However, when cells were exposed to CB+BaP in the presence of neutralizing antibody to TNF-alpha (lane 4), no evidence for DNA laddering was observed. These observations suggest that CB+BaP-induced apoptosis in RAW 264.7 cells may be mediated by TNF-alpha . Interestingly, RAW 264.7 cells exposed to either untreated CB or free BaP alone had a negligible effect on TNF-alpha secretion as assessed by ELISA, whereas the combination of CB and BaP induced a marked secretion of TNF-alpha (Fig. 4). Based on the observations (Figs. 1-3) that TNF-alpha may play an important role in mediating CB+BaP-induced apoptosis in RAW 264.7 cells, we hypothesized that CB or BAP alone would not induce apoptosis in RAW 264.7 cells due to their inability to induce TNF-alpha . This was confirmed by exposing RAW 264.7 cells to CB and BAP alone: no apoptosis as assessed by DNA-laddering assays was exhibited (Fig. 5).


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Fig. 2.   A: CB+BaP-induced apoptosis in RAW 264.7 cells. Genomic DNA was isolated from cells exposed to CB+BaP (2 µg/ml) for 24 and 48 h and fractionated in a 1.5% agarose gel electrophoresis as described in MATERIALS AND METHODS. MW, molecular-weight marker. B: TNF-alpha -induced apoptosis in RAW 264.7 cells. Genomic DNA was isolated from cells exposed to TNF-alpha (1 ng/ml) for 24 h and fractionated in a 1.5% agarose gel electrophoresis as described in MATERIALS AND METHODS. Data are representative of 4 independent experiments.


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Fig. 3.   Effect of neutralizing antibody to TNF-alpha on CB+BaP-induced apoptosis. A: genomic DNA was isolated from cells exposed to CB+BaP (2 µg/ml) for 24 h in absence and presence of neutralizing antibody to TNF-alpha (100 U) and then fractionated in a 1.5% agarose gel electrophoresis as described in MATERIALS AND METHODS. Lane 1, CTL; lane 2, CB+BaP alone; lane 3, TNF-alpha antibody alone; lane 4, CB+BaP + neutralizing antibody to TNF-alpha . Data are representative of 3 independent experiments. B: genomic DNA was isolated from cells exposed to CB+BaP (2 µg/ml) for 24 h in absence and presence of control isotype IgG antibody and then fractionated in a 1.5% agarose gel electrophoresis as described in MATERIALS AND METHODS. Lane 1, CTL; lane 2, CB+BaP alone; lane 3, IgG antibody alone; lane 4, CB+BaP + IgG antibody.


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Fig. 4.   TNF-alpha secretion is not affected in RAW 264.7 cells exposed to untreated CB or BaP. Supernatants were collected from cells after exposure to 24 h of untreated CB (2 µg/ml), BaP (2 µg/ml), or CB+BaP (2 µg/ml), and ELISAs were performed as described in MATERIALS AND METHODS. Data are means ± SE of measurements from 3 independent experiments. * P < 0.05 compared with CTL.


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Fig. 5.   Effect of CB or BaP on apoptosis in RAW 264.7 cells. Genomic DNA was isolated from cells exposed to 24 h of untreated CB (2 µg/ml), BaP (2 µg/ml), or CB+BaP (2 µg/ml) alone and fractionated in a 1.5% agarose gel electrophoresis as described in MATERIALS AND METHODS. Lane 1, CTL; lane 2, CB; lane 3, BaP; lane 4, CB+BaP. Data are representative of 3 independent experiments.

TNF-alpha activates MAPK (ERK1/ERK2) in RAW 264.7 cells. RAW 264.7 cells were treated with TNF-alpha (1 ng/ml), and p44/p42erk1/2 immunoprecipitation assays were performed with the transcriptional factor Elk1 as our substrate for measuring MAPK (ERK1/ERK2) activities. Figure 6A demonstrates that ERK1 and -2 are both activated after 1 h of TNF-alpha treatment, and their activities are maintained throughout the duration of the 24-h exposure. Furthermore, we investigated whether CB+BaP alone can also activate the MAPK (ERK1/ERK2) pathway. Figure 6B illustrates the activation of MAPK (ERK1/ERK2) in RAW 264.7 cells in response to CB+BaP.


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Fig. 6.   A: effect of TNF-alpha on mitogen-activated protein kinase (MAPK) activity (p44/p42) in RAW 264.7 cells. Cellular lysates were isolated at indicated times after treatment with TNF-alpha (1 ng/ml) and were analyzed for MAPK activity as described in MATERIALS AND METHODS. +CTL, positive control [extracellular signal-regulated kinase (ERK) 2] provided by manufacturer as described in MATERIALS AND METHODS. Data are representative of 4 independent experiments. B: effect of CB+BaP on MAPK activity (p44/p42) in RAW 264.7 cells. Cellular lysates were isolated after treatment with CB+BaP (2 µg/ml) and were analyzed for MAPK activity as described in MATERIALS AND METHODS. Lane 1, CTL; lane 2, 2-h CB+BaP.

Role of MAPK pathway in mediating CB+BaP-induced apoptosis in RAW 264.7 cells. Based on our observations that TNF-alpha or CB+BaP alone can stimulate MAPK activity (Fig. 6) and may mediate CB+BaP-induced apoptosis in RAW 264.7 cells, we investigated whether inhibiting the MAPK pathway would result in the attenuation of both CB+BAP-induced TNF-alpha release and CB+BAP-induced apoptosis in RAW 264.7 cells. Cells were treated with CB+BaP in the presence and absence of the MAPK kinase (MEK) inhibitor PD-098059. MEK is an upstream activator of ERK1/ERK2 and has been shown to regulate the activity of ERK1/ERK2 in response to TNF-alpha (24, 34, 35). We did not observe any differences in the amounts of TNF-alpha secreted between the control cells (untreated) and the cells treated with the MEK inhibitor alone (Fig. 7). Marked induction of TNF-alpha secretion was observed in cells treated with CP+BaP alone; however, RAW 264.7 cells exposed to CB+BaP in the presence of the MEK inhibitor exhibited a marked attenution of TNF-alpha release, levels comparable to control levels (Fig. 7). We then performed DNA-laddering assays from genomic DNA isolated from RAW 264.7 cells that were pretreated with the MEK inhibitor PD-098059 before exposure to CB+BaP (Fig. 8). There was no evidence of DNA laddering in control untreated cells (lane 1) or MEK inhibitor-treated cells (lane 2). DNA laddering is prominent in cells treated with CB+BaP alone (lane 3); however, we did not observe evidence of DNA laddering in cells exposed to CB+BaP in the presence of the MEK inhibitor (lane 4). Furthermore, we also examined the effect of the MEK inhibitor on TNF-alpha -induced apoptosis by DNA-laddering assays. As illustrated in Fig. 9, the MEK inhibitor attenuated TNF-alpha -induced apoptosis in RAW 264.7 cells.


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Fig. 7.   Effect of MAPK kinase (MEK) inhibitor (I) on CB+BaP-induced TNF-alpha secretion in RAW 264.7 cells. Cells were exposed to CB+BaP with and without pretreatment with MEK I PD-098059 (10 µM) for 24 h. Supernatants were collected from cells after exposure, and ELISAs were performed as described in MATERIALS AND METHODS. Data are means ± SE of measurements from 3 independent experiments. * P < 0.05 compared with CB + BAP. ** P < 0.05 compared with CTL.


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Fig. 8.   Effect of MEK I on CB+BaP-induced apoptosis in RAW 264.7 cells. Genomic DNA was isolated from cells exposed to CB+BaP (2 µg/ml) for 24 h in absence and presence of MEK I PD-098059 (10 µM) and then fractionated in a 1.5% agarose gel electrophoresis as described in MATERIALS AND METHODS. Lane 1, CTL; lane 2, MEK I PD-098059; lane 3, CB+BaP; lane 4, CB+BaP + MEK I PD-098059. Data are representative of 3 independent experiments.


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Fig. 9.   Effect of MEK I on TNF-alpha -induced apoptosis in RAW 264.7 cells. Genomic DNA was isolated from cells exposed to TNF-alpha (1 ng/ml) for 24 h in absence and presence of MEK I PD-098059 (10 µM) and then fractionated in a 1.5% agarose gel electrophoresis as described in MATERIALS AND METHODS. Lane 1, CTL; lane 2, MEK I PD-098059; lane 3, TNF-alpha ; lane 4, TNF-alpha  + MEK I PD-098059. Data are representative of 3 independent experiments.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Combustion of fossil fuels can be inefficient and results in the generation of airborne environmental PM consisting of a carbonaceous particle with layers of pollutant-adsorbed molecules (36). This carbonaceous PM is increasingly recognized as a major environmental air pollutant, contributing to the increasing morbidity and mortality of various respiratory diseases such as asthma (5, 6). The CB used in this study has defined surface properties that are similar to those of carbonaceous particles found in PM. If this CB is coated with a defined surface coverage of BaP, it represents a well-characterized and defined model for airborne PM.

We demonstrated that carbon particles such as CB+BaP induce the expression of an inflammatory cytokine, TNF-alpha , whereas macrophages exposed to untreated carbon particles alone or to a saturated free solution of BaP show no induction of TNF-alpha secretion. Interestingly, noncarbonaceous particles such as asbestos, quartz, and silica (17, 29) induce TNF-alpha expression and secretion without surface-adsorbed molecules. Moreover, it has been demonstrated that saturated solutions of BaP can be internalized without the presence of carbon particles (4, 13). These results show that the internalization of CBs or BaP separately do not trigger the secretion of TNF-alpha . Triggering the secretion of TNF-alpha appears to require that the macrophages be exposed to BaP adsorbed onto the surface of a particle. Because phagocytosis of CB+BaP is critical to elicit biological effects in macrophages (21, 23), including the induction of stress gene products (data not shown), and phagocytosis of PM is coupled to the production of reactive oxygen species, it is reasonable to propose that the surface-adsorbed BaP on the phagocytosed CB+BaP is activated by intracellular reactive oxygen species to trigger the induction of TNF-alpha . The precise biochemical mechanism(s) by which CB+BaP triggers TNF-alpha production will require further study. Interestingly, our observations of this differential effect of TNF-alpha secretion between carbon particles with and without adsorbed molecules on their surfaces have been reported in other models. For example, Dasenbrock et al. (7) observed differences in the rate of tumor formation between diesel PM and extracted diesel PM.

We report here that PM such as CB+BaP can induce apoptosis in cultured macrophages. Our findings are consistent with a recent report that alveolar macrophages undergo apoptosis when exposed to the urban particle residual oil fly ash (19). This study, however, did not examine the signaling pathways or mechanism(s) by which residual oil fly ash induces apoptosis. We believe that the TNF-alpha secretion induced by CB+BaP plays a major role in mediating CB+BaP-induced apoptosis based on the following observations: 1) TNF-alpha directly induces apoptosis in macrophages, and these cells treated with CB+BaP in the presence of a neutralizing antibody against TNF-alpha do not exhibit apoptosis; 2) inhibition of CB+BaP-induced TNF-alpha secretion by the MEK inhibitor also inhibits CB+BaP-induced apoptosis; and 3) cells treated with BaP or untreated carbon alone do not induce TNF-alpha release and do not induce apoptosis in macrophages.

The MAPK signaling pathway has been shown to play a major role in regulating a variety of cellular functions such as cell growth during proliferation and the stress response to DNA damage; cellular stimuli including inflammatory cytokines, hyperosmolality, and oxidative stress; and activation of transcription factors mediating downstream gene transcription and expression (31, 33). This study suggests that the ERK1/ERK2 MAPK pathway plays an important role in regulating both CB+BaP-induced TNF-alpha secretion and CB+BaP- or TNF-alpha -induced apoptosis based on our observations that inhibition of the ERK1/ERK2 pathway resulted in attenuation of both CB+BaP-induced TNF-alpha secretion and CB+BaP- or TNF-alpha -induced apoptosis. A schematic diagram illustrating the possible signaling pathways of CB+BaP-induced apoptosis in RAW 264.7 macrophage cells is provided to summarize our proposed model (Fig. 10). Moreover, recent data (2, 22) have also implicated the MAPK pathway in the regulation of programmed cell death or apoptosis. For example, in a nerve growth factor withdrawal model of apoptosis, both ERK1/ERK2 and c-Jun-NH2-terminal kinase MAPKs play an important role in modulating apoptosis in neuronal tissue. We have not examined rigorously the role of c-Jun-NH2-terminal kinase or p38, the other two MAPK signaling pathways in our CB+BaP model of apoptosis. These studies are currently being pursued.


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Fig. 10.   A proposed model for CB+BaP-induced apoptosis in RAW 264.7 macrophage cells.

Is there a functional significance of TNF-alpha -induced apoptosis in macrophages exposed to our model of environmental pollutant? Others (16) have speculated on the role of TNF-alpha in mediating apoptosis in parasitic worms, helminths, as "a protective immune response." These authors have argued that TNF-alpha induction of an intrinsic programmed cell death signal may serve to protect the host from potentially hazardous oxidants and/or radicals generated from parasitic material. The function of TNF-alpha in CB+BaP-induced apoptosis in macrophages may serve as a signal to minimize the inflammatory process during PM exposure. On the other hand, TNF-alpha mediates the recruitment of inflammatory cells such as polymorphonuclear leukocytes and macrophages, and additional autocrine/paracrine secretion of TNF-alpha induced by CB+BaP can further aid in the recruitment of even more inflammatory cells to the affected areas. This complex regulation of apoptosis by CB+BaP poses a challenge to a straightforward assessment of the physiological function of apoptosis in lung injury. Further investigations leading to the identification of cell types undergoing apoptosis in these models of PM exposure will be helpful in delineating the functional significance of apoptosis.

    ACKNOWLEDGEMENTS

A. M. K. Choi was supported by National Heart, Lung, and Blood Institute Grant HL-55330, National Institute of Allergy and Infectious Diseases Grant AI-42365; and American Heart Association (AHA) Established Investigator Award. T. H. Risby was supported by National Institute of Environmental Health Sciences Grant ES-03156. R. M. Strieter was supported by National Cancer Institute Grant CA-66180. M. E. Choi was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 5-K12-DK-0129809, AHA Grant GIA96015510, and Department of Veterans Affairs Career Development Award.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: A. M. K. Choi, Section of Pulmonary and Critical Care Medicine, Yale Univ. School of Medicine, 333 Cedar St., LCI 105, New Haven, CT 06520.

Received 21 May 1998; accepted in final form 30 July 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(5):L942-L949
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