Contribution of IL-1{beta} and TNF-{alpha} to the initiation of the peripheral lung response to atmospheric particulates (PM10)

Hiroshi Ishii,1 Takeshi Fujii,1 James C. Hogg,1 Shizu Hayashi,1 Hiroshi Mukae,1 Renaud Vincent,2 and Stephan F. van Eeden1

1James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital, University of British Columbia, Vancouver, British Columbia V6Z 1Y6; and 2Environmental Health Directorate, Health Canada, Ottawa, Ontario K1A 0L2, Canada

Submitted 26 August 2003 ; accepted in final form 2 March 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Alveolar macrophages (AM) play a key role in clearing atmospheric particulates from the lung surface and stimulating epithelial cells to produce proinflammatory mediators. The present study examines the role of "acute response" cytokines TNF-{alpha} and IL-1{beta} released by AM exposed to ambient particulate matter with a diameter of <10 µm (PM10) in amplifying the proinflammatory mediator expression by A549 cells and human bronchial epithelial cells (HBEC). The results showed that supernatants from human AM incubated 24 h with PM10 (100 µg/ml) contained more TNF-{alpha}, IL-1{beta}, granulocyte-macrophage colony stimulating factor, IL-6, and IL-8 than nonexposed AM supernatants. The 3-h treatment of A549 cells with PM10-exposed AM supernatants increased TNF-{alpha}, IL-1{beta}, IL-8, regulated on activation normal T-cells expressed and secreted (RANTES), and leukemia inhibitory factor mRNA compared with the treatment with nonexposed AM supernatants and, compared with untreated A549 cells, additionally increased ICAM-1 and monocyte chemotactic protein-1 mRNA. Preincubating PM10-exposed AM supernatants with anti-IL-1{beta} antibodies reduced all the above mediators as well as VEGF mRNA expression (P < 0.05), while anti-TNF-{alpha} antibodies were less effective (P > 0.05), and the combination of the two antibodies most effective. When HBEC were treated similarly, anti-TNF-{alpha} antibodies had the greatest effect. In A549 cells PM10-exposed AM supernatants increased NF-{kappa}B, activator protein (AP)-1 and specificity protein 1 binding, while anti-TNF-{alpha} and anti-IL-1{beta} antibodies reduced NF-{kappa}B and AP-1 binding. We conclude that AM-derived TNF-{alpha} and IL-1{beta} provide a major stimulus for the production of proinflammatory mediators by lung epithelial cells and that their relative importance may depend on the type of epithelial cell target.

ribonuclease protection assay; transcription factors; enzyme-linked immunosorbent assay; proinflammatory mediators; particulate matter with diameter < 10 µm


THERE IS ABUNDANT epidemiological evidence that exposure to ambient particulate matter with a diameter of <10 µm (PM10) produces cardiopulmonary disease (8, 26). Although the precise biological mechanisms are unclear, PM10 are known to stimulate the production of inflammatory mediators by alveolar macrophages (AM) (23, 31, 33), epithelial (3, 11, 12, 20), and other lung cells (18). Previous studies from our laboratory have shown that humans exposed to high levels of air pollution develop a systemic inflammatory response characterized by a leukocytosis (30) and increased proinflammatory mediators in the circulating blood (33). We have also shown that deposition of atmospheric particles in the lung results in a systemic inflammatory response that includes stimulation of the bone marrow to increase production and release of polymorphonuclear leukocytes into the circulation (10, 23, 30, 31, 33). There are three potential sources of these mediators. First, mediators released from AM as they phagocytize particles are critically important in inducing this systemic response and marrow stimulation (22). Second, lung epithelial cells also phagocytize inhaled particles (11, 23) and synthesize a variety of proinflammatory cytokines that could influence the local inflammatory response (11, 12, 20). These findings are supported by other studies showing that lung epithelial cells respond to particle exposure in vitro by modulating transcription factor activity and by releasing inflammatory mediators (3, 16). Third, several laboratories including our own have provided data suggesting that cytokines produced by AM as they phagocytize PM10 stimulate epithelial cells to produce inflammatory mediators (10, 15). We recently reported that human AM and bronchial epithelial cells (HBEC) exposed to PM10 in coculture interact to enhance the synthesis and release of a variety of proinflammatory mediators and that supernatants from this coculture instilled into rabbit lungs induced a systemic inflammatory response (10).

The present study was designed to determine whether mediators released by PM10-stimulated AM affect transcriptional regulation of inflammatory mediator genes in human alveolar epithelial (A549) cells and HBEC in the absence of physical contact between AM and epithelial cells. To accomplish this we measured cytokines released from human AM exposed to PM10 (EHC-93) and determined whether the supernatants from the stimulated AM enhanced inflammatory mediator release from both types of lung epithelial cells. We then determined the role of AM-derived TNF-{alpha} and IL-1{beta} in stimulating this response by using blocking antibodies to remove them from the AM supernatants. Finally, we assessed whether this modification of the supernatants modulated transcription factor binding activity in the nucleus of A549 cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Urban air particles (PM10). PM10 particles collected in an urban environment (EHC-93) were obtained from the Environmental Health Directorate, Health Canada, Ottawa, Ontario. A detailed analysis of the EHC-93 has been presented elsewhere (34). Particles were suspended at a concentration of 1 mg/ml in RPMI-1640 medium containing 10% FBS (Life Technologies, Rockville, MD) and sonicated three times for 1 min each at maximal power on a Vibra Cell VC-50 sonicator (Sonics and Materials, Danbury, CT) before being added to the cells. The endotoxin content of the PM10 suspension of 100 µg/ml was 6.4 ± 1.8 EU/ml or <3.0 ng/ml (10, 11). This dose of LPS has been shown not to activate either AM or lung epithelial cells to produce cytokines (11).

Isolation of human AM and exposure to PM10. Bronchoalveolar lavage (BAL) fluid was obtained from a total of six patients who underwent lobectomy or pneumonectomy for a small peripheral tumor at St. Paul's Hospital, Vancouver. Informed consent was obtained from all subjects, and these studies were approved by the Human Ethics Committee of the University of British Columbia. All subjects were current smokers and were asked to abstain from smoking for 6 wk before the operation. Their mean age was 57.0 yr (range 35–68 yr) (3 women and 3 men). Human AM were harvested from BAL fluid obtained from lung segments or lobes that were free of the tumor. BAL was performed three or four times using 60-ml aliquots of sterile physiological saline solution. The cells were washed twice in cold RPMI-1640 containing 10% FBS, antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin; Sigma, St. Louis, MO) and Fungizone (1 µg/ml, GIBCO BRL, Gaithersburg, MD). AM monocultures were suspended in RPMI-1640. The BAL fluid cells were >90% viable (trypan blue exclusion method) and consisted of 90–95% AM and <2% neutrophils. The remaining cells were lymphocytes. AM (1.0 x 107) were placed in 100-mm cell culture dishes and allowed to adhere to the plastic dish for 30 min in a humidified incubator with 5% CO2 at 37°C. The nonadherent cells (<1.0 x 106) were then removed by rinsing twice with RPMI-1640, and adherent AM (>98% AM as assessed by Wright-Giemsa stain) were incubated for 24 h in 10 ml of RPMI-1640 alone as control or RPMI-1640 with 100 µg/ml of PM10 (EHC-93) suspension.

ELISA measurements. Pairs of culture supernatants were collected after 24-h incubation of AM with medium alone or 100 µg/ml of PM10, centrifuged, filtered through a syringe filter with pore size of 0.22 µm (Corning, Cambridge, MA) to eliminate as much as possible any remaining particles, and stored at –70°C. TNF-{alpha}, IL-1{beta}, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-6, and IL-8 protein levels were measured by the Cytokine Core Laboratory (Baltimore, MD) using an ELISA based on a biotin-streptavidin-peroxidase detection system as previously described (11). The measurements for these cytokines in each of the pairs of supernatants from six experiments were done in triplicate, and values reported are the mean of six measurements.

Stimulation of A549 cells with supernatants from PM10-exposed AM. A549 cells (American Type Culture Collection, Manassas, VA), a human type II alveolar-like cell line originally derived from a patient with bronchiolo-alveolar carcinoma, were grown to 95–100% confluence in 60-mm cell culture dishes (~3.0 x 106 cells/dish) in MEM supplemented with 10% heat-inactivated FBS in 5% CO2 at 37°C, then incubated for 3 and 6 h in 2 ml of RPMI-1640 (control), control supernatant from nonexposed AM, or supernatant of PM10-exposed AM. In a separate experiment, the direct effect of particles on A549 cells was examined by adding fresh stock suspensions of 100 µg/ml of PM10 prepared in MEM to the cells.

Blocking antibody studies. As AM produce both TNF-{alpha} and IL-1{beta}, two primary acute response cytokines, when stimulated (10, 33), we selected to neutralize these cytokines in supernatants of PM10-exposed AM to determine their individual effect on mediator release from A549 cells. Supernatants of PM10-exposed AM were preincubated for 1 h at 4°C with either 1) control mouse IgG1 (2 µg/ml) (DAKO, Copenhagen, Denmark), 2) monoclonal mouse anti-human TNF-{alpha} antibody, 3) monoclonal mouse anti-human IL-1{beta} antibody, or 4) both TNF-{alpha} and IL-1{beta} antibodies (1 µg/ml, respectively) (R&D Systems, Minneapolis, MN) before we treated A549 cells with the supernatants. Blocking doses were selected using the following manufacturer's guidelines: neutralization doses of 0.04–0.08 µg/ml anti-TNF-{alpha} are required to yield 50% maximal inhibition of cytokine activity of 0.25 ng/ml of recombinant human TNF-{alpha} and 0.05–0.2 µg/ml anti-IL-1{beta} antibodies to yield 50% maximal inhibition of cytokine activity of 0.05 ng/ml of recombinant human IL-1{beta}.

To determine whether the TNF-{alpha} antibody was effective in blocking TNF-{alpha}, A549 cells were treated with 1 ng/ml recombinant human TNF-{alpha} (rhTNF-{alpha}) (Calbiochem), and the expression of inflammatory mediator mRNA by these cells was compared with cells incubated with the same rhTNF-{alpha} after it had been preincubated as above with 1 µg/ml anti-human TNF-{alpha} antibody.

RNAse protection assay. After 3- or 6-h treatment, total RNA was isolated from A549 cells using a single-step phenol-chloroform extraction procedure (Trizol, Life Technologies, Grand Island, NY). The levels of inflammatory mediator mRNA were determined using the RiboQuant multiprobe system (PharMingen, San Diego, CA) following the instructions of the supplier. A customized template set was used that included mRNAs of the following inflammatory mediators: human regulated on activation, normal T-cells expressed and secreted (RANTES), TNF-{alpha}, ICAM-1, IL-1{beta}, monocyte chemotactic protein (MCP)-1, IL-8, leukemia inhibitory factor (LIF), and VEGF. Internal controls included mRNAs of the ribosomal protein L32 and GAPDH. In brief, 10 µg of total cellular RNA was hybridized overnight to the [{alpha}-32P]UTP-labeled riboprobes that had been synthesized from the supplied template sets. Single-stranded RNA and free probe remaining after hybridization were digested by a mixture of RNase A and T1. The protected RNA was then phenolized, precipitated, and analyzed on a 5% denaturing polyacrylamide gel. After electrophoresis, the gel was dried under vacuum and subjected to autoradiography, and the quantity of protected labeled RNA was determined using densitometry and the NIH Image 1.63 software (National Institutes of Health, Bethesda, MD). Results were normalized to the expression of the internal control, GAPDH. For the densitometric analysis, each RNase protection assay (RPA) of the 3-h incubation period was repeated four or five times.

Stimulation of HBEC with supernatants from PM10-exposed AM. Primary HBEC were isolated from bronchial tissues of the same patients as the AM according to a previously described procedure (11) and grown to 95–100% confluence in 60-mm cell culture dishes. To determine whether TNF-{alpha} and IL-1{beta} in the supernatants from PM10-exposed AM also activate primary lung epithelial cells, these HBEC were incubated with supernatants from PM10-exposed AM before and after being blocked with TNF-{alpha} and IL-1{beta} antibodies as described above for A549 cells and with supernatants from nonexposed AM. Inflammatory mediator mRNA was measured by RPA as above.

EMSA. A549 cells grown to confluence in 60-mm dishes were exposed to RPMI-1640 (control), or control supernatants from nonexposed AM or supernatants of PM10-exposed AM for 1 or 2 h. The direct effect of particles on A549 cells was examined by adding fresh stock suspensions of 100 µg/ml of PM10 to the cells. In blocking studies, supernatants of PM10-exposed AM were preincubated with either mouse IgG1, TNF-{alpha}; or IL-1{beta} blocking antibodies or both TNF-{alpha} and IL-1{beta} antibodies before A549 cells were incubated with the supernatants. After being washed twice with PBS, the nuclear proteins were extracted as previously described except for the use of 1:500 diluted proteinase inhibitor cocktail (Sigma) instead of PMSF and leupeptin (Sigma) (17). EMSA of specific nuclear proteins was carried out as previously described (17). Briefly, 10 µg of the nuclear protein extract was incubated at 4°C for 30 min with four volumes of binding reaction buffer (10 mM Tris·HCl, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, and 4% glycerol) and 1.5 µg of poly(dI-dC) (Boehringer Mannheim) followed by the addition of [{gamma}-32P]ATP end-labeled double-strand consensus oligonucleotides for NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG-3'), activator protein (AP)-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3'), or specificity protein 1 (Sp1) (5'-ATT CGA TCG GGG CGG GGC GAG-3') (Promega, Madison, WI). Further incubation for 20 min allowed the formation of protein-DNA complexes. For competition assays, excess unlabeled double-stranded oligonucleotides (3.5 pmol) of sequence identical with or unrelated to that of the labeled probe was added to the nuclear extract before addition of the labeled probe. The protein-DNA complexes were separated on a nondenaturing 6% polyacrylamide gel. Gels were dried under vacuum and autoradiographed.

Statistical analysis. Data are expressed as mean values ± SE. The minimum number of replicates for all measurement was at least four. Differences in protein production by ELISA for matched pairs (control vs. PM10 treated) were compared by the Wilcoxon signed-ranked test. Differences between multiple groups were compared by one-way ANOVA. The post hoc test for a multiple comparison was the Fisher's protected least-significant-difference test. Significance was assumed at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of PM10 on the release of cytokines from AM. Figure 1 shows the protein levels of cytokines in supernatants of human AM incubated with medium alone or 100 µg/ml of PM10 suspension for 24 h. TNF-{alpha}, IL-1{beta}, GM-CSF, IL-6, and IL-8 were significantly higher in supernatants of PM10-exposed AM than in control supernatants (P < 0.05).



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Fig. 1. TNF-{alpha}, IL-1{beta}, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-6, and IL-8 protein levels in supernatants of alveolar macrophages (AM) after 24 h incubation with medium alone (open bars) or medium containing 100 µg/ml of particulate matter with a diameter of <10 µm (PM10) (filled bars). Values are means ± SE of 6 experiments. *P < 0.05 compared with control supernatants.

 
Effect of supernatants of AM on expression of mRNA in A549 cells. Figure 2A shows representative autoradiographs of proinflammatory mediator mRNA expression in A549 cells after 3- and 6-h incubation in medium alone (control) or supernatants of untreated AM (AM-control) or supernatants of AM incubated with 100 µg/ml of PM10 for 24 h (AM-PM10). A549 cells exposed for 3 h to supernatants of untreated AM had increased levels of ICAM-1, MCP-1, and IL-8 mRNA compared with medium-treated A549 cells (control). These differences were less after the 6-h exposure. When A549 cells were exposed for 3 h to supernatants of PM10-exposed AM, levels of RANTES, TNF-{alpha}, ICAM-1, IL-1{beta}, MCP-1, IL-8, LIF, and VEGF mRNA were increased compared with control or supernatants of untreated AM. Except for RANTES mRNA, these differences were less in the 6-h samples. The mean densitometric value for each mediator mRNA from five separate RPA experiments representing the 3-h incubation period (Fig. 2B) confirmed that only MCP-1 mRNA expression was significantly increased by untreated AM supernatants (P < 0.005), while supernatants of PM10-exposed AM significantly increased the levels of RANTES, TNF-{alpha}, ICAM-1, IL-1{beta}, MCP-1, IL-8, and LIF mRNA (P < 0.01) but not that of VEGF, compared with that in control A549 cells. Figure 2B also shows that supernatants from PM10-exposed AM significantly increased levels of RANTES, TNF-{alpha}, IL-1{beta}, IL-8, and LIF mRNA in A549 cells compared with those from nonexposed AM (P < 0.05). In contrast, direct exposure of A549 cells to 100 µg/ml of PM10 did not alter the mRNA expression of these inflammatory mediators (data not shown).



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Fig. 2. A: autoradiographs of RNase protection assay (RPA) showing the expression of proinflammatory mediator mRNAs by A549 cells after 3- and 6-h incubation in medium alone (control) or supernatants of untreated AM (AM-control) or supernatants of AM incubated with 100 µg/ml particulate matter with a diameter of <10 µm (PM10) for 24 h (AM-PM10). Incubation with AM-control for 3 h increased ICAM-1, monocyte chemotactic protein (MCP)-1, and IL-8 mRNA compared with control. These differences were less after the 6-h exposure. Compared with control or AM-control, incubation with AM-PM10 for 3 h increased regulated on activation normal T-cells expressed and secreted (RANTES), TNF-{alpha}, ICAM-1, IL-1{beta}, MCP-1, IL-8, leukemia inhibitory factor (LIF), and VEGF mRNA. Except for RANTES mRNA, these increases were less after the 6-h exposure. L32 and GAPDH were used as controls for lane loading. B: densitometric analysis of bands on autoradiographs such as that shown in A. The density of the bands representing the mediator mRNA was compared with that of the GAPDH mRNA band in the same lane and the resulting ratio (AM-control; hatched bars, AM-PM10; filled bars) is shown as the percentage change from control values (open bars). Values are means ± SE of 5 experiments representing the 3-h incubation period. *P < 0.01, **P < 0.005 compared with control. {dagger}P < 0.05 compared with AM-control group.

 
Effect of TNF-{alpha}- and IL-1{beta}-neutralizing antibodies on expression of A549 cell and HBEC mRNAs. To determine whether TNF-{alpha} or IL-1{beta} released from PM10-exposed AM influence mRNA expression by A549 cells, supernatants of PM10-exposed AM were preincubated with neutralizing antibodies of either TNF-{alpha}, IL-1{beta}, or a combination of both before addition to A549 cells. Figure 3A shows a representative autoradiograph of inflammatory mediator mRNA expression in A549 cells after 3-h incubation in medium alone (control) or in supernatants from PM10-exposed AM (AM-PM10) preincubated with mouse IgG1 (IgG), anti-TNF-{alpha} antibody (anti-TNF-{alpha}), anti-IL-1{beta} antibody (anti-IL-1{beta}), or both TNF-{alpha} and IL-1{beta} antibodies (anti-TNF-{alpha}, IL-1{beta}). Expression of RANTES, TNF-{alpha}, ICAM-1, IL-1{beta}, MCP-1, IL-8, LIF, and VEGF mRNA was compared with the control antibody, attenuated by the combination of the TNF-{alpha} and IL-1{beta} antibodies, and less drastically reduced by each of these antibodies alone. Compared with the untreated A549 cells, a residual level of ICAM-1, MCP-1, and IL-8 expression remained after treatment with the combined antibodies. Densitometric analysis of four separate RPA experiments shown in Fig. 3B confirmed that, compared with the control antibody, the IL-1{beta} antibody alone (P < 0.05) or a combination of TNF-{alpha} and IL-1{beta} antibodies (P < 0.005) significantly reduced the expression of all eight mRNAs, including RANTES, that was reduced to 31.2% (mean) of control values, while the TNF-{alpha} antibody decreased only RANTES to 76.5% (P < 0.05). In addition, while the IL-1{beta} antibody alone reduced the expression of ICAM-1 mRNA to 49.6% of control values (P < 0.05), the combination of TNF-{alpha} and IL-1{beta} antibodies significantly reduced this expression further to 26.2% (P < 0.005).



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Fig. 3. A: representative autoradiograph of RPA showing the expression of inflammatory mediator mRNAs in A549 cells after 3-h incubation in medium alone (control) or in neutralizing antibody-treated supernatants from PM10-exposed AM (AM-PM10). The supernatants were preincubated with mouse IgG1 (IgG), anti-TNF-{alpha} antibody (anti-TNF-{alpha}), anti-IL-1{beta} antibody (anti-IL-1{beta}), or both TNF-{alpha} and IL-1{beta} antibodies (anti-TNF-{alpha}, IL-1{beta}) before application to the A549 cells. Levels of RANTES, TNF-{alpha}, ICAM-1, IL-1{beta}, MCP-1, IL-8, LIF, and VEGF mRNA expression were attenuated by treating the supernatants with TNF-{alpha} or IL-1{beta} antibodies or both. The right 2 lanes represent RPA from A549 cells exposed to culture media containing 1 ng/ml of recombinant human TNF-{alpha} with either 1 µg/ml of mouse IgG1 (rhTNF-{alpha} + IgG) or TNF-{alpha} antibody (rhTNF-{alpha} + anti-TNF-{alpha}). TNF-{alpha} induced an increase in RANTES, ICAM-1, MCP-1, and IL-8 mRNA levels, a response that was markedly abolished in the presence of TNF-{alpha} antibody. B: densitometric analysis of bands on autoradiographs such as that shown in A. The density of the bands representing the mediator mRNA was compared with that of the GAPDH mRNA band in the same lane, and the resulting ratio (anti-TNF-{alpha}, hatched bars; anti-IL-1{beta}, gray bars, anti-TNF-{alpha}, IL-1{beta}, black bars) is shown as the percentage change from control values (mouse IgG1; open bars). Values are means ± SE of 4 experiments. *P < 0.05, {dagger}P < 0.005 compared with control. **P < 0.005 compared with anti-IL-1{beta} antibody alone.

 
To ensure that the TNF-{alpha} antibody was indeed neutralizing, we treated A549 cells with culture media containing 1 ng/ml of rhTNF-{alpha} preincubated with and without 1 µg/ml of anti-TNF-{alpha} antibodies. This concentration of TNF-{alpha} was higher than that found in the supernatants of AM treated with PM10. rhTNF-{alpha} was able to induce an increase in RANTES, ICAM-1, MCP-1, and IL-8 mRNA levels, a response that was almost completely abolished in the presence of TNF-{alpha} neutralizing antibody (Fig. 3A).

Figure 4 shows a representative autoradiograph of inflammatory mediator mRNA expression in HBEC after 3-h incubation in medium alone (control); supernatants from untreated AM; or supernatants from PM10-exposed AM preincubated with mouse IgG1, TNF-{alpha}; or IL-1{beta} antibody, or both TNF-{alpha} and IL-1{beta} antibodies. Expression of ICAM-1, IL-1{beta}, and IL-8 mRNA was, compared with the control antibody, attenuated by the TNF-{alpha} antibody and the combination of TNF-{alpha} and IL-1{beta} antibodies but less drastically reduced by IL-1{beta} antibody alone.



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Fig. 4. Representative autoradiograph of RPA showing the expression of inflammatory mediator mRNAs in human bronchial epithelial cells (HBEC) after 3-h incubation in medium alone (control) or in supernatants from untreated AM (AM-control) or PM10-exposed AM (AM-PM10) preincubated with mouse IgG1 (IgG), anti-TNF-{alpha} antibody (anti-TNF-{alpha}), anti-IL-1{beta} antibody (anti-IL-1{beta}), or both TNF-{alpha} and IL-1{beta} antibodies (anti-TNF-{alpha}, IL-1{beta}). Increased expression of ICAM-1, IL-1{beta}, and IL-8 mRNA as a result of incubation in PM10-exposed AM supernatants was attenuated by treating the supernatants with either TNF-{alpha} or IL-1{beta} antibodies as well as with both antibodies together.

 
Effect of supernatants of PM10-exposed AM on transcription factor binding activity. Representative autoradiographs of EMSA to detect NF-{kappa}B, AP-1, and Sp1 binding activity in nuclear extracts of A549 cells after 1-h incubation in medium alone (control), control supernatants from untreated AM (AM-control), or supernatants of AM incubated with 100 µg/ml of PM10 for 24 h (AM-PM10) are shown in Fig. 5. The NF-{kappa}B binding activity in A549 cells incubated with the supernatant from control AM was higher than that in medium-treated A549 cells, while supernatants from PM10-exposed AM further increased this binding activity (Fig. 5A). Despite the higher basal levels of AP-1 and Sp1 binding activity compared with that of NF-{kappa}B, binding of these two transcription factors was also increased when A549 cells were treated with the supernatants from PM10-exposed AM (Fig. 5, B and C) but not from control AM. Similar results were obtained when these experiments were repeated three times for NF-{kappa}B and Sp1 and twice for AP-1 after either 1- or 2-h incubation (data not shown). Specific binding was abolished by competition with excess unlabeled oligonucleotide specific for each transcription factor, whereas the nonspecific competitor was ineffective (right two lanes of Fig. 5, AC). The NF-{kappa}B (Fig. 6), AP-1, and Sp1 (data not shown) binding activities in A549 cells directly exposed to 100 µg/ml of PM10 were much lower than those of cells incubated with PM10-exposed AM supernatants. Also the binding activity of NF-{kappa}B (Fig. 6A) and AP-1 (Fig. 6B), but not Sp1 (data not shown), was reduced by TNF-{alpha} antibodies or the combination of TNF-{alpha} and IL-1{beta} antibodies.



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Fig. 5. Representative autoradiographs of EMSA showing NF-{kappa}B, activator protein (AP)-1, and specificity protein 1 (Sp1) binding activities in nuclei of A549 cells after 1-h incubation in medium alone (control) or control supernatants from untreated AM (AM-control) or supernatants of AM incubated with 100 µg/ml of PM10 for 24 h (AM-PM10). The level of NF-{kappa}B binding activity after incubation in supernatant from AM-control was higher than that in the control and further increased after incubation in supernatant from AM-PM10 (A). Likewise, the binding activities of AP-1 (B) and Sp1 (C) were increased by supernatants from AM-PM10. The specificity of the bands representing each transcription factor (arrow) was determined by adding excess unlabeled oligonucleotide with the same nucleotide sequence as the radiolabeled oligonucleotide (Competitor) or excess unlabeled nonspecific oligonucleotide (Noncompetitor).

 


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Fig. 6. Representative autoradiographs of EMSA showing NF-{kappa}B (A) and AP-1 (B) binding activities in nuclei of A549 cells after 1-h incubation in medium alone (control), 100 µg/ml of PM10 suspension (PM10), supernatants from untreated AM (AM-control) or PM10-exposed AM (AM-PM10) preincubated with mouse IgG1 (IgG), anti-TNF-{alpha} antibody (anti-TNF-{alpha}), anti-IL-1{beta} antibody (anti-IL-1{beta}), or both TNF-{alpha} and IL-1{beta} antibodies (anti-TNF-{alpha}, IL-1{beta}). NF-{kappa}B and AP-1 binding activities were attenuated by treating the supernatants with TNF-{alpha} antibodies or the combination of TNF-{alpha} and IL-1{beta} antibodies.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AM and lung epithelial cells play a key role in processing inhaled particulate matter. We have previously shown that both human AM and bronchial epithelial cells produce proinflammatory mediators when they phagocytize atmospheric particles and that an interaction between them amplifies the production of these mediators (10, 11, 33). The present study examines the effect of AM supernatants on the epithelial cells after removing the TNF-{alpha} and IL-1{beta} using specific blocking antibodies. The results show that the removal of TNF-{alpha} alone had a small effect on the mediator mRNAs produced by alveolar epithelial cells whereas the removal of IL-1{beta} significantly reduced mediator mRNA production (reduced RANTES to ~31.2% of the control values). Blocking both TNF-{alpha} and IL-1{beta} had an additive effect on reducing mediator production (including ICAM-1 mRNA expression) in A549 cells. While these two acute response cytokines are the major inflammatory gene activators in the macrophage supernatants, because the combination of antibodies blocking TNF-{alpha} and IL-1{beta} did not completely reduce inflammatory gene expression to levels found in the untreated A549 cells, other more minor inflammatory factors are likely present in the PM10-stimulated AM supernatant. Interestingly in HBEC, blocking TNF-{alpha} in supernatants of AM had a more pronounced effect than blocking IL-1{beta}, suggesting that TNF-{alpha} is more important in activating epithelial cells in the larger airways while IL-1{beta} might be important for activating the epithelium in the lower airways.

Jimenez and colleagues (15) have demonstrated that conditioned media from blood monocytes exposed to PM10 collected from the London and Edinburgh air particulate monitoring stations stimulate IL-8 release by A549 cells. Our results obtained by using Ottawa ambient particles and freshly isolated human AM support this finding and extend this observation. We showed that, while direct exposure of A549 cells to 100 µg/ml PM10 (EHC-93) did not alter the inflammatory mediator mRNA expression, exposure of these cells to supernatants from particle-exposed AM caused a significant increased expression of a large variety of proinflammatory mediator mRNAs including IL-8 but also RANTES, TNF-{alpha}, ICAM-1, IL-1{beta}, and LIF. We also confirmed previous findings (22, 33) that the supernatants of PM10-exposed AM contained elevated levels of IL-1{beta}, IL-6, IL-8, GM-CSF, and TNF-{alpha}. In contrast to our present results, our previous study (33) showed that TNF-{alpha} and IL-8 were increased by PM10 exposure, but due to the large variation in their levels, these increases were not statistically different. The inflammatory mediators produced by these lung cells as a direct and indirect consequence of PM10 exposure are those that regulate the local inflammatory response in the lung as well as contribute to the systemic inflammatory response.

Several studies have suggested that direct cellular contact between lung cells (e.g., epithelial cells, endothelial cells, and fibroblasts) is necessary for activation and cytokine expression (18, 19). However, other reports (4, 15, 27, 28) demonstrated that soluble mediators released from AM and alveolar epithelial cells in the absence of cell contact are an equally important pathway of enhancing the response to PM10 exposure. The present results show that direct contact between AM and A549 cells or HBEC is not necessary, but we suspect that close proximity of AM to lung epithelial cells is important in delivering the highest concentration of AM-generated mediators to these cells.

Of all the cytokines released from AM when they were exposed to atmospheric particles in this study (IL-1{beta}, IL-6, IL-8, GM-CSF, and TNF-{alpha}), TNF-{alpha} and IL-1{beta} are "acute response" cytokines that promote neutrophilic and eosinophilic inflammation. They do so either directly or indirectly by stimulating other cell types such as the lung epithelial cells to increase their synthesis of relevant secondary cytokines and cell adhesion molecules (7, 20). Previous studies (4, 27) have shown that TNF-{alpha} and IL-1{beta} released by AM in response to LPS stimulation amplified the expression of IL-6 and IL-8 by A549 cells while direct exposure of A549 cells to LPS did not. TNF-{alpha} and IL-1{beta} act in synergy to increase IL-8 expression. Standiford and colleagues (28) showed that conditioned media from LPS-stimulated AM increased MCP-1 expression in A549 cells. MCP-1 expression was reduced by IL-1{beta} neutralizing antibody but not TNF-{alpha} antiserum, and synergism was again demonstrated between TNF-{alpha} and IL-1{beta}. In the study by Jimenez and colleagues (15), conditioned media from PM10-exposed blood monocytes amplified IL-8 release from A549 cells via an elaboration of TNF-{alpha}. Our study demonstrated that while TNF-{alpha}-neutralizing antibody only reduced RANTES mRNA expression in A549 cells treated with supernatants of PM10-exposed AM, IL-1{beta}-neutralizing antibody or a combination of TNF-{alpha}- and IL-1{beta} antibodies reduced the expression of all eight candidate mRNAs measured, including that of IL-8. This finding underlines the importance of IL-1{beta} as a critically important regulator of the proinflammatory response of alveolar epithelial cells (A549 cells).

Our results show an increased expression in lung epithelial cells, both A549 and HBEC, of the key mediators IL-8 and ICAM-1 implicated in neutrophil migration as a consequence of macrophage exposure to PM10. IL-8 is one of the most potent chemoattractants and activators of these cells, while ICAM-1 assists neutrophil migration by promoting firm adhesion to the endothelium and epithelium. Besides its importance in the recruitment of neutrophils, IL-8 is also a potent chemotactic factor for eosinophils and T lymphocytes (9). Results from other laboratories have shown that IL-8 and ICAM-1 upregulation is a feature of the inflammatory response to diesel exhaust particles and silica as well as PM10 (3, 5, 13). Our results extend these findings by showing that IL-1{beta} and TNF-{alpha} are the key cytokines produced by AM in the initiation of this response from the epithelial cells.

NF-{kappa}B and AP-1 are two transcription factors commonly found to regulate many TNF-{alpha} (1)- and IL-1 (6)-induced genes, while Sp1 is required for basal constitutive expression (reviewed in Ref. 32). Sp1 has also been shown to be required to activate gene expression driven by other transcription factors (2, 25), including genes responding to IL-1{beta} (24). In the present study, although the cytokines secreted by PM10-stimulated AM increased the activity of Sp1 in nuclei of A549 cells, this increased activity was not altered by TNF-{alpha} or IL-1{beta} blocking. This result, together with the increased expression of a number of inflammatory mediators by these lung epithelial cells, specifically RANTES, ICAM-1, and MCP-1, all with potential Sp1 binding sites in their promoters (2, 21, 29), suggests either that the basal activity of Sp1 in the A549 cells is sufficient to assist in activating the promoters of these mediators or that inflammatory mediators other than TNF-{alpha} and IL-1{beta} are responsible for stimulating this transcription factor. The presence of other minor inflammatory factors in the PM10-stimulated AM supernatants is supported by the failure of the combination of TNF-{alpha} and IL-1{beta} blocking antibodies to completely reduce inflammatory gene expression to levels found in the untreated A549 cells. Therefore, the increase in Sp1 binding in the A549 cells as a result of exposure to supernatants from PM10-stimulated AM, although novel, most likely is not related to the TNF-{alpha}- and IL-1{beta}-stimulated expression of RANTES, ICAM-1, and MCP-1. In contrast to Sp1, increased NF-{kappa}B and AP-1 binding activities in A549 cells exposed to PM10-stimulated AM supernatant were reduced after blocking TNF-{alpha} and IL-1{beta}. These results suggest that NF-{kappa}B and AP-1 are key transcription factors regulating the increase in mediator expression by lung epithelial cells in response to indirect stimulation by environmental particles.

In summary, we have demonstrated that TNF-{alpha} and IL-1{beta} released from AM exposed to ambient particles (PM10) stimulates the expression of proinflammatory mediators by lung epithelial cells, most likely by promoting the binding of transcription factors to the enhancers of the mediator genes. TNF-{alpha} has a larger effect on bronchial epithelial cells, while the IL-1{beta} effect is more prominent on alveolar epithelial cells. This difference may be reflected in differences in mediator mRNA expression by these two lung epithelial cells. Our results further suggest that cellular communication between AM and lung epithelial cells via soluble mediators is a pivotal step in PM10-induced lung inflammation and possibly also the systemic inflammatory response induced by particulate matter air pollution.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The work was supported by a grant from the Canadian Institutes of Health Research (no. 4219) and the British Columbia Lung Association. S. F. van Eeden is the recipient of a Career Investigators Award from the American Lung Association and the William Thurlbeck Distinguished Researcher Award.


    ACKNOWLEDGMENTS
 
We thank Dr. E. Ogawa and Dr. W. M. Elliott for technical support and Health Canada for providing the EHC-93.

Present address of T. Fujii: Div. of Infectious Disease, Institute of Medical Science, Tokyo University, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108-8639, Japan.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. F. van Eeden, iCAPTURE Centre, St. Paul's Hospital, Univ. of British Columbia, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6 (E-mail: SVaneeden{at}mrl.ubc.ca).

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. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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