Ozone Enhances Diesel Exhaust Particles (dep)-induced Interleukin-8 (il-8) Gene Expression in Human Airway Epithelial Cells through Activation of Nuclear Factors-b (nf-b) and Il-6 (nf-il6)

Ozone, a highly reactive oxidant gas is a major component of photochemical smog. As an inhaled toxicant, ozone induces its adverse effects mainly on the lung. Inhalation of particulate matter has been reported to cause airway inflammation in humans and animals. Furthermore, epidemiological evidence has indicated that exposure to particulate matter (PM 2.5-10), including diesel exhaust particles (DEP) has been correlated with increased acute and chronic respiratory morbidity and exacerbation of asthma. Previously, exposure to ozone or particulate matter and their effect on the lung have been addressed as separate environmental problems. Ozone and particulate matter may be chemically coupled in the ambient air. In the present study we determined whether ozone exposure enhances DEP effect on interleukin-8 (IL-8) gene expression in human airway epithelial cells. We report that ozone exposure (0.5 ppm x 1 hr) significantly increased DEP-induced IL-8 gene expression in A549 cells (117 ± 19 pg/ml, n = 6, p < 0.05) as compared to cultures treated with DEP (100 Dg/ml x 4 hr) alone (31 ± 3 pg/ml, n = 6), or cultures exposed to purified air (24 ± 6 pg/ml, n = 6). The increased DEP-induced IL-8 gene expression following ozone exposure was attributed to ozone-induced increase in the activity of the transcription factors NF-B and NF-IL6. The results of the present study indicate that ozone exposure enhances the toxicity of DEP in human airway epithelial cells by augmenting IL-8 gene expression, a potent chemoattractant of neutrophils in the lung.


Introduction
Ozone and particulate matter (PM 2.5-10 ), including diesel exhaust particles (DEP) are major ambient air pollutants.The lung is the primary target for their toxic effect.Ozone, a highly reactive oxidant gas is a major component of photochemical smog.As an inhaled toxicant, ozone induces its adverse effects mainly on the lung [1].Inhalation of particulate matter has been reported to cause airway inflammation in humans and animals [2][3][4][5][6][7][8].Furthermore, epidemiological evidence has indicated that exposure to particulate matter (PM) in urban areas of the United States has been correlated with increased acute and chronic respiratory morbidity [9][10][11] and exacerbation of asthma [12].Previously, exposure to ozone or particulate matter and their effect on the lung have been addressed as separate environmental problems.
Ozone and particulate matter may be chemically coupled in the ambient air [13].Ozone, because of its high reactivity will react with the airway epithelial lining fluid and cell membrane (lipids and proteins) causing phospholipids remodeling and the generation of inflammatory mediators [14][15][16].Conversely, particulate matter will deposit and will be engulfed by the lung cells, or phagocytized by macrophages.Many studies have shown that the inflammatory response associated with ozone exposure is mediator-related.This view is supported by data demonstrating increased recovery of several soluble mediators of inflammation in bronchoalveolar lavage (BAL) fluid from humans exposed to ozone.Among the various pro-inflammatory mediators, increased levels of prostaglandin E 2 (PGE 2 ), interleukin-(IL-) 6 and IL-8 were consistently found in BAL from humans exposed to low levels of ozone [17][18][19].Airway epithelial cells have been proposed to play an important role in the ozone-induced inflammatory process [20].They are among the first cells to come in contact with inhaled ozone, and act as target cells for ozone-induced toxicity.
Particulate matter is released into the ambient air from the combustion of fossil products by industrial and agricultural processes, and transportation.Diesel exhaust particles generated and emitted from diesel engines are a major component of atmospheric PM.The pulmonary epithelium and resident macrophages are primary targets of inhaled particulate matter (PM 2.5-10 ).Increased morbidity and mortality from cardiopulmonary complications have been associated with exposure to PM 2.5-10 .Diesel exhaust particles are of the criteria air pollutants that are implicated in inducing lung disease and injury [21].Many studies have implicated fine particles, including DEP in airway inflammation and hyper-responsiveness [22] and in exacerbation of asthma and chronic obstructive pulmonary disease (COPD) [23][24][25].Benzo[a]pyrene, a major aromatic hydrocarbon constituent coupled with DEP was shown to induce the release of inflammatory cytokines in human airway epithelial cells [21].In vitro studies have indicated that airway epithelial cells and macrophages can bind and ingest various types of PM 2.5-10 [26].
Over-expression of pro-inflammatory genes in the lung is regulated at the transcription level [27,28].Many pro-inflammatory genes, for example, IL-8, IL-6, tumor necrosis factor-(TNF-), and granulocyte macrophagecolony stimulating factor (GM-CSF) have B sites in their 5'-flanking regions [29].Activation of several transcription factors, notably, NF-B results in the expression of many of these inflammatory genes [30].Increased activation of NF-B has been demonstrated in airways of asthmatics and in sputum macrophages [31].Moreover, Glucocorticoids that inhibit NF-B activation have been shown to reduce the survival of eosinophils, a characteristic in asthma [32].
Our objectives in the present study were to determine whether, ozone exposure enhances DEP-induced IL-8 release in small airway epithelial cells.IL-8, a potent inflammatory mediator has been demonstrated to play a key role in neutrophilia, a trademark of lung inflammation and COPD.

Materials
The human airway epithelial-type II-like cell line A549, F 12K basal medium, antibiotics and supplements, and fetal bovine serum (FBS) were obtained from American Type Culture Collection (Rockville, MD).Hank's buffered saline solution (HBSS containing 30 mM Hepes) was obtained from Clonetics (San Diego, CA).Double stranded NF-B and NF-IL6 consensus oligonucleotides were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).Lactate dehyrogenase (LDH) activity assay was obtained from Sigma Chemical (St.Louis, MO).Human recombinant sandwich enzyme-linked immunosorbent assay kits specific for IL-8 were purchased from Pierce-Endogen (Springfield, IL).Diesel exhaust particulates (DEP, SRM 2975) with a mean diameter of 0.3 Dm were purchased from the National Institute of Standards and Technology (NIST, Rockville, MD).

Cell Culture
A549 cells: A549 cell line, human pulmonary type-IIlike epithelial cells were utilized in the present study as an airway epithelial cell model.This cell line retains the features of type-II epithelial cells.A549 cells were cultured on the apical side of a Transwell-Col membrane system (Costar, Cambridge, MA) in F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Gibco BRL, Gaithersburg, MD).A549 cell were grown on collagencoated Transwell-Col membranes (25 mm diameter and 0.4 Dm pore size) until confluency [15].Before exposure to ozone (0.5 ppm x 1 hr) or to DEP (10, 25, or 100 Dg/ml x 4 hr), following ozone exposure, growth medium was aspirated and HBSS (with 30 mM Hepes) was added basolaterally to keep the cells hydrated.In studies that examined the effect of DEP alone, cells were grown on plastic plates (35 mm diameter).

Measurement of Lactate Dehydrogenase Levels
To determine the effect of combined ozone and PM 2.5- 10 exposure on membrane integrity of A549 cells, lactate dehydrogenase levels in supernatants of exposed cultures were used as an index of membrane leakage.Confluent cultures of A549 were exposed to DEP (10, 25, or 100 Dg/ml x 4 hr) and ozone (0.5 ppm x 1 hr) separately and simultaneously.Following exposure, levels of lactate dehydrogenase in supernatants were determined.

In vitro Ozone Exposure System
An in vitro ozone exposure system, previously described [33] is used in the present study.This system has been used to study the in vitro effects of ozone on various cell types, including airway epithelial cells [34,35].Briefly, the exposure system consists of two plexiglass chambers of 2.5L capacity each, through which humidified high purity air (negligible or nondetectable levels of hydrocarbons or nitrogen dioxide) with or without ozone is delivered through Teflon-made delivery lines (with 0.1 Dm particle removal filter) at 1.2L/min.Ozone is generated by passing humidified high purity air through a chamber equipped with an occluded UV pen lamp and regulated through a voltage output regulator.Ozone concentrations (0.1-1.0 ppm) were monitored continuously with an in-line ozone analyzer (Dasibi Environment, Glendale, CA).The ozone concentration was maintained within ± 10% of the desired concentration.Cultured A549 cells were exposed inside an incubator where the temperature was maintained at 37°C.The lack of CO 2 supply to the incubation system does not affect the pH of the medium.

Measurement of IL-8 Protein
To measure the effect of ozone or particulate matter on IL-8 production, confluent monolayers of A549 cells, grown on Transwell-Col membranes were exposed at 37°C to either air, ozone alone, or to ozone followed by DEP.Following the various exposures, the amount of IL-8 released to the apical or basolateral compartments was determined using a recombinant human IL-8 ELISA (Endogen) according to the manufacturer's directions.IL-8 protein levels in the culture supernatants were calculated from corresponding absorbances measured at 450 nm using a Bio-Tek EL311 autoplate reader (Bio-Tek, Winooski, VT) and standard calibration curves.The ELISA kit has a lower limit of detection of 5 pg/ml, is specific for IL-8, and does not cross react with IL-6 or other interleukins.

Cell Fractionation
Cells were processed to obtain cytosolic and nuclear fractions.Cells were pelleted and suspended again in icecold hypotonic buffer (10 mM Hepes, pH 7.5, 10 mM KCl, 3 mM NaCl, 3 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 2 mM DTT) containing protease and phosphatase inhibitors (60 DL per 5 x 10 6 cells).Cells were centrifuged at 500 x g for 10 minutes at 4 o C to collect the supernatant.This supernatant was labeled as cytosolic extract and was aliquoted and stored at 80 o C for further analysis.

Nuclear Factors -B (NF-B) and IL6 (NF-IL6) Activation Assays
A549 cells were cultured as described earlier in the methods section.Following the various exposure regimens to DEP alone, or DEP following ozone exposure, cells were harvested on ice and washed with phosphate buffered saline (PBS, pH 7.4) without calcium or magnesium (Kafoury et al., submitted).Cell suspensions were centrifuged at 1000 g at 4°C for 10 min.Peletted cells were suspended in lysing buffer [20 mM Hepes, pH 7.9, 125 mM NaCl, 5 mM MgCl2, 12% glycerol, 0.1% nonidet P-40, 5 mM dithiothreitol (DTT), 0.5 mM polymethyl sulphonylflouride (PMSF), 10 Dg/ml leupeptin] and left to swell on ice for 10 min.Following incubation on ice, cell suspensions were subjected to sonication (35% cycle) and microcentrifuged at 12000 rpm at 4°C for 10 min.Total protein extracts were determined using the Bradford method (Bio Rad, Richmond, CA) and the extracts were stored at -80°C.

Statistical Analysis
Data are expressed as mean ± standard error of the mean (SEM).Comparisons between DEP-exposed, air-, or ozone-exposed groups and control cultures were carried out using one-way analysis of variance (ANOVA) [36].

Measurement of the Cytotoxic Effect of DEP and Ozone Exposure
To determine the effect of DEP exposure on small airway epithelial cell membrane integrity, A549 cells were cultured in F12k medium, supplemented with antibiotics and 10 % fetal bovine serum (FBS).The cells were plated on plastic plates (35 mm diameter) at a density of 100 x 10 5 cells/well in 95% air, 5% CO 2 and humid environment.The cells reached confluency in 3 days.Monolayers of A549 cells were treated with DEP at concentrations of 10, 25, and 100 Dg/ml for 4 hr, and the cytotoxic effect of DEP was assessed by lactate dehydrogenase activity assay.Control cultures were treated with HBSS (containing 30 mM Hepes) alone.Figure 1 demonstrates that DEP exposure at concentrations of 10, 25, and 100 Dg/ml (x 4 hr) did not induce any significant effect on A549 cell membrane integrity, as determined by lactate dehydrogenase activity (5 ng/ml ± 2 ng/ml, n = 6, 7 ± 3 ng/ml, n = 6, and 11 ± 4 ng/ml, n = 3; p > 0.05, respectively).To determine the cytotoxic effect of ozone exposure on A 549 cells, cultures were grown on the apical side of collagen-coated Transwell-Col membranes (25 mm diameter and 0.4 Dm pore size) (Costar, Cambridge, MA) until confluency [15] Cells reached confluency in 3 days.Prior to exposure to ozone, growth media were aspirated and the cells were hydrated with 2 ml HBSS (with 30 mM Hepes) in the basolateral compartment.Following ozone exposure (0.5 ppm x 1 hr), 1 ml of HBSS was added apically and LDH levels were measured.Figure 2 shows that ozone exposure (0.5 ppm x 1 hr) did not induce any significant cytotoxic effect on A 549 cells (14 ± 3 ng/ml, n = 6, p > 0.05), as compared to air control cultures (7 ± 2 ng/ml, n = 6).

Effect of DEP on IL-8 Release
To determine the effect of DEP on IL-8 release in A549 cells, monolayers were exposed to DEP at a concentration of 100 Dg/ml for 4 hr and IL-8 release was measured using human recombinant IL-8 ELISA. Figure 3 shows that exposure of A549 cultures to DEP did not induce any significant release of IL-8 (31 ± 6 pg/ml, n = 6, p > 0.05), as compared to control cultures (23 ± 4 pg/ml, n = 6).

Ozone Exposure Increases DEP-Induced NF-B Binding Activity
Exposure of human airway epithelial cells, A 549, in culture to biologically-relevant concentrations of DEP (100 Dg/ml x 4 hr) following ozone exposure (0.5 ppm x 1 hr) showed that ozone induced an increase in the binding activity of NF-B, as determined by electrophoretic mobility shift assays of crude nuclear extracts (Fig. 4).In contrast, exposure of A549 cultures to DEP alone failed to induce any significant increase in NF-B binding activity (Fig. 4).

Figure 4: Activation of Nuclear Factor-B (NF-B) in A549 Cells by Ozone and DEP following Ozone Exposure.
Cells were exposed to ozone (0.5 ppm x 1 hr) or purified air.Following ozone exposure, A 549 monolayers were exposed to DEP at a concentration of 100 Dg/ml for 4 hr.Cells were scraped using rubber policeman, fractionated and nuclear extracts were subjected to EMSA using a labeled oligonucleotide containing an NF-B binding site.Lanes are as follows: 1, probe; 2, excess cold oligo + ozone (0.5 ppm x 1 hr); 3, Control; 4, ozone (0.5 ppm x 1 hr); 5, DEP (100 Dg/ml x 4 hr); 6, DEP (100 Dg/ml x 4 hr) following ozone exposure (0.5 ppm x 1 hr).

Ozone Exposure Increases NF-IL6-DNA Binding Activity
Figure 5 demonstrates that exposure of A 549 cultures to ozone resulted in a significant increase in NF-IL6-DNA binding activity, as demonstrated by DNA mobility assays.DEP exposure (100 Dg/ml x 4 hr) alone, however, failed to induce any significant increase in NF-IL6-DNA binding activity, as compared to control cultures (Fig. 5).

Discussion
Ambient air pollutants, including ozone and diesel exhaust particles have been linked to increased incidence of lung inflammation and exacerbation of COPD and asthma.Recent studies have shown increased levels of IL-8 and neutrophil counts during severe lung inflammation and exacerbation of COPD [37,38].Lung inflammation is characterized by an influx of neutrophils to sites of inflammation [39].Furthermore, COPD has been associated with neutrophilic inflammation [40].Neutrophil, lymphocyte, and eosinophil counts were reported to be significantly higher in humans with moderate to severe COPD than in healthy individuals [37].Moreover, increased levels of IL-8 were observed in patients with COPD [41] and severe asthma [42].IL-8 has been initially identified as a potent neutrophil chemo attractant in the lung [43].
Numerous stimuli and agonists have been shown to induce IL-8 gene expression in human bronchial epithelial cells, macrophages, fibroblasts, and lymphocytes [44,45,46].IL-8-stimulated neutrophils in vitro, were reported to release granule-derived factors, including cathepsins and elastases [47].Beta-2 agonists which are used in the treatment of COPD have been shown to inhibit IL-8 gene expression, elastase release, and migration of neutrophils in airway epithelial and endothelial cells, in vitro [48].Exposure to DEP has been shown to significantly increase IL-8 synthesis and release in human bronchial epithelial cells [49,50].However, limited data is available regarding the effect of DEP on IL-8 gene expression in distal small airway epithelial cells, major cells in the lower airways where DEP is likely to deposit.The results of the present study indicate that exposure to DEP did not induce any significant IL-8 gene expression in A 549 cells, type-IIlike human airway epithelial cells.However, DEP treatment of A549 cells following ozone exposure induced a significant increase in IL-8 gene expression (Fig. 3).Boland et al. [51] have reported that DEP carbonaceous core failed to induce any significant increase in IL-8 or Granulocyte macrophage-colony stimulating factor (GM-CSF) in human bronchial epithelial cells.The results reported by Boland and coworkers are in agreement with our findings.
Previously, exposure to ozone or particulate matter and their effect on the lung have been addressed as separate environmental problems.Ozone and PM 2-10 , including DEP may be chemically coupled in the ambient air [13].Ozone, because of its high reactivity will react with the airway epithelial lining fluid and cell membrane (lipids and proteins).Conversely particulate matter will deposit and bind to lung cells, or become phagocytized by macrophages.The molecular and cellular events underlying the effect of ozone, DEP, or the simultaneous exposure to these two airborne pollutants remain unclear.
In the present study, ozone exposure enhanced DEPinduced IL-8 release in the human airway epithelial cell line, A549 (Fig. 3).This increase in DEP-induced IL-8 release following ozone exposure may be related to either, increased binding of DEP to A549 cell membrane, or increased uptake of DEP following ozone exposure, especially that separate or simultaneous exposure to ozone or DEP did not have any significant effect on A549 membrane integrity (Figs. 1 and 2).IL-8 has been demonstrated to serve as neutrophil chemoattractant to sites of inflammation [44].Ozoneinduced increase in DEP-induced IL-8 gene expression may explain the role of DEP in lung inflammation and aggravation of COPD.Therefore, the results of the present study may provide information regarding the cellular and molecular mechanisms underlying exacerbation of COPD and asthma in response to DEP exposure.In the present study, DEP alone failed to induce any significant release of IL-8, an important chemotactic in the lung.However, exposure of A549 cultures to ozone (0.5 ppm x 1 hr) followed by DEP treatment (100 Dg/ml x 4 hr) resulted in a significant increase IL-8 release.Jaspers et al. [52] have shown that ozone exposure at 0.1 ppm induced significant release of IL-8 in A549 cells.The Increased IL-8 gene expression by ozone exposure was associated with activation of NF-B in A549 cells [52].Previously, we have shown that lipid ozonation products (LOP) generated from reaction of ozone with unsaturated fatty acids in airway epithelial cell induce significant release of IL-8 in cultured human bronchial epithelial cells [14].Furthermore, co-activation of NF-B (Fig. 4) and NF-IL6 (Fig. 5) was demonstrated to be responsible for LOP-induced IL-8 release in human bronchial epithelial cells [53].
Therefore, we propose that ozone can prime A549 cells to induce increased pro-inflammatory gene expression in response to DEP, and other PM 2.5-10 by activating DNA-binding transcription factors in lung target cells.The effect of ozone on simultaneous DEP exposure may aggravate lung inflammation, and exacerbate COPD and asthma.The molecular mechanisms underlying the observed DEP-mediated IL-8 release by lung cells remain to be further elucidated.Results in the present study indicate that activation of NF-B and its translocation to the nucleus, in addition to NF-IL6 activation appear to be responsible for regulating the release of IL-8 in response to DEP exposure in A549 cells following ozone exposure.Currently, studies to examine the signaling mechanisms responsible for activation of NF-B and NF-IL6 in DEP-induced IL-8 gene expression following ozone exposure are being conducted.

Figure 1 :
Figure 1: Cytotoxic Effect of DEP on A549 Cell Membrane Integrity.Confluent A549 cultures were treated with DEP at concentrations of 10, 25, and 100 Dg/ml for 4 hr and lactate dehydrogenase activity (LDH) was measured as an indicator of cell membrane integrity.Data (ng/ml) of LDH are presented as mean ± SEM (n = 6, each group).

Figure 2 :
Figure 2: Cytotoxic Effect of Ozone on A549 Cell Membrane Integrity.Confluent A549 cultures were exposed to ozone at a concentration of 0.5 ppm for 1 hr and lactate dehydrogenase activity (LDH) was measured as an indicator of cell membrane integrity.Data (ng/ml) of LDH are presented as mean ± SEM (n = 6, each group).

Figure 3 :
Figure 3: Effect of Exposure to DEP alone, Ozone, or DEP Following Ozone on IL-8 Release in A549 Cells.The effect of ozone exposure on DEP-induced IL-8, an inflammatory cytokine was measured.Confluent A549 monolayers grown on Transwell-Col membranes were exposed to ozone (0.5 ppm x 1 hr) prior to DEP exposure (100 Dg/ml of DEP for 4 hr).After the different treatment regiments, IL-8 was measured in the cell-free supernatants by ELISA.The data (pg/ml) are presented as the mean ± SEM (n = 6, each group).*Significant