Activation of Human Epithelial Lung A549 Cells by the Pollutant Sodium Sulfite: Enhancement of Neutrophil Adhesion

Martin Pelletier, Valérie Lavastre and Denis Girard1

INRS-Institut Armand-Frappier, Université du Québec, 245 Boulevard Hymus, Pointe-Claire, Québec, Canada H9R 1G6

Received March 21, 2002; accepted May 1, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Air pollutant exposure may induce deterioration of respiratory health. Concentrations of air particles, ozone, nitrogen dioxide, sulfur dioxide, and sulfate are among the players involved in the initiation and/or exacerbation of lung diseases. We have previously documented that the pollutant sodium sulfite (Na2SO3) is a human neutrophil agonist. To date, there is no evidence in the literature that Na2SO3 can activate epithelial lung cells. In the present study, we found that Na2SO3 (0.01–10 mM) induces tyrosine phosphorylation events and interleukin-8 production in human epithelial lung A549 cells. In addition, we found that Na2SO3 did not promote A549 cell apoptosis as assessed by the degradation of the cytoskeletal gelsolin protein and by FITC-annexin-V binding. Human neutrophil adhesion to Na2SO3-induced A549 cells was increased when compared with untreated A549 cells. As assessed by flow cytometry, cell surface expression of intercellular adhesion molecule (ICAM)-1, ICAM-3, and vascular cell adhesion molecule-1 (VCAM-1) on A549 cells was not affected by Na2SO3. We conclude that Na2SO3 can activate A549 cells. In addition, we conclude that neutrophil adhesion to Na2SO3-induced A549 cells is increased via an ICAM-1-, ICAM-3-, and VCAM-1-independent mechanism.

Key Words: inflammation; sodium sulfite; Na2SO3; lung cells; neutrophils; cell adhesion.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Occupational and environmental lung diseases are subjects of an increasing number of studies published in the scientific literature. In particular, the reported association between daily air pollution and daily hospital admissions strengthens the argument that air pollution may induce deterioration of respiratory health (Atkinson et al., 1999Go; Goldberg et al., 2001Go; Petroeschevsky et al., 2001Go; Wong et al., 2002aGo,bGo). Concentrations of air particles, ozone, nitrogen dioxide, sulfur dioxide, and sulfate are among the players involved in the initiation and/or exacerbation of lung diseases (Chen et al., 2001Go; Kodavanti et al., 2000Go; Lippmann, 1985Go). Airway inflammation, frequently associated with neutrophil inflammation, is a hallmark of numerous lung diseases including asthma, chronic bronchitis, adult respiratory distress syndrome, and pulmonary fibrosis (Blomberg et al., 1999Go; Graham et al., 1988Go; Sibille and Reynolds, 1990Go; Terashima et al., 1999Go; Toren et al., 1996Go).

Sodium sulfite (Na2SO3), a chemical used as an antidarkening food agent and widely used in the pulp and paper sector as a bleaching agent (Denisov and Tkachev, 1990Go; IARC Working Group, 1992Go), was recently found to activate neutrophils (Beck-Speier et al., 1993Go, 1994Go; Labbé et al., 1998Go; Mishra et al., 1995Go; Mitsuhashi et al., 1998Go; Pelletier, et al., 2000Go). Workers from pulp and paper mills develop some obstructive airway diseases in addition to hypersensitivity reactions and cardiovascular diseases (Toren et al., 1996Go). Sulfites, formed in the atmosphere as a reaction product of sulfur dioxide and water droplets, were reported as stimuli from acid fog, which may be capable of inducing bronchoconstriction (Balmes et al., 1989Go). Because of these observations, and knowing that many different sulfate products and derivatives can be transported by airborne particles (Siskos et al., 2001Go), Na2SO3 should be considered an important risk factor that may contribute to human lung diseases. In neutrophils, Na2SO3 is known to induce reactive oxygen species (ROS) production such as H2O2 and superoxide (O2-) (Beck-Speier et al., 1994Go; Pelletier et al., 2000Go) directly, and can also prime these cells to respond to the bacterial tripeptide fMLP (Labbé et al., 1998Go). Na2SO3 was found to induce ROS via protein kinase C and Ca2+ calmodulin pathways (Beck-Speier et al., 1993Go). Neutrophil production of ROS is of significant importance, as these products can act as potent antimicrobial agents involved in host defense (Ricevuti, 1997Go; Weiss, 1989Go). However, they can also contribute to tissue injuries and alter normal physiological functions of various organs, such as the lungs, when their production is unregulated. In addition to its ability to induce ROS production, Na2SO3 was found to induce the release of the potent neutrophil chemotactic factor interleukin (IL)-8 (Pelletier et al., 2000Go). In addition, Na2SO3 was induced tyrosine phosphorylation events in human neutrophils, but not in either immature or DMSO-differentiated promyelocytic HL-60 cells. Despite the fact that Na2SO3 induces neutrophil ROS production, it does not induce apoptosis and does not alter phagocytosis (Pelletier et al., 2000Go).

Although the above observations clearly indicate that human neutrophils are important targets of Na2SO3, further studies are required to better elucidate the role of this chemical in the inflammatory process. In particular, the role of Na2SO3 on epithelial lung cells is presently unknown, despite the fact that these cells represent another important potential target to Na2SO3. Because acute lung injury is frequently mediated by the interaction between neutrophils and epithelial lung cells (Sibille and Reynolds, 1990Go), and because recruitment of circulating inflammatory cells such as neutrophils into the airways involves soluble mediators such as cytokines and cell–cell adhesion molecules, we decided to study the role of Na2SO3 on human epithelial lung A549 cells as well as the effect of this chemical on neutrophil adhesion to these epithelial cells.

In the present study, we found that Na2SO3 (0.01–10 mM) induces tyrosine phosphorylation events and IL-8 production in human epithelial lung A549 cells. In contrast, Na2SO3 did not promote A549 cell apoptosis. In addition, we found that human neutrophil adhesion to Na2SO3-induced A549 cells was increased, but cell surface expression of intercellular adhesion molecule (ICAM)-1, ICAM-3, and vascular cell adhesion molecule (VCAM)-1 on A549 cells was not affected by Na2SO3.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and agonists.
Sodium sulfite (Na2SO3) was purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ont) and was freshly dissolved in HBSS and used at nonnecrotic concentrations as we have previously reported (Labbé et al., 1998Go; Pelletier et al., 2000Go). Epidermal growth factor (EGF), Viscum album agglutinin (VAA)-I was purchased from Sigma. Specific human IL-8 ELISA kit (sensitivity < 5 pg/ml) was purchased from Medicorp (Mtl, Qc). Monoclonal antibodies to human CD50 (ICAM-3), CD54 (ICAM-1), and CD106 (VCAM-1) were purchased from Serotec (Raleigh, NC). Fluorescein (FITC)-conjugated goat antimouse IgG, F(ab`)2 fragment-specific was purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Antihuman gelsolin (clone GS-2C4) was purchased from Sigma. Tumor necrosis factor (TNF)-{alpha} was purchased from R&D Systems Inc. (Minneapolis, MN).

Epithelial cell culture.
The human epithelial lung cell line A549 was purchased from the American Type Culture Collection (Rockville, MD) and was grown in RPMI-1640 supplemented with 10% fetal calf serum (FCS) and antibiotics. Cell viability was systematically evaluated before and after each treatment, and mortality never exceeded 5%.

Neutrophil isolation.
Neutrophils were isolated from venous blood of healthy volunteers by dextran sedimentation followed by centrifugation over Ficoll-Hypaque (Pharmacia Biotech Inc, QUE), as previously described (Lavastre et al., 2002Go; Pelletier et al., 2000Go). Blood donations were obtained from informed and consenting individuals who were not under drug treatment, according to institutionally approved procedures. Cell viability was monitored by trypan blue exclusion and the purity (> 98%) was verified by cytology from cytocentrifuged preparations colored by Diff-Quick staining.

Tyrosine phosphorylation events.
A549 cells (1 x 106 cells/ml in RPMI-1640) were incubated for 5, 15, or 30 min at 37°C with buffer, EGF, or Na2SO3 in a final volume of 120 µl. Reactions were stopped by adding 125 µl of 2X Laemmli's sample buffer, as we have described elsewhere (Pelletier et al., 2000Go, 2001Go). Aliquots corresponding to 1 x 106 cells were loaded onto 10% SDS-PAGE and transferred from gel to PVDF membranes (Millipore, Bedford, MA). Nonspecific sites were blocked with 1% BSA in TBS-Tween (25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.15% Tween-20) for 1 h at room temperature. After washing, the membranes were incubated with monoclonal antiphosphotyrosine UB 05-321 (1:4000) (UBI) for 1 h at room temperature. Membranes were then washed and incubated with a horseradish peroxidase-conjugated goat antimouse IgG + IgM (1:10,000) (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature in fresh blocking solution. Membranes were washed three times with TBS-Tween, and phosphorylated bands were revealed with the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham, Pharmacia Biotech Inc., Baie d'Urfé, Québec). Protein loading was verified by staining the membranes with Coomassie blue at the end of the experiments.

Apoptosis assessment.
Apoptosis was assessed by two different methods: degradation of the cytoskeletal gelsolin protein (Kothakota et al., 1997Go; Savoie et al., 2000Go; Stearns et al., 2001Go) and FITC-Annexin-V staining. For the degradation of gelsolin, cells (1 x 106 cells/ml for A549 cells or 10 x 106 cells/ml for neutrophils in 24-well plate) were incubated with or without Na2SO3, for 24 h, then harvested for the preparation of cell lysates in Laemmli's sample buffer. Aliquots corresponding to 225,000 cells were loaded and run on 10% SDS-PAGE and transferred from gel to PVDF membranes. Nonspecific sites were blocked with 1% BSA in TBS-Tween (25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.15% Tween-20) overnight at 4°C. Membranes were incubated with monoclonal antihuman gelsolin (1:1500), for 1 h at room temperature followed by washes, and incubated with a horseradish peroxidase-labeled sheep anti-mouse IgG (1:20,000) (Bio/Can) for 1 h at room temperature in fresh blocking solution as previously documented (Savoie et al., 2000Go; Stearns et al., 2001Go). Membranes were washed three times with TBS-Tween, and bands were revealed with the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham, Pharmacia Biotech Inc.). Protein loading was verified by staining the membranes with Coomassie blue at the end of the experiments (data not shown). Viscum album agglutinin (VAA)-I, was used as a positive control for human neutrophils (Lavastre et al., 2002Go; Savoie et al., 2000Go).

For assessment of apoptosis by FITC-Annexin-V staining, 1 x 106 A549 cells were washed twice with cold PBS and then resuspended in 100 µl of 1X binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). The cell suspension was incubated for 15 min at room temperature, light protected, after the addition of 3 µl FITC-Annexin-V (Pharmingen Canada, Mississauga, ONT). A volume of 250 µl of 1X binding buffer was added to each tube before fluorescence-activated cell sorting (FACS) analysis. Flow cytometric analysis (10,000 events) was performed using a FACScan (Becton Dickinson).

IL-8 production.
A549 cells (10 x 106 cells/ml in RMPI-1640 supplemented with 5% FCS) were stimulated with buffer, EGF, or Na2SO3 for 24 h. After the incubation, the supernatants were harvested and conserved at –80°C. The concentration of IL-8 in the supernatants was quantified with the use of the commercially available ELISA kit according to the manufacturer's instructions.

Neutrophil adherence assay.
A549 cells were grown on glass coverslips. When confluent, the cells were washed twice and treated with or without Na2SO3 for 30 min. Cells were washed twice after the treatment. In parallel, freshly isolated human neutrophils were labeled for 30 min with 5 µM calcein-AM (Molecular Probes, Inc., Eugene, OR) according to the manufacturer's recommendation. After the incubation, 1 ml of neutrophil suspension (5 x 106 cells/ml) was added to each well of a 12-well plate containing confluent A549 cells on a coverslip for 30 min. After the incubation, coverslips were extensively washed and mounted on a glass slide. The number of adherent neutrophils was calculated by counting the number of fluorescent cells from five randomly selected high-power fields (x400) observed with a photomicroscope Leica DMRE equipped with an ebq 100 dc epifluorescent condenser. Images were taken with a Cooke Sensicam High performance camera coupled to the Image Pro-plus® (version 4.0) program.

Cell surface expression of CD50, CD54, and CD106.
For these experiments, A549 cells were harvested from tissue culture flasks with the use of a rubber policeman and then transferred to 5 ml (75 x 12 mm) polypropylene tubes. Following the incubation period with Na2SO3, cells were suspended at 1.5 x 106 cells/ml, washed, and incubated with the different antibodies (1 µg/ml) for 1 h (4°C, light protected). TNF-{alpha} was used as a positive control, as it was previously found to induce CD54 expression on A549 cells (Chen et al., 2001Go). After two additional washes, cells were incubated with FITC-conjugated secondary antibody (1 µg/ml). Cells were then washed and fixed with paraformaldehyde (0.5%). Flow cytometric analysis (10,000 events) was performed using a FACScan (Becton Dickinson).

Statistical analysis.
Statistical analysis was performed with SigmaStat for Windows Version 2.0 with a one-way analysis of variance (ANOVA). Statistical significance was established at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Na2SO3 Induces Tyrosine Phosphorylation Events
Tyrosine phosphorylation is a rapid response that can occur after cell stimulation. Because we were interested in whether Na2SO3 can activate A549 cells, we decided to investigate its ability to induce such a response. As illustrated in Figure 1Go, Na2SO3 is a potent activator of A549 cells, as it induces tyrosine phosphorylation of several proteins in a concentration-dependent fashion. The response is maximal at 1 mM and then decreases. Although only the results obtained after 30 min are shown, we have performed time-course experiments (data not shown). The response was found to increase in time (1, 5, 15, and 30 min), but decreased after 60 min of stimulation (data not shown). EGF was used as a positive control and, as expected, A549 cells were responsive (Hauck et al., 2001Go). This indicates that Na2SO3 is an activator of A549 cells.



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FIG. 1. Effects of Na2SO3 on tyrosine phosphorylation events in human epithelial lung A549 cells. Cells were incubated (1 x 106 cells/ml) for 30 min with buffer (C) or the indicated increasing concentrations of EGF (ng/ml) or Na2SO3 (mM) and tyrosine phosphorylation assay was performed as describes under Materials and Methods. The right panel is the corresponding Coomassie blue stained membrane illustrating protein loading equivalence. Results are from one representative experiment out of four.

 
Na2SO3 Does Not Modulate A549 Cell Apoptosis
Knowing that Na2SO3 can induce a rapid response in A549 cells, we next investigated whether it can alter functions that require a longer period of incubation time. Results in Figure 2Go illustrate that Na2SO3 does not induce A549 apoptosis, as assessed by degradation of the cytoskeletal protein gelsolin, an event known to occur during apoptosis and to be mediated by caspases (Kothakota et al., 1997Go; Lavastre et al., 2002Go; Savoie et al., 2000Go). As expected, gelsolin was degraded in VAA-I–induced human neutrophils (Lavastre et al., 2002Go; Savoie et al., 2000Go). VAA-I treatment also induced A549 cell apoptosis as assessed by cytology (data not shown). Results were confirmed by FITC-Annexin-V binding. As indicated in Figure 2Go, the apoptotic rate never exceeded 15% at the highest Na2SO3 concentration used. Clearly, Na2SO3 is not a modulator of A549 cell apoptosis.



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FIG. 2. Effect of Na2SO3 on A549 cell apoptosis. A549 cells (10 x 106 cells/ml) were incubated for 24h in the presence of buffer (C or Ctrl), VAA-I (1000 ng/ml) or Na2SO3 and apoptosis was assessed by monitoring the degradation of gelsolin (left panel) or by staining with FITC-annexin-V (right panel) as described under Materials and Methods. Results are from one representative experiment out of 3 independent experiments.

 
Na2SO3 Induces IL-8 Production by A549 Cells
IL-8 production can be used as an excellent marker of pulmonary cell activation (Hauck, et al., 2001Go; Jaspers et al., 1998Go). To answer whether Na2SO3 can induce IL-8 production, we decided to incubate cells with an increasing concentration of the pollutant and measure the concentration of IL-8 in the supernatant by ELISA. As illustrated in Figure 3Go, Na2SO3 induced IL-8 production in a concentration-dependent fashion. The concentration of IL-8 was variable within the experiments. For simplicity, results from three separate experiments are presented. In each experiment, EGF was used as a positive control. These results indicate that A549 cells can produce IL-8 in response to Na2SO3. Knowing the important role of IL-8 in attracting inflammatory cells, our results are in agreement with prior experiments suggesting a potential proinflammatory role of Na2SO3.



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FIG. 3. Effect of Na2SO3 on IL-8 production. A549 cells (10 x 106 cells/ml in RMPI-1640 supplemented with 5% FCS) were stimulated for 24 h with buffer, EGF or Na2SO3 as indicated. The concentration of IL-8 in the supernatant was measured by ELISA, as described under Materials and Methods. Each line represents the result obtained from a different experiment (numbered 1, 2, or 3). The numbers in parentheses represent the IL-8 concentration obtained when cells were stimulated with 20 ng/ml EGF.

 
Enhancement of Neutrophil Adherence on Na2SO3-Induced A549 Cells
Adhesion of neutrophils to epithelial cells is an important part of the inflammatory process (Refsnes et al., 2001Go). We next decided to verify if treatment of A549 cells with the air pollutant Na2SO3 could influence the adherence of neutrophils. As illustrated in Figure 4Go, the adhesion of human neutrophils to Na2SO3-induced A549 cells was increased. Again, the maximal response was observed with a concentration of 1 mM. The insert illustrates portions of fields observed at a lower magnification (x100) than the results plotted in the bar graph (x400), to better appreciate the effect of Na2SO3. These results are in agreement with the fact that Na2SO3 possesses proinflammatory properties.



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FIG. 4. Role of Na2SO3 on neutrophil adherence. Confluent A549 cells were stimulated for 30 min with Na2SO3 and adhesion of calcein-labeled human neutrophils to A549 cells was measured as described under Materials and Methods. Results (mean ± SEM, n = 3) are expressed as the number of cells counted in 5 different high power fields under magnification x400. Ctrl, control; S1, 1 mM sulfite; S10, 10 mM sulfite). Inset, adherent fluorescent neutrophils probed with 5 µM calcein AM. Note that the pictures were taken at magnification x100 to better appreciate the effect of Na2SO3. *p <0.05 vs. control by ANOVA.

 
Effect of Na2SO3 on Cell Surface Expression of Adhesion Molecules
Because adhesion of neutrophils to epithelial cells occurs via adhesion molecules, we then decided to study the expression of ICAM-1 (CD54), ICAM-3 (CD50), and VCAM-1 (CD106) on the cell surface of Na2SO3-induced A549 cells using flow cytometry. As illustrated in Figure 5Go, the results were variable from experiment to experiment, but clearly, Na2SO3 did not increase cell surface expression of the tested molecules. This suggests that enhanced adhesion of human neutrophils to Na2SO3-induced A549 cells does not occur via upregulation of cell surface expression of CD50, CD54, and CD106.



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FIG. 5. Cell surface expression of CD50, CD54, and CD106 on human A549 epithelial lung cells following Na2SO3 treatment. A549 cells (1.5 x 106 cells/ml) were incubated for 60 min in the presence or absence (Ctrl) of an increasing concentration of Na2SO3 or in the presence of TNF-{alpha} (100 ng/ml). Cell surface expression of CD50, CD54 and CD106 was evaluated by flow cytometry as described under Materials and Methods. Results are means ± SEM (n = 3). *p < 0.05 vs. control (Ctrl) by ANOVA.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Further to our previous work (Labbé et al., 1998Go; Pelletier et al., 2000Go), we decided to elucidate the role of the air pollutant Na2SO3 in inflammation. Knowing that this contaminant was recently reported to activate human neutrophils, which play significant roles in the inflammatory response, we were particularly interested in its role on the cell physiology of human epithelial lung cells, an area of research that is poorly documented. This emanates from the fact that pulmonary cells are among the first to be in contact with air pollutants. When activated, they can participate in lung inflammation by producing various soluble factors, including cytokines, and by recruiting others cells (Sibille and Reynolds, 1990Go).

By studying the role of Na2SO3 on a rapid biological response, we found that the pollutant can effectively activate A549 cells, as several still-unidentified proteins are tyrosine phosphorylated even after brief exposure. Interestingly, some of these proteins comigrated with those recruited by the potent agonist EGF. EGF is known to induce tyrosine phosphorylation of several proteins in A549 cells, such as the two major 170 kD EGF receptor and the focal adhesion kinase proteins (Hauck et al., 2001Go). Whether Na2SO3 recruits these proteins remains to be determined. Although it was previously reported that several chemicals of environmental concern, including Na2SO3, could induce tyrosine phosphorylation events in neutrophils, this is the first study reporting that Na2SO3 induces such a response in A549 cells. Ozone, one of the most common air pollutants humans routinely inhale, was found to induce IL-8 production in A549 cells partly via tyrosine kinases (Jaspers et al., 1998Go). This is in agreement with the present report, as we found that Na2SO3 induces tyrosine phosphorylation events and IL-8 production.

The ability of Na2SO3 to markedly induce the production/release of the potent chemotactic factor IL-8 corresponds to its potential proinflammatory properties, although the biological effect(s) of such IL-8 production by A549 cells is unclear. However, our results indicate that this IL-8 increase is not involved in the modulation of apoptosis. This conclusion is not without precedent, as we have previously reported that although Na2SO3 induces human neutrophil IL-8 production, it does not alter the basal apoptotic rate of these cells (Labbé et al., 1998Go; Pelletier et al., 2000Go). IL-8 is known to delay spontaneous, cytokine- and Fas-induced neutrophil apoptosis (Celi et al., 1999Go; Leuenroth et al., 1998Go; Refsne et al., 2001). In contrast, others have reported that IL-8 does not alter neutrophil spontaneous apoptosis (Brach et al., 1992Go; Kettritz et al., 1998Go), attesting to the complexity of this biological response. It was recently reported that asbestos induces A549 cell apoptosis via caspase-3 activation (Colotta et al., 1992Go), the main caspase involved in the degradation of gelsolin (Kothakota et al., 1997Go; Lavastre et al., 2002Go; Savoie et al., 2000Go). As our results indicate that gelsolin is not degraded in Na2SO3-induced A549 cells (this report), it is clear that the biological effects of this pollutant are not mediated via activation of apoptosis.

In addition to the above observations, we found that neutrophil adhesion to Na2SO3-induced human A549 cells was increased when compared with adhesion onto untreated A549 cells. The ability of Na2SO3 to increase neutrophil adhesiveness corresponds with its proinflammatory properties. We were interested in studying the effect of Na2SO3 on the expression of cell surface molecules, knowing their potential involvement in neutrophil recruitment and sequestration during lung inflammation (Aljandali et al., 2001Go; Burns et al., 2001bGo) and during neutrophil adhesion to human airway epithelial cells (Burns et al., 2001aGo). The effect of air pollutants on the expression of cell surface molecules on A549 cells is not well documented. Results from one study have demonstrated that diesel exhaust particulates, a common air pollutant from diesel engine-powered car exhaust thought to cause chronic airway diseases, enhance eosinophil adhesion to nasal epithelial cells (Jagels et al., 1999Go). However, cell surface expression of adhesion molecules was not investigated. Our results indicate that Na2SO3 does not affect the modulation of CD50, CD54, and CD106 on the surface of A549 cells, despite the fact that neutrophil adhesion to Na2SO3-induced A549 is increased. The results were, however, variable from experiment to experiment. This may be related to the number of passages of the cells. Nevertheless, Na2SO3 did not increase the expression of the tested molecules. This is in agreement with one study reporting that the adhesion of human neutrophils to A549 cells was increased when these latter cells were treated with PMA by an ICAM-1-independent mechanism (Refsnes et al., 2001Go). This is in agreement with our results. We are aware that we cannot rule out the possibility that Na2SO3 alters the expression of other cell surface molecules on A549 cells; this remains to be investigated. Interestingly, results from a recent study performed with granulocytes other than neutrophils, the eosinophils, demonstrated that adhesion of these cells to IL-5–induced A549 cells was not increased despite an increase in ICAM-1 (CD54) expression (Terada et al., 1997Go), in contrast to what would be expected. In addition, these results indicate the complex interaction between neutrophils and epithelial A549 cells.

Among the observations and conclusions that have been reported from a series of elegant studies performed with beagle dogs was that in vivo exposure of respirable sulfur (IV) aerosols has the potential to induce epithelial alterations in the proximal alveolar region (Heyder et al., 1999Go; Kreyling et al., 1999Go; Maier et al., 1999Go; Schulz et al., 1999Go). This is of major importance, because this region is the primary target for air pollutants. These studies provide a rationale for the one present here, as we found that Na2SO3 can directly activate pulmonary A549 cells. This encourages us to further investigate the mode of action of Na2SO3 on both A549 and neutrophil cells in vitro. In contrast, we also conclude that it is very unlikely that respiratory diseases can be initiated by the inhalation of sulfur-related environmental air pollution, as the in vivo treatment presents no health risk to the healthy lung (Heyder et al., 1999Go). However, no information is presently available on individuals who initially present some lung dysfunctions. It is highly plausible that individuals who present some lung dysfunction will be more vulnerable to air pollution.

To date, we can conclude that the pollutant Na2SO3 possesses proinflammatory properties in vitro based on the following: (1) it activates both neutrophils and A549 cells, because it induces tyrosine phosphorylation events; (2) it induces the production of the potent chemotactic factor IL-8 by neutrophils and A549 cells; and (3) it enhances the adhesion of neutrophils to A549 cells. Based on these results, it is tempting to propose the following scenario. Exposure to this chemical may invoke an inflammatory response by epithelial lung cells by activating them to produce IL-8, which eventually attracts neutrophils to the site of inflammation. These neutrophils then produce IL-8, which recruits other neutrophils and other cells to the site. In addition, neutrophils adhere to epithelial lung cells to further provoke and prolong the inflammatory response.


    ACKNOWLEDGMENTS
 
This study was partly supported by Association Pulmonaire du Québec (APQ), Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR), FRSQ-Réseau de Recherche en Santé Environnementale. M.P. holds a Ph.D. FCAR-FRSQ Santé award, and V.L. holds a Ph.D. Fondation Armand-Frappier award. We thank Mary Gregory for reading the manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (514) 630-8850. E-mail: denis.girard{at}inrs-sante.uquebec.ca Back


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 ABSTRACT
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 RESULTS
 DISCUSSION
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