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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 697-704, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0115OC

Interaction between Respiratory Syncytial Virus and Particulate Matter in Guinea Pig Alveolar Macrophages

Philomena M. Kaan and Richard G. Hegele

UBC McDonald Research Laboratories and iCAPTUR4E Centre, St. Paul's Hospital, Vancouver, BC Canada

Address correspondence to: Richard G. Hegele, M.D., Ph.D., UBC McDonald Research Laboratories and iCAPTUR4E Centre, St. Paul's Hospital, 1081 Burrard Street, Vancouver, BC, V6Z 1Y6 Canada. E-mail: rhegele{at}mrl.ubc.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alveolar macrophages (AM) play a pivotal role in host lung defense mechanisms. Respiratory syncytial virus (RSV) stimulates secretion of proinflammatory cytokines in AM while it suppresses the cell's phagocytic ability. However, exposure of AM to ambient particulate matter (PM10) has been reported to inhibit RSV uptake. The mechanisms involved in the interaction between RSV and PM10 in AM are not known. We hypothesize that the cellular response of AM to RSV and PM10 is dependent on the sequence in which AM are exposed to these agents. In this study, we compared the sequential effect of RSV and PM10 exposure in vitro on the phagocytic function of guinea pig AM, the RSV Yield in AM, and the production of proinflammatory cytokines (interleukin [IL]-6, IL-8, and tumor necrosis factor [TNF]-{alpha}). The ability of AM to phagocytose PM10 was not affected by sequential exposure to RSV and PM10. RSV Yield was severely decreased in PM10-exposed AM, regardless of sequence of exposure, compared with AM that were not exposed to PM10 (P < 0.004). Exposure of AM to RSV and/or PM10 resulted in enhanced secretion of bioactive TNF-{alpha} compared with controls (P < 0.02), without synergistic or inhibitory interaction of these agents on TNF-{alpha} production. By contrast, exposure of AM to PM10 significantly decreased the production of RSV-induced IL-6 (P < 4 x 10-6) and IL-8 (P < 0.003). In summary, our findings suggest that PM10 exposure may interfere with mechanisms of RSV replication and viral-induced cytokine production in guinea pig AM, independent of the sequence of exposure to these agents.

Abbreviations: alveolar macrophages, AM • bronchoalveolar lavage, BAL • enzyme-linked immunosorbent assay, ELISA • fetal bovine serum, FBS • fluorescein isothiocyanate, FITC • interleukin, IL • modified Eagle's medium, MEM • mean fluorescence intensity, MFI • nitric oxide, NO • phosphate-buffered saline, PBS • plaque-forming units, pfu • particulate matter < 10 µm diameter, PM10 • respiratory syncytial virus, RSV • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Respiratory syncytial virus (RSV) is the most common cause of acute bronchiolitis, a serious lower respiratory tract infection, particularly in infants and young children (1). For unexplained reasons, the prevalence of hospitalization for acute bronchiolitis has increased more than 2-fold over the past two decades (2). Furthermore, children hospitalized for acute bronchiolitis are at increased risk for developing asthma-like symptoms and allergic sensitization that can persist well into childhood (3). The pathogenesis of RSV bronchiolitis is believed to involve a combination of direct cytopathic effects induced by viral replication and the resulting host response of production of proinflammatory cytokines (4). However, the relative importance of viral versus host factors in the pathogenesis of RSV bronchiolitis is not clearly understood.

The escalating prevalence of allergic respiratory diseases in recent decades has been largely attributed to environmental factors such as viral infections and air pollution (5). The issue of whether childhood viral infections provide protection against allergic sensitization (6, 7) or are associated with increased prevalence of atopic conditions (8, 9) is contentious. Similarly, epidemiologic evidence for both protective (10, 11) as well as pathogenic roles (12–14) for air pollution have been implicated in the development of pediatric asthma and allergy. The Hygiene Hypothesis (15) might provide a plausible explanation for the unfavorable trend in allergic respiratory diseases; however, the protective versus pathogenic roles of viral infections and air pollution in the development of respiratory allergic diseases remain controversial.

Alveolar macrophages (AM) play major roles in lung defense against microbial and viral infections and in response to air pollutants (16). As "gatekeepers," AM are responsible for clearance of microorganisms and small inhaled particulate material in the alveolar space. In vitro studies indicate that AM from humans (17), mice (18), and guinea pigs (19) are permissive to acute RSV infection. In addition to the production of infectious virus, RSV infection of AM also results in the secretion of proinflammatory cytokines involved in modulation of inflammatory and immune responses (20). However, the response of AM to the interaction of environmental factors such as viral infection and air pollution is unclear. Although some studies demonstrate that RSV infection severely diminished the phagocytic ability of AM (21), other studies indicate that exposure of AM to particulate matter inhibited the ability of these cells to take up RSV (22).

In this study, we examined the interactive effects of RSV and air pollution, using particulate matter < 10 µm in diameter (PM10), on guinea pig AM functions in vitro. We proposed that the AM response to RSV and PM10 is dependent on the sequence in which AM were exposed to these agents. Following sequential exposure of guinea pig AM to RSV and PM10, we analyzed the phagocytic ability of AM, the RSV Yield of AM, and the production of proinflammatory cytokines (interleukin [IL]-6, IL-8, and tumor necrosis factor [TNF]-{alpha}) by AM in response to these stimuli.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Design
AM were obtained by bronchoalveolar lavage (BAL) from female juvenile guinea pigs. Unless otherwise stated, all experiments were performed in quadruplicate. AM from each animal were subjected to five different treatments: exposure to PM10 followed by RSV infection (PM10+RSV), RSV infection followed by PM10 exposure (RSV+PM10), exposure to PM10 only (PM10), exposure to RSV only (RSV), and the negative control (NEG; i.e., exposure to neither PM10 nor RSV). At 24 h after treatment (20), the AM were used immediately for plaque assay to quantify RSV and flow cytometric analyses to determine the proportion of RSV-infected cells and cell scatter properties while the cell supernatants were stored at –70°C until used for enzyme-linked immunosorbent assay (ELISA) (IL-6 and IL-8 detection) and bioassay (TNF-{alpha}) analyses. In addition, the association of PM10 within AM was examined using both light and electron microscopy.

Isolation of Guinea Pig Alveolar Macrophages
Female juvenile Cam Hartley guinea pigs (22–29 d old, 250–300 g body weight) were purchased from Charles River Laboratories (Montreal, QC, Canada) and maintained in accordance with standards of the Canadian Council on Animal Care (23). The guinea pigs were killed by intraperitoneal administration of pentobarbital (Euthanyl; MTC Pharmaceuticals; Cambridge, ON, Canada) at a dose of 40 mg/kg body mass. The lungs underwent BAL in situ by intratracheal instillation and aspiration of 5-ml aliquots of sterile, nonpyrogenic normal saline solution (Baxter, Toronto, ON, Canada), prewarmed at 37°C with a total volume of 100 ml. BAL cells underwent centrifugation at 500 x g for 10 min at 4°C, plated onto 6-well plates (2 x 106 cells/well in modified Eagle's medium [MEM]) and allowed to adhere for 30–60 min. Nonviable AM were removed by aspiration and viable AM were immediately subjected to different treatments described in the experimental design. The viability of the AM, as determined by trypan blue dye exclusion test, was > 90% in all groups. Experiments using a commercially available monoclonal antibody (MR-1; Serotec Ltd., Mississauga, ON, Canada) established that all adherent cells within wells that were isolated by this method had a macrophage/monocyte phenotype (19).

Preparation of RSV Stocks for Infection
Human RSV (Long strain/lot 18D) was purchased from American Tissue Culture Collection (Bethesda, MD) and was propagated on monolayers of HEp-2 cells in MEM supplemented with 2% fetal bovine serum (FBS) and 50 µg/ml gentamicin. RSV stocks were harvested at the peak of syncytia formation, usually 5–7 d after infection. Concentrated stocks of RSV were prepared by mechanical disruption of the syncytia-filled monolayers of HEp-2 cells using sterile 3-mm glass beads (Fisher Scientific, Nepean, ON, Canada) over a vortex for 30 s. The cell suspension underwent centrifugation at 1,500 x g, 4°C for 15 min to sediment cellular debris. The clear supernatant (containing free virus and soluble macromolecules such as inflammatory mediators) was applied onto Centriplus concentrators (Amicon, Beverly, MA) with molecular cutoff at 100,000 Daltons to concentrate the virus as well as to remove soluble macromolecules synthesized during viral infection of HEp-2 cells. Final preparations had undetectable levels of endotoxin by Limulus E-Toxate assay (24) and levels of IL-6, IL-8, or TNF-{alpha} were below detection limits of cytokine assays (see below). The clear supernatant was applied to the concentrator unit and underwent centrifugation at 3,000 x g, 25°C for 75 min. The virus-enriched retentate was stored in 1 ml aliquots at –70°C until use. The titer of the RSV stocks (107–108 plaque-forming units [pfu]/ml) was determined by plaque assay before infection of guinea pig AM. To determine the extent of virus aggregates resulting from preparation of viral stocks from the Centriplus concentrators, aliquots of RSV stocks underwent negative staining transmission electron microscopy as follows: samples were mixed with latex beads (173 nm diameter) and diluted with phosphate-buffered saline (PBS) 1:5, with 50–200 µl of diluted sample placed in a centrifuge tube (Polyallomer, 5 x 20 mm; Beckman Airfuge, Fullerton, CA). A copper grid (200–300 mesh covered with Formvar and carbon) was placed at the bottom of the tube. Tubes underwent centrifugation at 120,000 x g for 5 min, and grids were recovered with tweezers. Grids were then placed in a drop of 3% phosphotungstic acid (pH 6.0), for 60 s, dried, and visualized by transmission electron microscopy. The number of "small" (2–10 particles/sample), "medium" (11–100 particles/sample) and "large" (> 101 particles/sample) aggregates were counted, with dispersion of virus particles being considered adequate with higher percentage of small aggregates.

In Vitro RSV Exposure
AM in 6-well plates (2 x 106/well) were exposed to RSV at a multiplicity of infection (m.o.i.) of 3, in which 1 ml of RSV stock (6 x 106 pfu/ml MEM) was applied to each well containing adherent AM. Viral adsorption with intermittent agitation of 6-well plates was performed over 90 min. The wells were washed with PBS and fresh MEM containing 5% FBS was added (2 ml/well) at the end of treatment according to the experimental design.

PM10
The urban air dust preparation, PM10, used in this study was obtained from Environmental Health Canada, Ottawa (EHC-93; kindly provided by Dr. R. Vincent, Health Canada, Ottawa, ON, Canada). Airborne urban particles were collected from the outdoors using a single-pass air-purificator by vacuum and were passed through a 36-µm mesh filter. The median diameter of these fine particles was 0.35 µm, with a maximal diameter of 15 µm. A detailed analysis of the chemical components of EHC-93 has been previously reported and consisted mainly of polycyclic aromatic hydrocarbons, ions and metals (25). The individual components of this complex mixture of organic and inorganic compounds responsible for adverse health effects have not been definitively identified. However, the soluble metal components in ambient PM10 have been proposed as the major contributor in stimulating the production of cytokines by AM (26). In addition, it has been suggested that the PM10-associated metals with redox potential (for example, iron, vanadium, or copper) play significant roles in contributing to the toxicity of particulate matter (27).

PM10 Exposure
The EHC-93 particles were prepared fresh as 100 µg/ml particulate suspensions using MEM media supplemented with 2% FBS and gentamicin. This dose of PM10 was selected based on results from preliminary studies (28, 29). A trace amount of endotoxin (0.05 EU/100 µg PM10) was detected in the batch of particulates used in this study. However, exposure of guinea pig AM to equivalent amount of endotoxin in vitro did not induce a cytokine response (29). Furthermore, there was minimal toxic effect on the AM (> 90% cell viability by trypan blue exclusion test) and a maximal cytokine response was obtained when AM were treated with this dose (28). To minimize aggregates of particles, the PM10 suspensions underwent sonication using a probe sonicator (VibraCell; Sonics and Materials, Danbury, CT) three times at 5-min intervals before use. Five hundred microliters of PM10 particulate suspensions were added to 6-well plates containing ~ 2 x 106 adherent AM per well. The cells were exposed to PM10 particulates for 60 min in a 5% CO2 incubator at 37°C. Following exposure, the excess PM10 suspensions were removed by washing twice with PBS before subsequent in vitro RSV infection. Two milliliters of fresh medium was added to wells with no RSV infection.

Light and Electron Microscopy
Following exposure to PM10, as described above, the association of PM10 within AM was examined using both light and electron microscopy. Cytocentrifuge preparations of PM10-exposed AM were subjected to hematoxylin-eosin staining and examined under the light microscope. For visualization by a Philips 400 transmission electron microscope (Philips, Amsterdam, The Netherlands), PM-10 exposed AM were fixed in 2.5% glutaraldehyde-containing sodium cacodylate buffer (0.1 M, pH 7.4) for 1 h at 4°C, postfixed in osmium tetroxide for 1 h at room temperature, and embedded in epoxy resin following standard methods (30).

Flow Cytometry
Flow cytometry was utilized for the measurement of cell parameters (cell size and granularity) and the expression of RSV antigens was assessed by labeling with fluorescent-conjugated antibodies. The measurement of side scatter intensity, which is proportional to the granularity of the cell and a reflection of particle ingestion, was obtained simultaneously with the fluorescence intensity of the fluorescent-labeled RSV-infected cells. The photomultiplier tube voltage and compensation were set using cell surface staining controls (i.e., IgG of equivalent protein concentration) and the same quadrant markers were used for all experiments to facilitate inter-experiment comparisons. Data were saved to zip disks and analyzed with WinMDI (version 2.8) graphics software (WinMDI is a freeware obtained from the World Wide Web at http://facs.scripps.edu). Using WinMDI, histograms and bivariate dot plots were generated upon data analysis to display the mean fluorescence intensities (MFI) and the frequency of AM with expression of surface and intracellular RSV antigens.

Immunofluorescent Staining of RSV Antigens
AM harvested by gentle scraping at 24 h after treatment were washed in PBS and centrifuged for 8 min at 1,000 x g. A cell pellet, obtained by rapid decanting, was resuspended in 100 µl PBS. 10 µl of fluorescein isothiocyanate (FITC) conjugated anti-RSV antibody (NCL-RSV3-FITC; Novacastra Laboratories, New Castle upon Tyne, UK) was added to the cells and incubated for 30 min to allow detection of surface RSV antigens. Excess antibodies were washed off and the cells were fixed in 4% paraformaldehyde (Ted Pella Inc., Redding, CA) in PBS for 5 min. The cells were washed and incubated in 0.5% Triton-X (Sigma, St. Louis, MO) for 10 min. Intracellular RSV antigens were labeled by a second incubation with the NCL-RSV3-FITC antibody for another 30 min. The cells were washed in PBS and 0.2 ml crystal violet (2 mg/ml PBS; BDH Chemicals, Toronto, ON, Canada) was added to quench autofluorescence (31). After a final wash with PBS, the cells were resuspended in 500 µl PBS and stored on ice. All incubations were performed at room temperature unless otherwise stated. Flow cytometry was performed on the AM using the FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) within 2 h at the Flow Cytometry Facility, UBC Biomedical Research Center (Vancouver, BC, Canada).

Viral Plaque Assay
AM were harvested at 24 h after treatment following removal of cell supernatant for cytokine analyses. The cells were gently scraped with sterile cell scraper (Fisher Scientific), collected into Eppendorf tubes and vortexed with glass beads to release cell-associated virus. Virus titer was determined by incubation of serial dilutions (10-1 to 10-3) of sonicated AM on HEp-2 cell monolayers. All samples were plated in duplicate. After a 90-min incubation at 37°C, 5% CO2, the supernatant was removed. The monolayer was washed with PBS, overlaid with 1 ml of medium–agarose mixture (1:1, 2x MEM with 2% FBS:1% agarose) and incubated until syncytia were visible (7–10 d). After fixation with 4% paraformaldehyde (Ted Pella) at room temperature for 30 min, the cells were stained with 0.1% neutral red (Life Technologies, Burlington, ON, Canada). The syncytia were counted under an inverted light microscope.

RSV Yield
In the literature, some groups measure the extent of RSV infection based on immunostaining and others by plaque assay (17, 19, 20). Here we introduce the term "RSV Yield" where we define it as the amount of viral replication per RSV-immunopositive cell; it is calculated from the following equation: RSV Yield = (# pfu/106 AM) ÷ (% RSV-immunopositive AM)

RSV Yield can therefore be considered as a reflection of the robustness of viral replication within an infected cell.

Detection of IL-6 and IL-8 by ELISA
Production of IL-6–like and IL-8–like proteins by AM was quantified using a sandwich ELISA technique, modified from the protocol of Denis and coworkers (32). In the IL-6 assay, a mouse anti-human IL-6 monoclonal antibody (4 µg/ml) was used as a coating antibody in conjunction with biotinylated goat polyclonal antibody to human IL-6 (25 ng/ml). Likewise, a mouse anti-human IL-8 monoclonal antibody (4 µg/ml) was used as a capture antibody and a second biotinylated goat polyclonal antibody to IL-8 (20 ng/ml) was used for detection of guinea pig IL-8–like proteins. Recombinant human IL-6 and IL-8 were used in the respective assays for calculation of standard curves that were generated for each set of samples assayed. All antibodies and recombinant proteins for ELISA were obtained from R&D Systems (Minneapolis, MN). A streptavidin–horse radish peroxidase conjugate was used to detect biotinylated antibodies and the substrate used for detection consisted of 1:1 H2O2:tetramethylbenzidine (San Carlos, CA). All samples were analyzed in duplicate. Standardization of the proteins was included with each assay performed using the respective recombinant human IL-6 and IL-8. The equation of the best-fit line was determined and the concentration of the cytokine was extrapolated from this equation and its absorbance reading at 405 nm.

Detection of TNF-{alpha} by Bioassay
TNF-{alpha} activities of guinea pig AM were detected in a bioassay on actinomycin D-treated (Sigma) L929 cells (a kind gift from Dr. R.R. Schellenberg, MRL, Vancouver, BC, Canada) as described by Horiuchi and colleagues (33). Recombinant human TNF-{alpha} (R&D Systems) was used as a positive control as well as a standard for calibration. To confirm the production of bioactive TNF-{alpha} by guinea pig AM, a neutralization test was performed by using polyclonal rabbit anti-mouse TNF-{alpha} neutralizing antibody (Genzyme, Cambridge, MA).

Statistical Analyses
Data for viral plaque assays, cell granularity, %AM showing RSV immunopositivity, RSV Yield, and results of cytokine assays were expressed as the mean value ± SD. Experimental groups were compared by use of ANOVA, with a P value < 0.05 (two-tailed) considered as statistically significant. Post hoc t tests were then performed to determine the source of statistical significance between the experimental groups, after correction for multiple comparisons by a Bonferroni method (34).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Negative Staining Transmission Electron Microscopy of Virus Stocks
Figure 1 shows an electron micrograph of a negatively-stained preparation of RSV stock examined by transmission electron microscopy. The RSV particles appeared to be in good condition and were generally well dispersed, with only occasional small aggregates composed of virus particles or virus particles with small cell debris. No medium or large aggregates were observed in the virus stocks prepared by Centriplus concentrators.



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  		Figure 1. Electron micrograph from a negatively-stained preparation of RSV stock prepared by Centriplus concentrator. Note the distinct irregularly-shaped RSV particles (arrows) and 173-nm-diameter latex bead (*), with minimal cellular and proteinaceous debris in the preparation. Bar represents 150 nm.

 
Effect of RSV–PM10 Interaction on Phagocytic Ability of AM
Figure 2 shows representative light and electron micrographs of PM10-exposed AM. Under the light microscope, PM10 particles were localized within cytoplasmic regions of PM10-exposed AM. The PM10 particles were contained within lysosomal compartments of the cell as revealed by electron microscopy. More than 95% of AM exposed to PM10 had engulfed particulate matter. Phagocytosis of PM10 by AM was also reflected by results of flow cytometric measurement of side scatter that is proportional to cell granularity. The effect of RSV–PM10 interaction on AM granularity is summarized and presented in Figure 3. AM that were exposed to PM10 showed a significant increase in mean side scatter in comparison to the negative control AM (P < 0.05) and in comparison to RSV-infected AM (P < 0.04). There were no significant differences in mean side scatter between negative control AM and RSV-infected AM. In addition, there were no significant differences in mean side scatter between AM that were exposed only to PM10 and AM that were exposed to both agents.



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  		Figure 2. Cytocentrifuge preparations of PM10-exposed AM were examined under light and transmission electron microscopes. (A) Light micrograph of AM with particulates (arrow) located within cytoplasmic regions. Bar represents 25 µm. (B) Electron micrograph of AM containing PM10 (arrow) within a lysosome. Bar represents 100 nm.

 


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  		Figure 3. Exposure of AM to PM10 results in increased cell granularity. AM were exposed to 100 µg/ml PM10, or no particles (NEG), for 1 h according to experimental design. Cells were collected at 24 h after treatment and side scatter, a reflection of cell granularity, was determined by flow cytometry. The bars represent the mean ± SD of guinea pig AM (n = 4) subjected to different treatments. *P < 0.05 NEG versus PM10+RSV or RSV+PM10 or PM10. {dagger}P < 0.04 RSV versus PM10+RSV or RSV+PM10 or PM10.

 
Effect of RSV–PM10 Interaction on RSV Immunopositivity
Typical histograms of flow cytometric analyses of RSV antigens on HEp-2 cells and guinea pig AM with and without RSV infection are presented in Figure 4. The results of the effect of RSV–PM10 interaction on RSV immunopositivity are shown in Figure 5. All RSV-treated groups showed a significantly greater proportion of RSV-immunopositive cells compared with negative control AM (P < 0.007). AM in the PM10+RSV group showed a significantly smaller proportion of RSV-immunopositive cells compared with the RSV group (P < 0.05). By contrast, the proportions of RSV-immunopositive cells in the RSV+PM10 group versus the RSV group were similar. In addition, the proportion of RSV-immunopositive AM was influenced by the sequence of exposure to RSV and PM10 (P < 2 x 10-5). A minor population of AM (1.98% ± 0.37% [mean ± SD]) in the PM10 only group showed nonspecific labeling with anti-RSV antibody.



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  		Figure 4. Representative histograms of HEp-2 cells and guinea pig AM with and without RSV infection. For both types of cells infected with RSV, m.o.i. = 3. The MFI of uninfected HEp-2 cells (A) and guinea pig AM (B) labeled with either anti-RSV antibodies (Red) or IgG1 (equivalent isotype control; Black line, no fill.) are very similar. The MFI of RSV-immunopositive HEp-2 cells (> 85%) increased by more than 10-fold compared with the respective IgG1 control (C; anti-RSV versus IgG1: 53.2 versus 4.3). The spread of this cell population indicates that some HEp-2 cells are more heavily infected than others. D Shows a right shift of a minor proportion (< 20%) of RSV-immunopositive guinea pig AM when labeled with anti-RSV antibodies (MFI; anti-RSV versus IgG1: 17.9 versus 1.6).

 


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  		Figure 5. PM10 exposure inhibits RSV immunopositivity. AM were exposed to RSV and/or PM10 as described in MATERIALS AND METHODS. RSV immunopositivity was determined by flow cytometric analysis of RSV antigens stained with a fluorescein-conjugated anti-RSV antibody (RSV3-NCL). The bars represent percentage of RSV-immunopositive AM (mean ± SD) from four separate experiments. *P < 0.007 NEG versus all other groups. {dagger}P < 0.05 PM10+RSV versus RSV. {ddagger}P < 2 x 10-5 PM10+RSV versus RSV+PM10.

 
Effect of RSV–PM10 Interaction on RSV Replication
The quantity of RSV progeny isolated from AM, as determined by plaque assay, is shown in Figure 6. AM that were exposed to both agents produced 3- to 9-fold less RSV progeny compared with AM that were exposed to RSV alone (PM10+RSV versus RSV, P < 0.005; RSV+PM10 versus RSV, P < 0.02). However, the quantity of RSV progeny was not significantly affected by the sequence of exposure to RSV and PM10. Negative control AM and those that were exposed to PM10 alone did not propagate RSV progeny.



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  		Figure 6. PM10 exposure suppressed RSV replication. AM were exposed to RSV and/or PM10 as described in MATERIALS AND METHODS. RSV replication was determined by plaque assay. The bars represent the quantity of RSV progeny scored per million AM (mean ± SD) of four separate experiments. *P < 0.005 PM10+RSV versus RSV. {dagger}P < 0.02 RSV+PM10 versus RSV.

 
Effect of RSV–PM10 Interaction on RSV Yield
The data for the effect of RSV-PM10 interaction on RSV Yield are summarized in Table 1. AM that were exposed to RSV alone produced the highest RSV Yield (P < 0.04). When AM were exposed to both RSV and PM10, a 5-fold decrease in RSV Yield was observed. This reduction in RSV Yield was independent of the sequence by which AM were exposed to both RSV and PM10.


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  		TABLE 1 Effect of PM10-RSV interaction on RSV Yield

 
Effect of RSV–PM10 Interaction on Cytokine Production by AM
The levels of IL-6–like, IL-8–like, and TNF-{alpha} proteins secreted by AM into culture supernatants are shown in Figure 7. Consistent with previous findings in human AM (20, 35), RSV infection of guinea pig AM also stimulated the production of all three cytokines measured compared with their respective negative controls (P < 0.02).



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  		Figure 7. Effect of RSV-PM10 interaction on cytokine production by guinea pig AM. Supernatants were collected at 24 h after treatment and assessed for production of (A) IL-6, (B) IL-8, and (C) TNF-{alpha}. Bars represent cytokine production (mean ± SD) by guinea pig AM from four separate experiments. (A) Exposure of AM to PM10 results in decrease production of RSV-induced IL-6 expression. *P < 0.0002 RSV versus all other groups. {dagger}P < 4 x 10-6 PM10+RSV versus RSV. {ddagger}P < 1 x 10-6 RSV+PM10 versus RSV. §P < 3 x 10-6 PM10+RSV versus RSV+PM10. (B) Exposure of AM to PM10 results in decrease production of baseline and RSV-induced IL-8 expression. *P < 0.009 RSV versus all other groups. {dagger}P < 0.009 PM10 versus NEG. {ddagger}P < 0.003 PM10+RSV versus RSV. §P < 0.003 RSV+PM10 versus RSV. (C) Exposure of AM to RSV and/or PM10 stimulates TNF{alpha} production. *P < 0.02 NEG versus all other groups.

 
Figure 7A shows that the exposure of AM to PM10 significantly decreased RSV-induced IL-6 production (PM10+RSV versus RSV, P < 4 x 10-6; RSV+PM10 versus RSV, P < 1 x 10-6). Furthermore, IL-6 production by guinea pig AM was affected the sequence of exposure to PM10 and RSV (PM10+RSV versus RSV+PM10, P < 3 x 10-6).

Exposure of AM to PM10 resulted in significant decreases of RSV-induced IL-8 production (P < 0.003) as well as that of baseline (PM10 versus NEG, P < 0.009). In contrast to IL-6 expression, IL-8 expression by guinea pig AM was not influenced by the sequence of exposure to PM10 and RSV (Figure 7B).

TNF-{alpha} released into cell supernatants were detected by bioassay of TNF-sensitive L929 cells. The data as shown in Figure 7C indicate that AM production of TNF-{alpha} was significantly increased when these cells were exposed to RSV, PM10, or a combination of both agents (P < 0.02). However, there were no differences in the production of TNF-{alpha} by AM among different treatments. AM exposed to both RSV and PM10 did not exhibit either a synergistic or inhibitory effect on TNF-{alpha} levels from interaction of the two agents.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of this study was to examine the sequential effect of RSV–PM10 interaction on guinea pig AM functions in vitro. Our data indicate that sequential exposure of guinea pig AM to RSV and PM10 has no effect on the phagocytic ability of these cells to take up PM10. Exposure of AM to PM10 resulted in reduced RSV Yield as well as levels of RSV-induced production of IL-6–like and IL-8–like proteins, regardless of sequence of exposure to RSV and PM10. Exposure of AM to RSV and/or PM10 resulted in increased expression of bioactive TNF-{alpha} with no synergistic effect from the interaction of these two agents.

The ability of AM to phagocytose PM10 was measured indirectly by the granularity (side scatter) of the cell. Contrary to results of previous studies (21), our data indicate that RSV infection did not affect phagocytosis of PM10 particles by these cells. Species specificities of AM and the type of particulate matter being phagocytosed are two possible factors that might explain these apparent differences. Furthermore, similar mean cell granularity of the cells in all three groups that were exposed to PM10 indicates that the ability of AM to phagocytose PM10 was not affected by sequential exposure to RSV and PM10.

In an effort to better understand the interaction between RSV and AM, we considered the ability of these cells to synthesize viral proteins as well as support viral replication as one entity. We defined the RSV Yield of a cell as the amount of viral replication (as determined by plaque assay) per RSV-immunopositive cell (as determined by RSV immunostaining). Regardless of the sequence of exposure, AM subjected to both RSV and PM10 demonstrated a significantly lower RSV Yield compared with cells that were exposed to RSV alone.

In comparison with the RSV group, the apparently lower RSV Yield and quantity of RSV progeny in the PM10+RSV group may be the consequence of a lower proportion of RSV-immunopositive AM. This observation is consistent with previous findings in which exposure of AM to environmental particles has been previously reported to impair phagocytosis of RSV (22). Furthermore, ingestion of environmental particles by AM has been demonstrated by others to impair subsequent phagocytosis of silica particles at the attachment and internalization steps (36). However, the mechanisms responsible for reduction of RSV-immunopositive cells in PM10+RSV versus RSV groups remain unclear. Early time point experiments examining the binding and entry of RSV into cells, expression of RSV mRNA and proteins in AM, and activity of host factors including double-stranded RNA nucleases that could potentially affect the RSV life cycle, might provide more insights on phagocytosis of RSV following PM10 exposure.

The above explanation, however, could not be extended to explain the findings RSV+PM10 group. Intriguingly, despite similar proportions of RSV-immunopositive cells between the RSV+PM10 and RSV groups, subsequent exposure of AM to PM10 resulted in significant inhibition of viral replication in these cells when compared with the RSV group. Because synthesis of viral mRNA generally occurs within one hour after infection (20), our data suggest that PM10 exposure of RSV-infected AM could suppress viral propagation despite initiation of viral protein synthesis. Although the mechanisms for this effect are unclear, a particle-induced immune response has been proposed to constitute an antiviral effect in vivo (37).

We studied the cytokine response of AM following exposures to RSV and PM10 by examining the production of IL-6, IL-8, and TNF-{alpha}. These cytokines were examined because of their roles implicated in RSV-induced inflammation (20). Consistent with findings in other animal models (20, 21), our data show that RSV infection of guinea pig AM results in increased expression of IL-6, IL-8, and TNF-{alpha}, and further studies are required to determine whether these cytokines were secreted only by RSV-infected AM, or whether uninfected cells may also have contributed to secretion of cytokines observed in culture supernatants.

Stimulation of IL-6, IL-8, and TNF-{alpha} by environmental particulates had been demonstrated in AM from humans and rats (22, 38). By contrast, guinea pig AM responded to PM10 by the production of TNF-{alpha} but not IL-6 or IL-8. Although the environmental particulates used in this study and those used by Becker and coworkers (22, 38) originated from the same source, the batch of PM10 in our hands contained relatively minute traces of endotoxin (0.05 EU/100 µg PM10). This amount of endotoxin has been shown insufficient to induce a cytokine response in guinea pig AM (29). This suggests that the endotoxin contamination cannot explain the TNF-{alpha} response of guinea pig AM to particulate stimulation. Soluble metal components of PM10, especially those with redox potential, have been proposed as major contributors in stimulating cytokine production by AM (26, 27). Differences in the particulate-induced response of IL-6 and IL-8 observed in this study compared with results of previous studies (22, 38) are likely due to differences in the animal species used by different groups of investigators and endotoxin levels in the preparation of environmental particulates.

The levels of IL-6 and IL-8 proteins were significantly lower in AM exposed to both agents compared with AM in the RSV group. It has been reported that RSV stimulates expression of IL-6 and IL-8 in a virus-dose dependent fashion (20). Because the proportion of RSV-immunopositive cells and RSV Yield of RSV-infected AM following PM10 exposure (PM10+RSV group) were significantly lower than AM in the RSV group, reduced virus load may be one mechanism by which PM10 particles suppress virus-induced IL-6 and IL-8 expression. Alternatively, because transcription of IL-6 and IL-8 in AM in the RSV+PM10 group was likely initiated within 1 h after RSV interaction (20), at a time when PM10 was introduced to the AM, decreased expression of these cytokines suggest that PM10 inhibition of these proteins could target the translation stage of cellular protein synthesis.

The levels of measured production of bioactive TNF-{alpha} by AM were similar regardless of exposure to RSV, PM10, or a combination of both agents. It is plausible that TNF-{alpha} may have an indirect antiviral effect. TNF-{alpha} has been shown to induce nitric oxide (NO) expression in RSV-treated as well as PM10-treated cells (39, 40). The antiviral role for NO has been reported in numerous in vitro virus models (41). Experiments designed to examine NO production in AM exposed to both agents might provide new insights to understanding mechanisms of viral inhibition.

In summary, environmental particles could alter the ability of guinea pig AM to take up RSV depending on the sequence of exposure to these agents. However, regardless of the sequence of exposure, guinea pig AM exposed to both RSV and PM10 results in a reduction of RSV Yield and decreased production of proinflammatory cytokines. These findings may have important implications for further investigations testing the Hygiene Hypothesis, and in particular, the controversy over the protective versus pathogenic roles of viral infections and air pollution in the prevalence and development of respiratory allergic diseases.


   Acknowledgments
 
This work was supported by The Canadian Institutes of Health Research and The British Columbia Lung Association. The authors thank Dr. Renaud Vincent, Environmental Health Canada, for providing PM10 particles; Mr. Stuart Greene and Mr. Dean English for expert photography assistance; Ms. Danyi Zhou for technical assistance in ELISA experiments; Ms. Diane Minshall and Ms. Lynne Carter for assistance in handling of experimental animals; and Dr. Stephan van Eeden for constructive discussion. Dr. Robert Alain, INRS-Institut Armand-Frappier, (Laval, QC, Canada) performed negative staining transmission electron microscopy of RSV stocks.

Received in original form July 12, 2002

Received in final form December 16, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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