© 2003 American Thoracic Society DOI: 10.1165/rcmb.2002-0100OC Increased Susceptibility to RSV Infection by Exposure to Inhaled Diesel Engine EmissionsAsthma and Pulmonary Immunology Program, and Experimental Toxicology Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico Address correspondence to: Kevin S. Harrod, Ph.D., Asthma and Pulmonary Immunology, Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr. SE, Albuquerque, NM 87108. E-mail: kharrod{at}lrri.org
Although epidemiologic data strongly suggest a role for inhaled environmental pollutants in modulating the susceptibility to respiratory infection in humans, the underlying cellular and molecular mechanisms have not been well studied in experimental systems. The current study assessed the impact of inhaled diesel engine emissions (DEE) on the host response in vivo to a common pediatric respiratory pathogen, respiratory syncytial virus (RSV). Using a relatively resistant mouse model of RSV infection, prior exposure to either 30 µg/m3 particulate matter (PM) or 1,000 µg/m3 PM of inhaled DEE (6 h/d for seven consecutive days) increased lung inflammation to RSV infection as compared with air-exposed RSV-infected C57Bl/6 mice. Inflammatory cells in bronchoalveolar lavage fluid were increased in a dose-dependent manner with regard to the level of DEE exposure, concomitant with increased levels of inflammatory mediators. Lung histology analysis indicated pronounced peribronchial and peribronchiolar inflammation concordant with the level of DEE exposure during infection. Mucous cell metaplasia was markedly increased in the airway epithelium of DEE-exposed mice following RSV infection. Interestingly, both airway and alveolar host defense and immunomodulatory proteins were attenuated during RSV infection by prior DEE exposure. DEE-induced changes in inflammatory and lung epithelial responses to infection were associated with increased RSV gene expression in the lungs following DEE exposure. These findings are consistent with the concept that DEE exposure modulates the lung host defense to respiratory viral infections and may alter the susceptibility to respiratory infections leading to increased lung disease.
Abbreviations: bronchoalveolar lavage fluid, BALF • Clara cell secretory protein, CCSP • diesel engine emissions, DEE • enzyme-linked immunosorbent assay, ELISA • interferon-
Respiratory infections constitute the greatest health impact to children worldwide. Respiratory viral infections in children are the primary cause of lung disease leading to diminished lung function and capacity (1). Viral infections early in life have been linked to the increased incidence of lung disease, particularly asthma, throughout childhood (2). Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in young children, infecting virtually every child by the age of two (3). RSV infections of the lower respiratory tract often lead to hospitalization in this age group and can cause severe or chronic bronchitis and wheezing. Importantly, infection in young children does not induce strong viral immunity against secondary and subsequent infections, which have hampered traditional vaccine prophylaxis. The mechanisms of lung pathogenesis to RSV have not been fully elucidated, but lung-specific host defense appears to be important in modulating lung pathogenesis during infection (4). The impact of environmental pollutants and poor air quality on respiratory infections has been suggested primarily based upon epidemiology studies examining clinical data during episodes of air pollution (5). Hospitalizations, physician visits, school absenteeism, and asthmatic episodes have been correlated in children during times of elevated particulate matter (PM) or ozone in urban settings (6, 7). Engine emissions continue to have a tremendous impact on air quality in the United States. Yet, the biological health effects of exposure to diesel engine emissions (DEE) have not been fully assessed, particularly to acute respiratory disease. In experimental models, PM from DEE exacerbates the host response to bacterial infections of the lung, likely occurring through mechanisms of altered bacterial clearance (8, 9). However, many of these studies have only examined filter-captured components of DEE (10–12) and do not examine others, such as gaseous-phase emission components. In the current study, whole mixtures of inhaled DEE were used to assess the effect of DEE exposure on the host response to subsequent RSV infection in vivo. Multiple levels of DEE exposure were assessed in a relatively RSV-resistant mouse strain at the time of peak viral titers following infection. The DEE exposure concentrations resemble levels ranging from high ambient to high levels occupational settings, thus providing information regarding possible health effects of DEE in human disease. Following exposure to DEE, lung inflammation and pathogenesis to RSV infection were increased concordant with decreased RSV clearance in lung tissues. These studies provide the first evidence of increased lung disease to RSV by prior exposure to DEE in vivo, and suggest a role for ambient DEE in the increased susceptibility to respiratory viral infection.
Reagents All reagents were obtained from Sigma Chemicals (St. Louis, MO) unless otherwise noted. For protein analysis studies, surfactant protein antibodies were purchased from Chemicon (Temecula, CA). PCR primer oligonucleotides were obtained from Sigma Genosys (The Woodlands, TX). All antibody and detection reagents for immunohistochemistry were purchased from Vector Laboratory (Burlingame, CA). Secondary antibodies for Western blot analysis were purchased from Calbiochem (San Diego, CA), and chemiluminescence reagents were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).
DEE Generation and Exposure Exposure atmospheres were monitored daily for the concentration of PM and Nitrogen Oxides (NOx), which is the sum of nitrogen oxide (NO) and nitrogen dioxide (NO2). These measurements were used both to define the exposure concentration and to provide information for adjusting system dilutions to achieve the target exposure concentrations. PM concentration was monitored by deposition on 47-mm Pallflex (Pall-Gelman, Ann Arbor, MI) filters. Pre- and post-filter weights were measured using a Mettler MT5 balance (Mettler, Columbus, OH). A static discharger was used before weighing to avoid any interference from electrical charge on the filters. Filter samples were collected three times (every 2 h) from the high-level and twice (every 3 h) from the low-level exposures. One filter sample per day was collected from the control chamber. Total NOx were measured using a chemiluminescent analyzer (API Model 200A NOx Analyzer; Teledyne Instr., San Diego, CA). Carbon monoxide was determined using a Photoacoustic Gas Analyzer (Innova 1312; California Analytical Instruments, Irvine, CA). Both NOx and CO analyzers were calibrated before each study using National Institute of Standards and Technology traceable standards. SO2 was collected utilizing potassium carbonate impregnated filters and analyzed by ion chromatography (500DX; Dionex, Sunnyvale, CA). Particle size was measured using a 10-stage micro-orifice–uniform deposit impactor (MOUDI; MSP Corp. St. Paul, MN). The MOUDI was operated at a flow rate of 30 liters/min, providing particle size resolution from 0.05–10 µm in aerodynamic diameter.
RSV Culture and Titration
Animal Husbandry and Intratracheal Administration of RSV
Reverse Transcriptase–Polymerase Chain Reaction Analysis of Viral Gene Expression
Inflammatory Cells in Bronchoalveolar Lavage Fluid
Lung Histology and Immunohistochemical Analysis For mucous cell metaplasia (MCM) studies, lung sections were stained with Periodic Acid Schiff and Alcian Blue as described previously (16). For quantification of MCM, first- or second-generation intrapulmonary bronchi were analyzed for the volume of mucosubstances by Scion Image software (NIH, Bethesda, MD), and mucosubstance calculations normalized to mm2 of basal lamina as described previously (17). For immunohistochemical studies, lung sections were prepared as described previously (18, 19). Briefly, lung sections (5 µm) were taken at equivalent distances from the histologic reference point and were stained overnight with primary rabbit antibodies against selected proteins. Slides were rinsed in physiologic buffer and incubated with secondary goat anti-rabbit antibodies conjugated to biotin. A streptavidin-conjugated peroxidase detection system (Vector Laboratory) was used to visualize antibody-binding complexes following incubation with diaminobenzidine. Multiple sections from each tissue block were analyzed under light microscopy. Clara cell secretory protein (CCSP) staining was quantified by adaptation of MCM staining procedure as described previously (17).
Inflammatory Cytokine Analysis
Western Blot Analysis
Statistical Analysis
DEE Exposure Characterization Table 1 shows a summary of exposure concentrations of PM, NOx, CO, and SO2 for DEE exposures as described herein. Diesel PM is the total PM minus the PM measured in the control chamber. Although air flowing through the control chamber is particle free, small amounts of PM that arise from suspension of rodent dander are measured in the control chamber. Assuming that the dander contribution to PM is the same in each exposure chamber, the control PM value is subtracted from the exposure chamber PM to yield "Diesel PM." The low-level DEE exposure was actually 38.8 µg/m3, whereas the high-level DEE exposure was 1,027.3 µg/m3. The gaseous co-pollutants shown in Table 1 ranged from 2.0–43.3 ppm for NOx (  90% = NO), 0.94–29.0 ppm CO, and 8.3–364.9 ppb SO2. The particle size measurements (not shown in Table 1) showed aerodynamic mass median diameters between 0.1 and 0.2 µm at both the low-level and high-level target exposure levels.
Increased Viral Gene Expression in the Lungs by Prior Exposure to Inhaled DEE C57Bl/6 mice (8-10 wk of age) were exposed to DEE by aerosol inhalation for 6 h/d for seven consecutive days, whereby RSV infection was subsequently introduced by intratracheal administration of 106 pfu under light anesthesia. To assess the impact of DEE exposure on lung viral gene expression, total RNA was isolated from the lungs of mice 4 d after RSV infection. RT-PCR analysis of the viral-specific genes RSV-F and RSV-G was performed using total RNA and visualized by ethidium bromide–stained gel electrophoresis. RT-PCR analysis of endogenous ß-actin mRNA steady-state levels was used as loading and assay controls. At 4 d after infection, RSV-F gene expression was not readily apparent in the lungs of air-exposed, infected animals, whereas RSV-G gene expression was detectable, albeit at very low levels (Figure 1A). In comparison, RSV-F and RSV-G gene expression was readily discernible and markedly elevated in the lungs of infected animals following exposure to either low- or high-level DEE. The increase in RSV gene expression following DEE exposure was not discernible between animals exposed to either low- or high-level DEE. ß-Actin mRNA steady-state levels were not changed in the lungs of DEE-exposed animals as compared with air-treated control animals. Densitometric analysis of RSV-F and RSV-G mRNA levels is shown in Table 2, indicating a 10- to 20-fold induction in the level of RSV-G mRNA in the lungs of DEE-exposed mice as compared with air-exposed controls.
DEE Exacerbated Lung Inflammation to RSV Infection Morphologic staining and hemacytometer counting of inflammatory cells were determined in BALF from the lungs of air- or DEE-exposed, RSV-infected, and uninfected mice. Cell numbers in BALF from the lungs of air-treated, vehicle-instilled animals were not changed from naive animals, consisting of normally resident alveolar macrophages (data of cell-type specific staining not shown). Following RSV infection, cell numbers in the BALF from air-treated animals were not elevated (Figure 1B). Exposure to low-level DEE did not induce a statistically significant increase in cell numbers as compared with those of air-treated, infected animals. However, exposure to high-level DEE caused an increase in cell numbers in BALF following RSV infection, as compared with that of air-treated, RSV-infected mice. Increased cell numbers were also detected in BALF from uninfected animals exposed to high-level DEE, as compared with uninfected animals exposed to air. In all groups, alveolar macrophages were the predominant cell type (>95%), and no substantial changes in infiltrating cell populations by exposure to DEE were noted (Table 3). Statistical power analysis of low-level DEE samples indicates that larger sample sizes (n  20) may be required to delineate a statistical increase in inflammatory cells in BALF from uninfected mice exposed to low-level DEE exposure alone, as compared with that of uninfected, air-treated mice.
Lung Inflammation and Airway Epithelial Remodeling to RSV Infection Are Augmented by DEE To assess histologic changes in the lungs following DEE exposure and RSV infection, lung sections (5 µm) were stained by standard hematoxylin-eosin procedures and scored blindly under light microscopy. Lung sections from air- or DEE-exposed, uninfected groups did not exhibit any observable change when examined (Figure 2A). At 4 d following RSV infection, lung sections of air-treated animals did not show discernible changes in either lung infiltrates or the lung epithelium. Increased inflammatory cell infiltration was noted in peribronchial regions in the lungs of RSV-infected animals following low-level DEE exposure as compared with that of air-exposed, RSV-infected animals. Areas of consolidated inflammatory cell infiltrates were distinct. Furthermore, airway epithelial structure was altered, consisting of loss of the normal cuboidal appearance of nonciliated bronchiolar epithelial (Clara) cells. Occasional regions exhibited columnar epithelial appearance and increased thickening of the myofibril layer in the basal compartments of the airway. These changes were further apparent in the lungs of RSV-infected animals exposed to high-level DEE. Lung inflammation, particularly surrounding bronchi and bronchioles, was strikingly apparent in lung sections of RSV-infected, high-level DEE animals. Morphologic changes in airway epithelial cells were readily observable, with the increased appearance of pseudostratified, columnar epithelial cell morphology and apparent airway epithelial cell sloughing. Both lung inflammation and airway epithelial morphology were further changed in the lungs of high-level DEE-exposed mice as compared low-level DEE exposure following RSV infection, indicating a dose-related increase in lung histopathology to RSV infection by prior exposure to DEE. Scoring of the histologic changes as assessed by light microscopy is presented in Figure 2B. Comparison of the inflammatory cell populations in the lungs of DEE-exposed, RSV-infected mice as determined by BALF studies or histologic analysis is shown in Table 3. In short, both analyses indicate that the macrophages and monocytes were the predominant inflammatory cell type in the lungs of DEE-exposed mice at 4 d following RSV infection.
DEE-induced Inflammatory Mediators during RSV Pathogenesis The proinflammatory cytokines TNF-  and IFN- are important regulators of lung pathogenesis and antiviral immune mechanisms to respiratory viral infection. ELISA analysis of lung homogenates from air- or DEE-exposed, RSV-infected mice indicated that RSV infection of air-treated animals did not induce TNF- or IFN- protein expression at 4 d after infection (Figures 3A and 3B). In contrast, both TNF- and IFN- protein levels were markedly increased in the lungs of infected mice following either low- or high-level DEE exposure. Increased TNF- following infection was elevated to similar levels in the lungs of low-level DEE-exposed animals as compared with infected animals exposed to high-level DEE. Induction of IFN- during RSV infection by prior exposure to inhaled DEE exhibited a more dose-related increase in IFN- levels in the lung, with IFN- levels in high-level DEE-exposed, infected animals greater than those exposed to low-level DEE. Lung total protein content, as assessed by Bradford assay, in RSV-infected animals was not elevated by prior exposure to DEE as compared with air-exposed animals (data not shown).
Mucous Cell Metaplasia during RSV Infection Induction of airway epithelial mucus production at 4 d after infection was assessed by Periodic Acid Schiff and Alcian Blue staining in the lungs of mice following air or DEE exposure and subsequent RSV infection. DEE exposure did not alter mucus staining in the lungs of uninfected animals (data not shown). In contrast, mucus staining was apparent in the airways following air treatment and subsequent RSV infection (Figure 4), with intermittent regions of mucus-producing cells observed by light microscopy in larger airways (airway generations 1–3). Mucous production in the airways of infected animals exposed to low-level DEE was more apparent, with increased numbers of cells staining in larger airways and increased mucosubstances in airway epithelial cells. Mucous staining was further increased in the airways of RSV-infected mice exposed to high-level DEE, with markedly apparent staining throughout the airway epithelium and increased size of mucosubstances within the epithelial lining. MCM in the lungs of DEE-exposed, RSV-infected animals was further assessed by quantification of the volume of mucosubstances in the proximal airways (Table 4). Analysis of airway mucosubstances indicated a dose-related increase in MCM in the lungs of DEE-exposed mice following RSV infection. Mucous staining of airway epithelial cells in more distal airways was also occasionally observed in the airways following high-level DEE exposure and RSV infection, suggesting that high-level DEE exposure induced MCM to RSV infection to more distal regions of the tracheobronchial tract.
Attenuation of CCSP Production in the Airway Epithelium To assess other airway epithelial functions that may be further exacerbated during RSV infection by prior exposure to inhaled DEE, CCSP (also called CC-10 or CC-16), an abundant immunomodulatory protein produced by nonciliated airway epithelial (Clara) cells, was assessed by immunohistochemical staining of lung sections (Figure 5A), and CCSP staining quantified by photomicrograph imaging (Table 5). By image analysis, CCSP staining was more intense in the small as compared with larger airways. DEE exposure alone did not alter CCSP staining in larger airways in any groups; however, a mild decrease in CCSP staining in the terminal airways of high-level, but not low-level, DEE-exposed animals was detected by image analysis. Following air exposure and subsequent RSV infection, CCSP was readily apparent in both proximal and distal airways of the lung at 4 d after infection. Clara cell morphology exhibited the normal cuboidal appearance, with a uniform monolayer appearance of the airway epithelium. Following exposure to low-level DEE and subsequent RSV infection, the numbers of CCSP-producing cells, thus Clara cells, were diminished throughout the airways. In addition, qualitative assessment of the intensity of CCSP staining within Clara cells following low-level DEE exposure and RSV infection suggested decreased production of CCSP by Clara cells of the airway epithelium. Following exposure to high-level DEE and RSV infection, CCSP staining was further decreased as compared with exposure to low-level DEE before infection. As observed under higher magnification by light microscopy, the airway epithelium, in particular Clara cells, exhibited an altered morphology, with diminished cytoplasmic projections into the airway lumen and regions of disorganized airway epithelial monolayers (Figure 5A, panel i). Quantitative assessment of CCSP in the lungs of DEE-exposed mice at 4 d after RSV infection indicated a marked decrease in both large and distal airways by prior exposure to low-level DEE (Table 5). CCSP staining was further decreased in both large and small airways of infected mice following high-level DEE as compared with the low-level DEE exposed animals.
As a further quantitative measure of CCSP levels in the airway epithelium, lung homogenates from low- or high-level DEE exposure and subsequent RSV infection were analyzed by Western Blot (Figure 5B). CCSP levels were not altered by RSV infection alone as compared with vehicle-instilled, air-exposed samples. Prior exposure to low-level DEE caused a discernible decrease in CCSP levels in the lung following RSV infection. CCSP levels were further decreased by RSV infection in lung homogenates following high-level DEE exposure. DEE exposure or infection status did not change total lung protein content. Densitometric analysis of CCSP protein levels, as measured by immunoblot assay, is shown in Table 6. Similar to findings shown in Figure 5B, CCSP levels were decreased in the lungs of DEE-exposed mice at 4 d after RSV infection. CCSP levels were not changed by exposure to DEE before RSV infection (Table 6).
Surfactant Protein Dysregulation following DEE Exposure and RSV Infection Surfactant protein B is a critical regulator of alveolar surfactant homeostasis and function (20). To assess putative alterations in alveolar function during RSV infection by prior exposure to inhaled DEE, surfactant proprotein B (proSP-B) levels were assessed by immunohistochemical analysis (Figure 6). ProSP-B staining was not altered by DEE exposure alone in either distal airways or alveolar type II cells (Figure 6A). Following RSV infection alone, proSP-B staining was observed by light microscopy specifically in type II cells of the alveoli and alveolar ducts (Figure 6B, panel a), with no discernible decrease when compared with that of uninfected animals. Staining in the airway epithelium, particularly in Clara cells, in the more distal regions of the airways was also detected. Following exposure to low-level DEE, RSV infection caused a moderate decrease in proSP-B staining in alveolar regions of the lung, with increased staining in resident inflammatory cells. Following exposure to high-level DEE, proSP-B staining in alveolar regions was further diminished as compared with prior exposure to low-level DEE. As described previously, proSP-B staining was associated with inflammatory cells in alveolar regions of the lung following RSV infection. Furthermore, proSP-B staining in the airway epithelium following exposure to high-level DEE and subsequent RSV infection was diminished as compared with RSV infection alone or low-level DEE exposure and RSV infection.
Decreased SP-A during RSV Infection following DEE Exposure SP-A is a member of the collectin family of host defense proteins and an important opsonin for bacteria and viruses infecting the lung. To assess the effect of prior inhaled exposure of DEE on SP-A levels at 4 d following RSV infection, immunohistochemical analysis of SP-A was performed and analyzed under light microscopy (Figures 7A and 7B). SP-A staining in either distal airways or alveolar type II cells was not altered by DEE exposure alone (Figure 7A). SP-A staining in alveolar type II cells and airway epithelium was readily detectable in the lungs of untreated and RSV-infected, air-treated mice, with no discernible changes in SP-A levels. In comparison, prior exposure to high- and low-level DEE decreased SP-A staining in alveolar type II cells and airway epithelial cells during RSV infection (Figure 7B). Differences in SP-A staining were not apparent between the DEE exposure levels. Changes in SP-A staining in the airway epithelium were concordant with the altered airway epithelial cell morphology to pseudostratified and/or columnar cell shape. Changes in alveolar type II cell morphology were not apparent in association with decreased SP-A staining.
The impact of ambient air pollution on acute respiratory disease has not been fully elucidated. Given the extent to which engine emissions contribute to poor air quality, particularly in urban settings, this study examined the role of inhaled DEE in the altered susceptibility to subsequent respiratory viral infection. The levels of inhaled DEE used in the current study represent plausible human exposure concentrations found in ambient settings (5, 21). Furthermore, the current study examined the impact of air pollutants on susceptibility to RSV, a common viral pathogen that infects nearly every child in infancy and early childhood (3). Herein, prior exposure to inhaled DEE increased RSV gene expression in the lungs of mice. Increased lung inflammation and morphologic changes in the airways were concordant with increased viral transcriptional activity. Markers of airway epithelial function indicated increased mucus response to infection and decreased levels of immunomodulatory proteins, such as expression of CCSP and SP-A. Furthermore, increased lung injury and diminished host defense mechanisms to infection were exacerbated by prior exposure to DEE. These findings are consistent with our hypothesis that inhaled DEE exposure increases the susceptibility to lung disease in response to respiratory viral infection in vivo. Recently published results have shown that C57Bl/6 strain mice are relatively resistant to RSV infection, having markedly diminished lung viral titers as compared with other, more susceptible strains such as AKR or 129/J strain mice (22). Peak RSV titers in the lungs of multiple strains of mice typically occur at Day 4 after infection, as well as in other experimental animal species, with greatly reduced lung viral titers found at Days 2 and 6 after inoculation (13, 23, 24). Mice were used in the current study because of the ease in performing exposure studies in a small rodent model. Although the study of human RSV in an unnatural host may have limitations, mouse studies of human RSV infection have been useful for delineating immune mechanisms of pathogenesis to RSV vaccines (25). The use of natural rodent paramyxoviruses, such as Sendai virus, may yield new information regarding altered host responses during respiratory viral infection by DEE exposure. Furthermore, the use of more susceptible mouse strains to RSV, such as AKR or 129J strains, may yield information regarding changes in adaptive immune responses to RSV following DEE exposure. In the current study, viral gene expression was detectable in the lungs of air-exposed C57Bl/6 mice following infection, albeit at low levels. Following exposure to inhaled DEE, RSV gene expression was increased in the lungs of C57Bl/6 mice at Day 4 after infection. The RSV genes F and G were both readily detectable in the lungs of DEE-exposed mice, suggesting that increased viral gene expression was not limited to a particular viral gene subset, but likely reflects an increase in viral replication and/or persistence. Viral gene expression was used in the current study as an indirect assessment of viral lung burden, and has the advantage of allowing repeated analysis when compared with traditional plaque titer assays, which, in the case of RSV, are severely attenuated by repeated freezing and thawing of samples. Both low (30 µg/m3 PM) and high (1,000 µg/m3 PM) DEE exposure levels increased lung viral gene expression at 4 d after infection, with no apparent difference between inhaled DEE exposure levels. The increase in lung viral gene expression during infection by prior exposure to inhaled DEE may result from increased persistence of the initial viral inoculate or diminished antiviral innate mechanisms of viral clearance in the lung. The host molecular determinants that regulate RSV infection and replication have not been fully elucidated at the molecular level in in vivo settings.
Lung inflammation and proinflammatory mediators were increased in the lung following RSV infection by prior exposure to inhaled DEE. The increase in lung inflammation coincided with the increase in lung RSV gene expression following inhaled DEE exposure. Previous studies have shown an association with increased viral persistence and increased lung inflammation in mice rendered more susceptible to respiratory infection. In SP-A-/- gene-targeted mice, lung inflammation is increased concordant with decreased clearance of RSV (4), as well as adenovirus and influenza A infection of the respiratory tract (26, 27). Not surprisingly, proinflammatory mediators such as TNF- RSV-related disease in the respiratory tract primarily involves aberrant airway epithelial responses. In addition, the deposition of inhaled particles, such as those found in inhaled engine emissions, likely occurs in the airway epithelium. Following exposure to inhaled DEE, airway-associated lung inflammation to RSV was increased in a manner dependent upon the level of DEE exposure. Furthermore, airway epithelial cell morphology was distinctly altered in the lungs of DEE-exposed, RSV-infected mice, involving proximal and distal airways. Concordant with altered airway epithelial cell changes was the induction of extensive MCM to RSV by prior DEE exposure. In the case of high-level DEE exposure, MCM was observed in more distal airway regions of the lung. Undoubtedly, lung inflammation and inflammatory mediators contributed to the induction of MCM in the airways. It is unclear how persistent viral gene expression in the current setting may contribute to airway epithelial remodeling and mucus responses to RSV exacerbated by inhaled DEE.
Nonciliated bronchiolar epithelial cells, otherwise known as Clara cells, comprise the largest percentage of the distal airway epithelial cell population (32). Although their function is not well understood, Clara cells secrete proteins into the lumen of the airways that may have important host-defense or immunomodulatory functions. CCSP is an abundant, 10- to 16-kD protein secreted by Clara cells with important immunoregulatory functions (33). Our previous studies have shown that CCSP deficiency in gene-targeted mice produces increased inflammatory responses to respiratory infections, including RSV (18, 34–36). Likewise, Clara cells produce other molecules important in host defense, including SP-A (33). In the current study, DEE exposure reduced CCSP and SP-A in the airway epithelium following RSV infection, whereas infection alone had little or no effect. Importantly, a mild decrease in CCSP abundance in distal airways was detected in mice exposed to high-level DEE before RSV infection, whereas SP-A staining did not appear to be affected. As stated previously, SP-A deficiency can render the lung more susceptible to respiratory viruses (4, 26, 27). The importance of decreased CCSP and SP-A, collectively as well as independently, in the current experimental setting will require further study and may reflect altered airway epithelial cell function to RSV. Indeed, TNF- In the current study, alveolar surfactant protein responses were altered during RSV infection by prior exposure to inhaled DEE. Both proSP-B and SP-A were diminished in alveolar type II cells, whose primary function is the production and regulation of surfactant homeostasis in the pulmonary alveoli. Furthermore, the appearance of proSP-B and SP-A staining in alveolar phagocytes and inflammatory cells suggests aberrant regulation of type II cell secretion and/or production of surfactant. Similar features of altered surfactant protein regulation have been observed in other inflammatory settings in alveoli (19, 38).
The induction of TNF- The exposure concentrations used in this study represent a range of plausible human exposures in occupational and environmental settings to diesel DEE. The high-level DEE is consistent with published exposure concentrations in occupations such as miners and forklift operators (42). The low-level DEE exposure used herein is in the range of an occupational exposure or a high-end environmental exposure in circumstances such as driving on busy freeways or standing on street corners near bus stops impacted by idling buses (42). Although the exposures used in the current study are based on human exposure levels, the extrapolation of human levels to rodents is difficult to determine. The composition of the exposures generated for the current studies was consistent with that of diluted DEE generated in laboratory settings (43).
Previous investigations have assessed the host response and antimicrobial defenses against PM and filter-captured engine emission componenets, having shown that bolus instillation of these components can attenuate bacterial clearance by lung alveolar macrophages (8–11, 44) and altered responses to allergens (45, 46). With respect to viral infections, exposure to DEE can exacerbate the host response to infection and diminish clearance of influenza virus (47, 48). In addition, exposure to ambient air particles may induce lung injury and inflammation per se (5). The results shown herein indicate that a mild but discernible induction of lung inflammation was detectable with inhaled DEE exposure before RSV infection. In contrast to previously reported studies, however, the proinflammatory cytokines TNF- In the present studies, prior exposure to inhaled DEE exacerbated the lung inflammation and injury to subsequent RSV infection, rendering a relatively resistant mouse strain more susceptible to RSV-induced lung disease. RSV gene expression was increased in the lungs following DEE exposure, concurrent with increased lung inflammation and altered airway epithelial cell remodeling. Airway and alveolar epithelial markers indicate that both lung compartments were affected by DEE exposure, including mucous cell responses and surfactant protein homeostasis during RSV infection. These studies present novel findings regarding the impact of DEE on pulmonary responses to a common viral pathogen. Furthermore, the findings herein may provide insight into the pulmonary epithelial determinants that regulate the host response to RSV infection and exposure to ambient air pollutants.
The authors wish to thank the National Environmental Respiratory Center (NERC) for operation of the diesel engine exposure systems. Richard K. White constructed the exposure system under the supervision of Edward B. Barr. Jose Madrid, Nick Sylvas, and Terry Zimmerman operated the system. This work was performed in conjunction with the NERC, at the Lovelace Respiratory Research Institute, with funds from multiple government and industry sponsors, including the US EPA. This article is not intended to represent the views or policies of any NERC sponsor. This work was also funded in part by a Pilot Project Award from the NIEHS Development Center Grant (P20-ES09781-04) (K.S.H.); the Health Effects Institute, an organization jointly funded by the US EPA, and automotive manufacturers (K.S.H); and the National Institutes of Health grant HL-66964 (K.S.H.). Lovelace Respiratory Research Institute is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Received in original form July 2, 2002 Received in final form October 2, 2002
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Abstract
Introduction
0.05. For non-normal distributed data sets, statistical analysis using nonparametric tests or logarithmic transformation of data to normal distribution sets and subsequent ANOVA was performed. 
