Inhaled Diesel Engine Emissions Reduce Bacterial Clearance and Exacerbate Lung Disease to Pseudomonas aeruginosa Infection In Vivo

Kevin S. Harrod*,1, Richard J. Jaramillo*, Jennifer A. Berger*, Andrew P. Gigliotti{dagger}, Steven K. Seilkop{ddagger} and Matthew D. Reed{dagger}

* Asthma and Pulmonary Immunology Program, {dagger} Experimental Toxicology Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico and {ddagger} SKS Consulting Services, Siler City, North Carolina

1 To whom correspondence should be addressed at Infectious Disease Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr. SE, Albuquerque, NM 87108. Fax: (505) 348-8567. E-mail: kharrod{at}LRRI.org

Received April 20, 2004; accepted September 26, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite experimental evidence supporting an adverse role for air pollution in models of human disease, little has been done in the way of assessing the health effects of inhalation of whole mixtures from defined sources at exposure levels relevant to ambient environmental exposures. The current study assessed the impact of inhaled diesel engine emissions (DEE) in modulating clearance of Pseudomonas aeruginosa (P.a.) and the adverse effects of infection to the pulmonary epithelium. At DEE concentrations representing from high ambient to high occupational exposures, mice were exposed to DEE continuously for one week or six months (6 h/day), and subsequently infected with P.a. by intratracheal instillation. At 18 h following P.a. infection, prior exposure to DEE impaired bacterial clearance and exacerbated lung histopathology during infection. To assess the airway epithelial cell changes indicative of lung pathogenesis, markers of specific lung epithelial cell populations were analyzed by immunohistochemistry. Both ciliated and non-ciliated airway epithelial cell numbers were decreased during P.a. infection by DEE exposure in a concentration-dependent manner. Furthermore, the lung transcription regulator, thyroid transcription factor 1 (TTF-1), was also decreased during P.a. infection by prior exposure to DEE concordant with changes in airway populations. These findings are consistent with the notion that environmental levels of DEE can decrease the clearance of P.a. and increase lung pathogenesis during pulmonary bacterial infection.

Key Words: respiratory infection; pulmonary toxicology; air pollution.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute respiratory infections are the most common cause of illness and disease worldwide. Increasing epidemiologic evidence strongly correlates respiratory disease with episodes of poor air quality or proximity to major roadways (Brauer et al., 2002Go; Dockery and Pope, 1994Go). Hospitalizations, physician visits, school absenteeism, and asthmatic episodes have shown an association with elevated levels of particulate matter (PM) or ozone in urban settings (Dockery and Pope, 1994Go; Pope, 1991Go; von Klot et al., 2002Go; Ware et al., 1986Go; Zanobetti et al., 2000Go). Furthermore, numerous epidemiologic studies have found that hospital admissions for pneumonia, asthma, and bronchitis—diseases that are strongly associated with respiratory infections—are increased during periods of increased ambient air pollutants (Dockery and Pope, 1994Go; Pope, 1991Go; Ware et al., 1986Go; Zanobetti et al., 2000Go). Both epidemiologic and experimental data suggest that air pollutant components likely exacerbate disease to respiratory infection (Gilmour et al., 2001Go; Jakab, 1993Go; Pope, 1991Go; Schwartz, 1994aGo,bGo).

In urban settings, engine emissions constitute a major portion of air pollution (Lloyd and Cackette, 2001Go). Diesel engine emissions (DEE) are a complex and varied mixture containing organic and inorganic emissions in gas, vapor, semi-volatile, and PM phases, and these components have been shown to cause health effects at various levels in rodent models (Cohen and Nikula, 1999Go). To begin to define the molecular and cellular mechanisms by which DEE may exacerbate respiratory infection, studies were performed using a bacterial infection model following inhalation exposure to DEE. Concentrations of emissions were used that represent plausible human exposure concentrations, including occupational and environmental exposures. Furthermore, a relevant bacterial pathogen to respiratory disease, Pseudomonas aeruginosa (P.a.), an important causative pathogen in antibiotic-resistant, community-acquired pneumonia in the elderly, was used as the infective agent (Bodey et al., 1983Go; El-Solh et al., 2002Go). The use of 109 colony forming units (cfu) is a standard inoculum of P.a. and has been used experimentally. Despite the large inoculum, this represents a sublethal challenge to mice and mice fully recover, as we have published previously (Hayashida et al., 2000Go). Inhaled DEE decreased the clearance of P.a. and increased lung inflammatory responses concordant with airway epithelial cell changes during infection.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male C57Bl/6 mice were purchased from Charles River Laboratories (Raleigh, NC) and were approximately 10–12 weeks of age at the beginning of exposure. Mice (n = 10 for each exposure condition including filtered air [FA] control exposure) were housed in Hazleton H2000 exposure chambers (Hazleton Systems, Maywood, NJ) for the duration of a two-week quarantine period and throughout exposures. Mice were fed Teklad certified rodent diet (Harlan Teklad, Madison, WI) ad libitum except during the 6-h exposure period. Water was available ad libitum throughout the duration of the study. Mice from each experimental group were serologically tested (BioReliance, Rockville, MD) and confirmed negative for common rodent pathogens, including CAR bacillus (CARB), epizootic diarrhea of infant mice (EDIM), Theiler's mouse encephalomyelitis virus (TMEV/GDVII), lymphocytic choriomeningitis virus (LCM), mouse hepatitis virus (MHV), Mycoplasm pulmonis (M. pol.), mouse parvovirus (MPV, MVM), pneumonia virus of mice (PVM), and Sendai virus (Sendai).

Diesel exhaust generation, exposures, and characterization. Diesel exhaust was generated alternately from two 2000 model 5.9-l Cummins ISB turbo diesel engines using certification diesel fuel (No. 2 National Certification Fuel, Chevron Phillips Chemical Co., Borger, TX) and 15W-40 lubrication oil (Rotella T, Shell, Houston, TX). Crankcase oil and filters (Fleetguard LF3349, Cummins, Columbus, IN) were changed after every 200 h of engine operation. The engines were mated to eddy current dynamometers (Alpha-240, Kiel, FRG) and operated on repeated, slightly modified transient engine cycles based on the U.S. Environmental Protection Agency (EPA) Engine Dynamometer Schedule for Heavy-Duty Diesel Engines (U.S. Code of Federal Regulations, Title 40, Appendix I). Further details on the engine/exposure system and the duty cycle are described elsewhere (McDonald, 2001Go; McDonald et al., 2004Go; Reed et al., 2004Go).

Exposures were conducted in 2 m3 H2000 Hazleton flow-through exposure chambers operated at a flow rate of 500 l/min as described previously (McDonald, 2001Go; McDonald et al., 2004Go; Reed et al., 2004Go). The exposures included five treatment groups: four dilutions of engine exhaust and high-efficiency particulate air (HEPA)-filtered clean air (control). Target exposures were set at 30 (low), 100 (mid-low), 300 (mid-high), and 1000 (high) µg/m3 PM. Exposures were conducted seven days per week, 6 h/day. Mice were exposed to DEE for either one week or six months. The one-week exposure study was repeated on a separate occasion. The exposure atmosphere was characterized for over 1000 separate analytes, and the summary results have been reported in detail elsewhere (McDonald, 2001Go; McDonald et al., 2004Go; Reed et al., 2004Go). Studies of uninfected air- and DEE-exposed mice (n = 6–8 per group) were conducted in parallel with the studies of P.a. infection. Samples from uninfected groups were acquired immediately following the final exposure period.

Clinical observations. Mice were examined for abnormal clinical signs twice daily, before and after exposure. More detailed clinical examinations were performed prior to the beginning of exposure, weekly for the first month of exposure, and monthly thereafter.

Instillation of P.a. Immediately following the final DEE exposure, mice were anesthetized with a mixture 5% halothane in 50:50 carrier gas mixture of nitrous oxide and oxygen and 1 x 109 cfu of P.a. in 100 µl of phosphate-buffered saline (PBS) administered intratracheally as previously described (Hayashida et al., 2000Go). P.a. was generated from a frozen culture of a mucoid strain of a clinical isolate.

Lung bacterial titer analysis. Lungs were acquired at 18 h following instillation of bacteria for analysis of lung bacterial titers. Homogenates of the right apical and cardiac lung lobes were serially diluted to three dilutions and plated onto 2 x YT agar plates, thus yielding bacterial titer data from three dilutions for each animal. Bacterial plates were incubated at 37°C overnight. Following colony formation (18–24 h), colonies were counted for each dilution, and the bacterial concentration per ml of lung homogenate was determined.

Lung histology and immunohistochemical analysis. Right lung lobes were inflation-fixed with 4% paraformaldehyde in PBS and embedded in paraffin blocks as described previously (Harrod et al., 2003Go). Lung sections (5 µm) were taken starting 100 µm from a designated reference point, and lung sections were collected at 100 µm intervals. Lung pathology was assessed on hematoxylin and eosin-stained sections and scored on a severity scale from 0–4 (normal = 0, minimal = 1, mild = 2, moderate = 3, marked = 4) for the following parameters: suppurative alveolitis, perivascular hemorrhage, perivascular cuffing, and bronchiolitis and/or peribronchiolar cuffs. In addition, black/brown particulate material within alveolar macrophages was assessed but excluded in the histopathology scoring analysis.

For immunohistochemical analysis of ß-tubulin, SCGB1A1, and TTF-1, lung sections were stained as previously described (Harrod et al., 2003Go). The SCGB1A1 primary antibody was a generous gift from Barry Stripp, University of Pittsburgh. The ß-tubulin and TTF-1 primary antibodies were purchased from Biogenex (San Ramon, CA) and Novacastra (Newcastle upon Tyne, U.K.), respectively. For quantitative analysis of immunohistochemical staining, duplicate slides from 4–6 animals per experimental group were selected randomly, and 8–10 airways were analyzed. For analysis of large airways, typically 2–3 were analyzed per lung section. Staining intensity was assessed using MetaMorph software 6.1 (Universal Imaging Corp., Downington, PA) and normalized to basal lamina length in mm. Statistical significance of immunohistochemical scores was determined by analysis of variance of mean scores and Tukey's and Bonferroni post hoc tests for pair-wise comparisons.

Statistical analysis. Bacterial clearance and lung histopathology data were screened prior to statistical analyses for normal distribution, and parametric or non-parametric statistical analyses applied accordingly. Animals instilled with bacteria but having no observed lung bacterial counts and no corresponding histopathology were removed from statistical analyses under the assumption that bacterial instillation was not successful in these animals. This represented 3% of the animals involved in these studies.

Bacterial count data, which exhibited non-parametric distribution and standard deviations that scaled with sample means, were log transformed, after adding 0.5 to each count (in order to include zero counts in the analysis). An analysis of variance (ANOVA) was performed on the log-transformed data, followed by Dunnett's multiple comparison procedure (Dunnett and Crisafio, 1955Go). For the two 1-week exposure blocks, bacterial counts differed significantly between the two exposure blocks (p ≤ 0.001), and the results were analyzed by blocked ANOVA to accommodate these temporal differences. This analysis yields an appropriate measure of variability to test differences in means pooled across the two exposure blocks, which is the basis for the estimated confidence limits on the mean values that are presented.

Histopathology data were scaled according to severity. Weighted least squares analysis of variance (Grizzle et al., 1969Go) was applied to the graded response data. Because there was clear evidence of a different pattern of response in the two 1-week exposure blocks, separate one-way ANOVAs were performed for each block. The CATMOD procedure (SAS, Cary, NC) was utilized to perform the analyses. Comparisons of mean scores in exposed versus control groups within each block were adjusted for multiple comparisons using the modified Bonferroni procedure. A summary histopathology score was computed as the mean of the scores for all findings except black/brown particulates within alveolar macrophages.

Statistical significance was assessed at p < 0.05 and p < 0.01 (two-tailed). The statistical criteria that were used to determine a significant exposure-related effect required both a significant (p < 0.05) trend across exposure groups (including controls) and mean values for one or both of the two highest exposure groups that differed significantly (p < 0.05) from the control group mean. ANOVA was used to assess exposure-related trends in response across control and exposed groups.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Clearance
Lung bacterial clearance was measured 18 h following intratracheal instillation of P.a. to the respiratory tract. P.a. was rapidly cleared from the lungs of control mice, as indicated by the marked reduction in bacterial titers in the lungs of control mice at 18 h following infection (Fig. 1A). One-week exposure at all levels of DEE decreased lung bacterial clearance as compared to controls. Exposure to 30 and 100 µg/m3 PM DEE caused a concentration-related decrease in lung bacterial clearance at 18 h following infection. Increased concentrations of DEE at 300 or 1000 µg/m3 PM did not further exacerbate lung bacterial clearance. Parametric and non-parametric statistical analyses indicated a concentration-related effect with regard to lung bacterial clearance as well as an overall DEE effect versus the control group.



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 1. DEE exposure reduces P.a. clearance. P.a. lung titers were assessed 18 h following intratracheal infection in mice exposed to DEE for one week (6 h/day) (A) and six months (B). DEE exposure markedly decreased lung P.a. clearance in mice exposed for one week but not six months. Data represent bacterial counts per ml of lung homogenate. n = 20 for each group in one-week data, and n = 10 per group for the six-month data. Asterisks indicate statistically significant differences (*p ≤ 0.05, **p ≤ 0.01) from control group geometric means as assessed by Dunnett's multiple comparison procedure. Trend 1 wk, p = 0.002, six months p = 0.453. p values for trends of all treatment groups as measured by the linear term of the ANOVA.

 
Similar studies were performed in mice with a six-month exposure to DEE and subsequent infection with P.a. Lung bacterial clearance was analyzed 18 h following infection. Exposure to DEE at all concentrations did not appreciably affect lung bacterial clearance at 18 h following infection (Fig. 1B). As compared to one-week exposures, lung bacterial counts in all groups were substantially higher (greater than ten-fold), likely indicating an age-related effect with regard to bacterial clearance. In light of these results, six-month exposures were not studied hereafter.

Lung Histopathology
Lung inflammation and lung histopathology were assessed in histologically stained lung sections under light microscopy. Lung inflammation and pathology were not apparent in the lungs of uninfected DEE exposed or control animals (data not shown). At 18 h following infection, lung inflammation and histopathology were markedly apparent in all mice in all exposure groups following infection, including the air control group (Fig. 2). Both cumulative scores for all histopathology criteria and summary scores for each histological criterion were increased in the lungs of mice exposed to DEE at all exposure concentrations as compared to that of air-exposed, infected animals. A concentration-related effect of DEE on lung inflammation and lung histopathology was not apparent as scores between DEE-exposed groups were not different. Lung inflammation was diffuse and consisted primarily of neutrophils with resident alveolar macrophages noted to a lesser degree. Inflammation was observed around both large and small bronchi and bronchioles, surrounding blood vessels, and in surrounding parenchymal tissue. Focal regions of neutrophilic consolidation were apparent in all tissues but appeared more frequently in groups exposed to higher DEE concentrations.



View larger version (94K):
[in this window]
[in a new window]
 
FIG. 2. Lung histopathology and lung inflammation are increased during P.a. infection by prior exposure to DEE. Lung histology (5 µm was scored by the histological criteria listed in the Materials and Methods section. Lung inflammation was increased from P.a. infection following exposure to DEE at any level. Photomicrographs shown are representative of air-exposed (sham), P.a.-infected (A and D); 30 µg/m3 PM-exposed, P.a.-infected (B and E); and 1000 µg/m3 PM-exposed, P.a.-infected (C and F). Both low magnification (A–C) at ~100x and high magnification (D–F) at ~400x are shown.

 
Summary of the cumulative scoring and the individual scoring parameters are shown in Figures 3A and 3B. Generally, histopathology scores following P.a. infection exhibited an exposure dependent increase at lower DEE concentrations (30 and 100 µg/m3 PM) as compared to that of air-exposed, P.a.-infected lungs, with either little further change or a reduction at higher DEE concentrations (300 and 1000 µg/m3 PM). The scoring of individual parameters within each exposure condition did not differ substantially from the cumulative scores for each group. The scores of lung histopathology from uninfected animals following DEE exposure did not reach statistical significance by pair-wise or trend analyses as compared to that of the air control group.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 3. Scoring of histopathology in mice exposed to DEE. Lung histopathology scoring (Scale 0–4) is shown for a total summary score (A) and with each individual scoring criteria (B). DEE exposure increased lung histopathology scores in all DEE-exposed groups as compared to controls following P.a. infection. Cumulative scores of all DEE-exposed, P.a.-infected groups was statistically significant (n = 10 per group) versus control groups (data not shown). Asterisks indicate statistically significant differences (*p ≤ 0.05, **p ≤ 0.01) from control group as assessed by weighted least square ANOVA and Dunnett's multiple comparison procedure. Trend (A) Summary p = 0.006 (B) Bronchiolitis p = 0.748, Perivascular hemmorage p = 0.001, Perivascular cuffing p = 0.119, and Suppurative alveolitis p = 0.016. No significant trends (p ≤ 0.05) were observed in uninfected exposed vs. control groups. p values for trends of all treatment groups as measured by the linear term of the ANOVA.

 
Particle Deposition
Particle deposition in the lungs of DEE-exposed, uninfected mice at one week of exposure was assessed by light microscopy on lung sections acquired immediately following the final DEE exposure period. Particle deposition was apparent in lung phagocytes of all groups exposed to DEE (Fig. 4). Furthermore, a strong concentration-related effect was observable with greater numbers of particle-laden macrophages in higher DEE concentration groups. DEE particles in lung phagocytes were readily apparent in the 30 µg/m3 PM DEE exposure group.



View larger version (38K):
[in this window]
[in a new window]
 
FIG. 4. Particle deposition in the lungs of DEE-exposed mice prior to infection with DEE. Histological sections (5 µm) were assessed in the lungs of air or DEE-exposed mice prior to P.a. infection. Lungs exposed to filtered air (A) had no observable particle deposition in lung phagocytes (arrows). Both DEE exposure at 30 µg/m3 PM (B) and 1000 µg/m3 PM (C) were found to have observable particle deposition in lung phagocytes (arrowheads). Representative micrographs at high magnification (~400x) are shown.

 
Ciliated Cell Changes in the Airway Epithelium
Mucociliary clearance is an important mechanism in the clearance of foreign particles and pathogens from the lungs, including P.a. As such, ciliated cells play an important role in host defense against bacterial infection. To assess the effect of DEE on ciliated cell populations during infection, immunohistochemical staining of ß-tubulin, a protein component of ciliary axonemes in the airway epithelium, was assessed in both the proximal and distal airways of mice exposed to DEE for one week prior to P.a. infection. Ciliary structures were readily apparent in the lungs of uninfected, control mice (Figs. 5A and 5E). ß-tubulin staining was not altered in either large or small airways in the lungs of control mice following infection (Figs. 5B and 5F) as compared to uninfected control mice. Exposure to 30 µg/m3 DEE PM decreased ciliated cell populations in large airways of P.a.-infected mice (Fig. 5C), but no changes were observed in small airways (Fig. 5G). An even greater reduction in ciliated cell populations was observed in the lungs of P.a.-infected mice following exposure of 1000 µg/m3 PM DEE (Fig. 5D). Furthermore, ß-tubulin staining was markedly decreased in the small distal airways of infected mice exposed to 1000 µg/m3 PM DEE (Fig. 5H). Changes in ciliated cell populations were more discernible in airways with marked bronchiolar inflammation as compared to those airways with lesser degrees of bronchiolar inflammation. Quantitative analysis of ß-tubulin staining is shown for both large (Fig. 5I) and small airways (Fig. 5J).



View larger version (69K):
[in this window]
[in a new window]
 
FIG. 5. Ciliated cell populations are diminished during P.a. infection by prior exposure to DEE. Lung sections (5 µm) were stained by immunohistochemical techniques for ß-tubulin. ß-tubulin (red) staining is readily apparent in both large (A–D) and small (E–H) airways. The lungs of air-treated, control mice (A and E) indicate abundant ciliated cell populations in the airway epithelium. Air-exposed P.a.-infected airways exhibit no apparent alteration in ciliated cell populations (B and F). DEE exposure at 30 µg/m3 PM (C and G) or 1000 µg/m3 PM (D and H) exhibited decreased ciliated cell populations in both proximal and distal airways following P.a. infection. Ciliated cell numbers are decreased by DEE exposure as indicated by quantitative scoring (mean ± SE) of ß-tubulin staining in either large (I) or small (J) airways. For micrographs A–D magnification is ~400x; for micrographs E–H magnification is ~200x. *Denotes statistical significance at p ≤ 0.05 by ANOVA.

 
Non-Ciliated Bronchiolar Epithelial Changes
Non-ciliated bronchiolar (Clara) cells comprise a substantial portion of the airways of rodents and secrete a number of host defense or immunomodulatory proteins (Singh and Katyal, 1997Go; Wright, 1997Go). To assess Clara cell injury in the lungs of mice following DEE and P.a. infection, the level of the Clara cell marker secretoglobin 1A1 (SCGB1A1, also known as CCSP, CC-10, or CC-16) was determined by immunohistochemical staining. SCGB1A1 was decreased in the lungs of P.a.-infected air control mice as compared to SCGB1A1 staining in the lungs of uninfected animals (Figs. 6A and 6B), in concordance with previously published data (Hayashida et al., 2000Go). Exposure to DEE reduced SCGB1A1 staining following P.a. infection as compared to control animals following infection (Figs. 6C and 6D). Decreased SCBG1A1 was observed in both low DEE exposure (30 µg/m3 PM) and high DEE exposure (1000 µg/m3 PM) concentrations to a relatively equal extent. Altered staining was most notable in distal airway epithelial cells as opposed to proximal airways. Reduced staining was observed concordant with altered Clara cell morphology from a normal cuboidal appearance to a flattened, squamous appearance (Figs. 6E and 6F). Quantitative scoring of SCGB1A1 staining is shown in Figure 6G.



View larger version (41K):
[in this window]
[in a new window]
 
FIG. 6. SCGB1A1 levels are decreased in the distal airway epithelium in P.a.-infected mice following DEE exposure. Lung sections (5 µm) were stained by immunohistochemistry for SCGB1A1, a marker of non-ciliated bronchiolar epithelial cells. SCGB1A1 levels were readily apparent in air-exposed, control mice (A). P.a. infection of air treated mice (B) caused a mild but discernable decrease in CCSP staining. DEE exposure decreased SCGB1A1 staining at both 30 µg/m3 PM (C) and 1000 µg/m3 PM (D) exposure concentrations as indicated by quantitative scoring (G). *Denotes statistical significance at p ≤ 0.05 by ANOVA. Altered Clara cell morphology, observed as a flattened appearance, during P.a. infection is shown in lung sections from air control, uninfected animals (E) and in lung sections from mice following 30 µg/m3 PM DEE exposure and P.a. infection (F). Representative micrographs are shown at ~200x magnification (A–D) and at ~600x (E and F).

 
Thyroid Transcription Factor-1 in the Airway Epithelium
Thyroid transcription factor-1 (TTF-1, also Nkx 2.1) is a critical regulator of lung-specific gene expression, including a number of lung-specific genes that have homeostatic and host defense functions (Korfhagen et al., 1996Go). To assess changes in Clara cell transcriptional activity by DEE exposure in the lungs of infected mice, immunohistochemical staining of TTF-1 levels was determined. TTF-1 was readily apparent in the lungs of naïve animals following air exposure (data not shown). Characteristic nuclear localization of TTF-1 following P.a. infection was mildly decreased but readily apparent following P.a. infection in control animals (Fig. 7A). DEE exposure markedly decreased TTF-1 staining in the airway epithelium of mice following P.a. infection. Low (30 µg/m3 PM) DEE exposure prior to infection caused only a mild decrease in TTF-1 staining (Fig. 7B). TTF-1 expression in alveolar epithelial cells was also markedly reduced (data not shown). High (1000 µg/m3 PM) DEE concentrations induced a further decrease in TTF-1 in the distal airways of P.a.-infected mice (Fig. 7C) as indicated by quantitative scoring of TTF-1 staining (Fig. 7D). DEE exposure did not alter TTF-1 staining in uninfected animals (data not shown).



View larger version (97K):
[in this window]
[in a new window]
 
FIG. 7. TTF-1 abundance is decreased during P.a. infection prior to DEE exposure. Lung sections (5 µm) were stained by immunohistochemistry for the lung transcriptional factor TTF-1. TTF-1 is readily apparent in the airway epithelium of air treated, P.a.-infected lungs (A). DEE exposures at 30 µg/m3 PM (B) or 1000 µg/m3 PM (C) exposures decreased TTF-1 staining in the distal airway epithelium. Representative micrographs are shown at ~200x magnification. Mean scores of quantitative TTF-1 staining are shown (D). *Denotes statistical significance at p ≤ 0.05 by ANOVA.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prior exposure to DEE impaired P.a. clearance from the lungs of mice in a concentration-dependent manner. Likewise, lung inflammation and morphologic changes in the airway epithelium were increased concordant with impaired bacterial clearance. Markers of airway epithelial cell populations indicated alterations in both ciliated and non-ciliated bronchiolar epithelial cell populations following DEE exposure, suggesting enhanced airway epithelial cell injury. Furthermore, TTF-1, an important transcriptional regulator of the lung epithelium, was decreased in P.a. infection by prior exposure to DEE. These findings indicate that inhaled DEE increases susceptibility to lung bacterial infection, and augments lung inflammation and pulmonary epithelial cell changes.

P.a. is a common, gram-negative bacteria that causes a variety of pulmonary infections in compromised hosts and is a common cause of nosocomial infections (Bryan and Reynolds, 1984Go). The dose of P.a. chosen for these studies represents a standard, sublethal experimental inoculum (Hayashida et al., 2000Go; LeVine et al., 1998Go). Despite the large dose of P.a. typically used in experimental studies, P.a. is rapidly cleared from the lungs of mice, as indicated by the substantially lower bacterial titers (in the range of 103 cfu) at 18 h following instillation. The results presented here demonstrate effective clearance of P.a. by murine host defenses with impaired bacterial clearance from the lungs of mice following exposure to DEE.

In the current study, P.a. clearance was diminished by a one-week exposure to DEE. Overall, both concentration-related and exposure-related effects (i.e., air versus DEE at all levels) on bacterial clearance were observed. Importantly, impaired bacterial clearance was observed at the lowest DEE exposure level group studied. These findings are consistent with findings from a previous study in which viral clearance and viral pathogenesis in the lung were altered by inhaled DEE at the same lowest exposure level (Harrod et al., 2003Go). The lowest exposure condition used in both studies, 30 µg/m3 PM DEE, represents a plausible ambient exposure level of inhaled air pollutants, particularly in urban settings during peak episodes of poor air quality (Cohen and Nikula, 1999Go). Similar findings have also been reported using other bacterial pathogens following exposure to inhaled air particulates or instillation of air pollutant PM, albeit at higher bolus concentrations (Castranova et al., 2001Go; Hatch et al., 1985Go; Jakab, 1993Go; Yang et al., 2001Go).

Lung inflammation was increased during P.a. infection by prior exposure to inhaled DEE. The increased lung inflammation and histopathology coincided with the decrease in lung bacterial clearance following inhalation exposure. Lung inflammation was primarily associated with bronchial and bronchiolar regions of lung; however, at higher DEE concentration exposures, lung histopathology was also associated with neutrophilic infiltration into the lung parenchyma. Neutrophil infiltration, the hallmark feature of lung disease to P.a. infection, is commonly associated with damage to the pulmonary epithelium, commonly through secretion of cytotoxic substances from neutrophils (Weiss, 1989Go). Increased lung inflammation to P.a. infection was associated with altered morphology of the airway epithelium during P.a. infection by prior DEE exposure. An additional histological finding was the observation of particle deposition in the lung phagocytes following DEE exposure. Particle deposition was apparent in the lungs of mice exposed to the lowest DEE concentration, 30 µg/m3 PM, indicating that environmentally relevant concentrations of engine emissions may produce an observable histological effect in the respiratory tract.

Ciliated bronchiolar epithelial cells comprise the largest percentage of the epithelial cell populations in the proximal airways. Ciliated cells, while likely performing many biological functions, are primarily thought to facilitate mucociliary clearance mechanisms through the biophysical action of ciliated membranes on the luminal surface. In the current study, cilia staining on the luminal airway surface of the airway epithelium cells was decreased by prior DEE exposure during bacterial infection. The findings in this study do not address alterations in mucociliary function, although these results could be consistent with decreased ciliary function in the airways of DEE-exposed lungs. Regardless, decreased ciliated cell numbers are strongly suggestive of airway epithelial injury and/or remodeling following exposure to DEE.

Non-ciliated bronchiolar epithelial cells, otherwise known as Clara cells, comprise the largest percentage of epithelial cells in the distal airways (Singh and Katyal, 1997Go). In light of the increased epithelial injury following P.a. infection by prior DEE exposure, two markers of Clara cells, SCGB1A1 (also known as CCSP) and TTF-1 were assessed. Previous studies have shown that CCSP deficiency in gene-targeted mice produces increased inflammatory responses to respiratory infections, including P.a. (Hayashida et al., 2000Go). In the current study, DEE exposure reduced SCGB1A1 in the distal airway epithelium following infection, representing a mechanism by which clearance of infectious agents may be compromised. DEE exposure alone has been shown to decrease SCGB1A1 levels in distal but not proximal airways (Harrod et al., 2003Go). TTF-1 is an important transcriptional regulator of a number of lung-specific genes, including SCGB1A1, and lung proteins with host defense properties such as surfactant proteins A and B (SP-A and SP-B) (Korfhagen et al., 1996Go). TTF-1 levels during P.a. infection were decreased by prior exposure to DEE, concordant with the decrease in SCGB1A1 levels. Collectively, the findings in the current study suggest that DEE exposure exacerbates epithelial cell injury in both proximal and distal airways during P.a. infection and may compromise host defense in both airway regions.

Numerous studies have elucidated the effects of air pollutants on the pulmonary response to respiratory infections. In experimental studies, exposure to air pollutants from a variety of sources has been shown to impair the clearance of pathogens from the respiratory tract and alter the host immune response to infection, often leading to exacerbated lung disease (Cohen and Nikula, 1999Go; Gilmour et al., 2001Go; Hatch et al., 1985Go). High concentrations of carbon black suppressed the ability of rodents to kill Staphylococcus aureus and to clear Listeria monocytogenes and influenza A virus (Jakab, 1993Go). Acute and chronic inhalation exposure of mice to diesel emissions caused increased mortality from respiratory infection with Streptococcus pyogenes (Campbell et al., 1981Go). Other investigators have found that either diesel or gasoline emissions altered respiratory bacterial infections in rodents (Campbell et al., 1979Go; Coffin and Blommer, 1967Go; Pepelko and Peirano, 1983Go). Diesel engine emissions and their particulate components have been shown to impair clearance of the respiratory viruses influenza and respiratory syncytial virus (Hahon et al., 1985Go; Harrod et al., 2003Go), as well as a number of bacteria species including Streptococcus, Bacillus (Calmette-Guerin), and Listeria (Castranova et al., 2001Go; Hatch et al., 1985Go; Saxena et al., 2003Go; Yang et al., 2001Go). Furthermore, diesel engine pollutant exposure augments lung inflammation to infection (Castranova et al., 2001Go; Harrod et al., 2003Go; Saxena et al., 2003Go). Prior exposure to DEE has been shown to downregulate host defense and innate immune mechanisms during respiratory infections (Castranova et al., 2001Go; Harrod et al., 2003Go; Yin et al., 2004Go) suggesting that modulation of early innate host defense may be central to the mechanisms of air pollutant-mediated exacerbation of respiratory infections. The findings presented herein provide evidence of diminished epithelial cell mechanisms of host defense that may be important in exacerbation of lung disease to infection by DEE. In addition, these studies provide the first observation of air pollutant impairment of Pseudomonas clearance from the respiratory tract. Further studies are necessary to clarify the distinct host defense and immune mechanisms modulated by air pollutant exposure and their importance in either the impaired clearance of respiratory pathogens or exacerbation of lung disease to respiratory infection.

In the present studies, prior exposure to inhaled DEE decreased lung bacterial clearance and exacerbated lung inflammation and injury to subsequent P.a. infection following a one-week exposure. However a six-month exposure failed to impair bacterial clearance or alter lung inflammation to P.a. infection, possibly due to age-related effects as indicated by slower clearance of P.a. in air-exposed animals after the six-month exposure compared with bacterial clearance in animals exposed to air for the one-week exposure studies. Multiple cell populations in the airway epithelium were shown to be affected by DEE during P.a. infection. In light of the epithelial cell changes observed in these studies, as well as those published previously, we can speculate that changes in pulmonary epithelial cell function may, in part, play a role in the impaired clearance of pathogens following DEE exposure in experimental models. In turn, the impaired clearance may provide the impetus for the increased lung inflammation observed. These studies present novel findings regarding the impact of DEE on pulmonary responses to a common bacterial pathogen that affects the elderly and those with compromised lung function. Furthermore, the findings herein may provide insight into the pulmonary epithelial determinants that regulate the host response to bacterial infection and exposure to ambient air pollutants.


    ACKNOWLEDGMENTS
 
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 U.S. EPA. This paper 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.) and the National Institutes of Health grant HL071547 (K.S.H.). Lovelace Respiratory Research Institute is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. The authors would like to thank Dr. Joe Mauderly and Ms. Leigh Schutzberger for critical reading of the manuscript.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bodey, G. P., Bolivar, R., Fainstein, V., and Jadeja, L. (1983). Infections caused by Pseudomonas aeruginosa. Rev. Infect. Dis. 5, 279–313.[ISI][Medline]

Brauer, M., Hoek, G., Van Vliet, P., Meliefste, K., Fischer, P. H., Wijga, A., Koopman, L. P., Neijens, H. J., Gerritsen, J., Kerkhof, M., Heinrich, J., Bellander, T., and Brunekreef, B. (2002). Air pollution from traffic and the development of respiratory infections and asthmatic and allergic symptoms in children. Am. J. Respir. Crit. Care Med. 166, 1092–1098.[Abstract/Free Full Text]

Bryan, C. S., and Reynolds, K. L. (1984). Bacteremic nosocomial pneumonia. Analysis of 172 episodes from a single metropolitan area. Am. Rev. Respir. Dis. 129, 668–671.[ISI][Medline]

Campbell, K. I., George, E. L., and Washington, I. S. (1979). Enhanced susceptibility to infection in mice after exposure to diluted exhaust from light duty diesel engines. In International Symposium on Health Effects of Diesel Engine Emissions, Cincinnati, OH.

Campbell, K. I., George, E. L., and Washington, I. S. (1981). Enhanced susceptibility to infection in mice after exposure to dilute exhaust from light duty diesel engines. Environ. Int. 5, 377–382.[CrossRef]

Castranova, V., Ma, J. Y., Yang, H. M., Antonini, J. M., Butterworth, L., Barger, M. W., Roberts, J., and Ma, J. K. (2001). Effect of exposure to diesel exhaust particles on the susceptibility of the lung to infection. Environ. Health Perspect. 109(Suppl. 4), 609–612.[ISI][Medline]

Coffin, D. L., and Blommer, E. J. (1967). Acute toxicity of irradiated auto exhaust. Its indication by enhancement of mortality from streptococcal pneumonia. Arch. Environ. Health 15, 36–38.[ISI][Medline]

Cohen, A. J., and Nikula, K.J. (1999). The health effects of diesel exhaust: laboratory and epidemiology studies. In Air Pollution and Health (J. M. Samet, S. T. Holgate, H. S. Koren, and R. L. Maynard, Eds.), pp. 707–745. Academic Press, London.

Dockery, D. W., and Pope, C. A., 3rd (1994). Acute respiratory effects of particulate air pollution. Annu. Rev. Public Health 15, 107–132.[CrossRef][ISI][Medline]

Dunnett, C. W., and Crisafio, R. (1955). The operating characteristics of some official weight variation tests for tablets. J. Pharm. Pharmacol. 7, 314–327.[ISI][Medline]

El-Solh, A. A., Aquilina, A. T., Dhillon, R. S., Ramadan, F., Nowak, P., and Davies, J. (2002). Impact of invasive strategy on management of antimicrobial treatment failure in institutionalized older people with severe pneumonia. Am. J. Respir. Crit. Care Med. 166, 1038–1043.[Abstract/Free Full Text]

Gilmour, M. I., Daniels, M., McCrillis, R. C., Winsett, D., and Selgrade, M. K. (2001). Air pollutant-enhanced respiratory disease in experimental animals. Environ. Health Perspect. 109(Suppl. 4), 619–622.[ISI][Medline]

Grizzle, J. E., Starmer, C. F., and Koch, G. G. (1969). Analysis of categorical data by linear models. Biometrics 25, 489–504.[ISI][Medline]

Hahon, N., Booth, J. A., Green, F., and Lewis, T. R. (1985). Influenza virus infection in mice after exposure to coal dust and diesel engine emissions. Environ. Res. 37, 44–60.[ISI][Medline]

Harrod, K. S., Jaramillo, R. J., Rosenberger, C. L., Wang, S. Z., Berger, J. A., McDonald, J. D., and Reed, M. D. (2003). Increased susceptibility to RSV infection by exposure to inhaled diesel engine emissions. Am. J. Respir. Cell Mol. Biol. 28, 451–463.[Abstract/Free Full Text]

Hatch, G. E., Boykin, E., Graham, J. A., Lewtas, J., Pott, F., Loud, K., and Mumford, J. L. (1985). Inhalable particles and pulmonary host defense: In vivo and in vitro effects of ambient air and combustion particles. Environ. Res. 36, 67–80.[ISI][Medline]

Hayashida, S., Harrod, K. S., and Whitsett, J. A. (2000). Regulation and function of CCSP during pulmonary Pseudomonas aeruginosa infection in vivo. Am. J. Physiol. Lung Cell Mol. Physiol. 279, L452–459.[Abstract/Free Full Text]

Jakab, G. J. (1993). The toxicologic interactions resulting from inhalation of carbon black and acrolein on pulmonary antibacterial and antiviral defenses. Toxicol. Appl. Pharmacol. 121, 167–175.[CrossRef][ISI][Medline]

Korfhagen, T. R., Bruno, M. D., Ross, G. F., Huelsman, K. M., Ikegami, M., Jobe, A. H., Wert, S. E., Stripp, B. R., Morris, R. E., Glasser, S. W., Bachurski, C. J., Iwamoto, H. S., and Whitsett, J. A. (1996). Altered surfactant function and structure in SP-A gene targeted mice. Proc. Natl. Acad. Sci. U.S.A. 93, 9594–9599.[Abstract/Free Full Text]

LeVine, A. M., Kurak, K. E., Bruno, M. D., Stark, J. M., Whitsett, J. A., and Korfhagen, T. R. (1998). Surfactant protein-A-deficient mice are susceptible to Pseudomonas aeruginosa infection. Am. J. Respir. Cell Mol. Biol. 19, 700–708.[Abstract/Free Full Text]

Lloyd, A. C., and Cackette, T. A. (2001). Diesel engines: Environmental impact and control. J. Air Waste Manag. Assoc. 51, 809–847.[ISI][Medline]

McDonald, J. D. (2001). Gas and particle phase distribution at four dilutions of diesel exhuast. In Annual Meeting of the American Association of Aerosol Research.

McDonald, J. D., Barr, E. B., White, R. K., Chow, J. C., Schauer, J. J., Zielinksa, B., and Grosjean, E. (2004). Generation and characterization of four dilutions of diesel engine exhaust for a subchronic inhalation study. Environ. Sci. Technol. 38, 2513–2522.[CrossRef][ISI][Medline]

Pepelko, W. E., and Peirano, W. B. (1983). Health effects of exposure to diesel engine emissions. A summary of animal studies conducted by the U.S. Environmental Protection Agency's Health Effects Research Laboratories at Cincinnati, OH. J. Am. Coll. Toxicol. 2, 253–306.[ISI]

Pope, C. A., 3rd (1991). Respiratory hospital admissions associated with PM10 pollution in Utah, Salt Lake, and Cache Valleys. Arch. Environ. Health 46, 90–97.[ISI][Medline]

Reed, M. D., Gigliotti, A. P., McDonald, J. D., Seagrave, J. C., Seilkop, S. K., and Mauderly, J. L. (2004). Health effects of subchronic exposure to environmental levels of diesel exhaust. Inhal. Toxicol. 16, 177–193.[CrossRef][ISI][Medline]

Saxena, R. K., Saxena, Q. B., Weissman, D. N., Simpson, J. P., Bledsoe, T. A., and Lewis, D. M. (2003). Effect of diesel exhaust particulate on bacillus Calmette-Guerin lung infection in mice and attendant changes in lung interstitial lymphoid subpopulations and IFNgamma response. Toxicol. Sci. 73, 66–71.[Abstract/Free Full Text]

Schwartz, J. (1994a). Air pollution and daily mortality: A review and meta analysis. Environ. Res. 64, 36–52.[CrossRef][ISI][Medline]

Schwartz, J. (1994b). What are people dying of on high air pollution days? Environ. Res. 64, 26–35.[CrossRef][ISI][Medline]

Singh, G., and Katyal, S. L. (1997). Clara cells and Clara cell 10 kD protein (CC10). Am. J. Respir. Cell Mol. Biol. 17, 141–143.[Free Full Text]

von Klot, S., Wolke, G., Tuch, T., Heinrich, J., Dockery, D. W., Schwartz, J., Kreyling, W. G., Wichmann, H. E., and Peters, A. (2002). Increased asthma medication use in association with ambient fine and ultrafine particles. Eur. Respir. J. 20, 691–702.[Abstract/Free Full Text]

Ware, J. H., Ferris, B. G., Jr., Dockery, D. W., Spengler, J. D., Stram, D. O., and Speizer, F. E. (1986). Effects of ambient sulfur oxides and suspended particles on respiratory health of preadolescent children. Am. Rev. Respir. Dis. 133, 834–842.[ISI][Medline]

Weiss, S. J. (1989). Tissue destruction by neutrophils. N. Engl. J. Med. 320, 365–376.[ISI][Medline]

Wright, J. R. (1997). Immunomodulatory functions of surfactant. Physiol. Rev. 77, 931–962.[Abstract/Free Full Text]

Yang, H. M., Antonini, J. M., Barger, M. W., Butterworth, L., Roberts, B. R., Ma, J. K., Castranova, V., and Ma, J. Y. (2001). Diesel exhaust particles suppress macrophage function and slow the pulmonary clearance of Listeria monocytogenes in rats. Environ. Health Perspect. 109, 515–521.[ISI][Medline]

Yin, X. J., Dong, C. C., Ma, J. Y., Antonini, J. M., Roberts, J. R., Stanley, C. F., Schafer, R., and Ma, J. K. (2004). Suppression of cell-mediated immune responses to listeria infection by repeated exposure to diesel exhaust particles in brown Norway rats. Toxicol. Sci. 77, 263–271.[Abstract/Free Full Text]

Zanobetti, A., Schwartz, J., and Dockery, D. W. (2000). Airborne particles are a risk factor for hospital admissions for heart and lung disease. Environ. Health Perspect. 108, 1071–1077.[ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
83/1/155    most recent
kfi007v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Harrod, K. S.
Articles by Reed, M. D.
PubMed
PubMed Citation
Articles by Harrod, K. S.
Articles by Reed, M. D.