* Asthma and Pulmonary Immunology Program, Experimental Toxicology Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico and
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
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ABSTRACT |
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Key Words: respiratory infection; pulmonary toxicology; air pollution.
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INTRODUCTION |
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In urban settings, engine emissions constitute a major portion of air pollution (Lloyd and Cackette, 2001). 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, 1999
). 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., 1983
; El-Solh et al., 2002
). 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., 2000
). Inhaled DEE decreased the clearance of P.a. and increased lung inflammatory responses concordant with airway epithelial cell changes during infection.
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MATERIALS AND METHODS |
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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, 2001; McDonald et al., 2004
; Reed et al., 2004
).
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, 2001; McDonald et al., 2004
; Reed et al., 2004
). 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, 2001
; McDonald et al., 2004
; Reed et al., 2004
). Studies of uninfected air- and DEE-exposed mice (n = 68 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., 2000). 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 (1824 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., 2003). 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 04 (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., 2003). 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 46 animals per experimental group were selected randomly, and 810 airways were analyzed. For analysis of large airways, typically 23 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, 1955). 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., 1969) 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.
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RESULTS |
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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.
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DISCUSSION |
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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, 1984). The dose of P.a. chosen for these studies represents a standard, sublethal experimental inoculum (Hayashida et al., 2000
; LeVine et al., 1998
). 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., 2003). 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, 1999
). 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., 2001
; Hatch et al., 1985
; Jakab, 1993
; Yang et al., 2001
).
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, 1989). 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, 1997). 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., 2000
). 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., 2003
). 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., 1996
). 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, 1999; Gilmour et al., 2001
; Hatch et al., 1985
). High concentrations of carbon black suppressed the ability of rodents to kill Staphylococcus aureus and to clear Listeria monocytogenes and influenza A virus (Jakab, 1993
). Acute and chronic inhalation exposure of mice to diesel emissions caused increased mortality from respiratory infection with Streptococcus pyogenes (Campbell et al., 1981
). Other investigators have found that either diesel or gasoline emissions altered respiratory bacterial infections in rodents (Campbell et al., 1979
; Coffin and Blommer, 1967
; Pepelko and Peirano, 1983
). 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., 1985
; Harrod et al., 2003
), as well as a number of bacteria species including Streptococcus, Bacillus (Calmette-Guerin), and Listeria (Castranova et al., 2001
; Hatch et al., 1985
; Saxena et al., 2003
; Yang et al., 2001
). Furthermore, diesel engine pollutant exposure augments lung inflammation to infection (Castranova et al., 2001
; Harrod et al., 2003
; Saxena et al., 2003
). Prior exposure to DEE has been shown to downregulate host defense and innate immune mechanisms during respiratory infections (Castranova et al., 2001
; Harrod et al., 2003
; Yin et al., 2004
) 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.
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ACKNOWLEDGMENTS |
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