Grantee Research Project Results
Final Report: Effects of Airborne Particles on Allergic Airway Disease
EPA Grant Number: R829216Title: Effects of Airborne Particles on Allergic Airway Disease
Investigators: Harkema, Jack , Sioutas, Constantinos
Institution: Michigan State University , University of Southern California
EPA Project Officer: Chung, Serena
Project Period: October 31, 2001 through October 30, 2004 (Extended to October 30, 2006)
Project Amount: $854,702
RFA: Health Effects of Particulate Matter (2001) RFA Text | Recipients Lists
Research Category: Human Health , Particulate Matter , Air
Objective:
The overall objective of this project was to conduct atmospheric and toxicologic research designed to understand the adverse effects of airborne particulate matter (PM) of various size fractions (coarse, fine, and ultrafine particles) on pulmonary airways with pre-existing allergic airway disease. The aims of this project have not changed from our original proposal. We tested the following hypotheses: 1) that PM exposure exacerbates the airway injury associated with allergic airway disease; 2) that the magnitude of PM-induced airway toxicity is dependent on particle size; 3) that PM in transported (“aged”) air pollution is more toxic to airways than that in locally generated air pollution; and 4) that PM-induced airway toxicity is most severe during periods of intense photochemical activity.
Summary/Accomplishments (Outputs/Outcomes):
For three consecutive years we have conducted inhalation toxicology studies in the Los Angeles Basin (LAB) at two different locations to distinguish the effects of locally generated versus transported particulate matter. In October 2001 and January 2002, we conducted studies using our mobile laboratory in a residential community in Claremont, CA in the northeast LAB, which served as our receptor site for transported PM. In following years, we conducted similar autumn and winter exposures in urban Los Angeles in the central LAB near the University of Southern California campus, which served as our source site of locally generated particulate air pollution. The potential health effects of co-exposures to urban air pollutants and airborne allergens have not been thoroughly investigated. The purpose of our studies was to determine the effects of inhalation exposure of various size fractions of concentrated ambient particles (CAPs) on the lungs of rats that were concurrently exposed to a pulmonary allergen (ovalbumin; OVA). A state-of-the-art mobile air research laboratory, equipped with inhalation exposure chambers and ambient particle concentrators, was used to conduct the inhalation toxicology studies. Our mobile laboratory was moved from its home site at the Michigan State University Engine Research Laboratory in Okemos, MI to a residential site in Claremont, CA, or near the University of Southern California campus in downtown Los Angeles, CA to these community-based inhalation toxicology studies in the early fall and winter months, as mentioned above.
Year 1: Studies in Claremont, CA (PM Receptor Site)
Study 1 Exposures: In Claremont, OVA-sensitized, male, Brown Norway rats (10-12 wks of age) were exposed to filtered air (controls), concentrated ambient coarse (2.5–10 μm; CCAPs), fine (0.1–2.5 μm; FCAPs) or ultrafine (0.01–0.15 μm; UFCAPs) particles, 5 h/day (11am - 4pm), for three consecutive days. Concentrated particle mass
and number concentrations and chemical speciation during the animal inhalation exposures are presented in Table
1. Immediately prior to each daily inhalation exposure, the rats were intranasally challenged with saline alone or a 0.5% solution of OVA in saline. Rats were exposed to average mass concentrations of 554, 515, and 45μg/m3for CCAPs, FCAPs, and UFCAPs, respectively. Twenty-four hours after the end of the exposures, rats were sacrificed, their pulmonary airways lavaged with saline, and their lung lobes processed for light microscopic or mRNA analyses. All of the animal sacrifices and necropsies were conducted by the laboratory staff of Dr. Harkema (Michigan State University) in the laboratories of Dr. Michael Kleinman (member of the Southern California Particle Center and Supersite) at the University of California in Irvine, CA.
Results: OVA-instilled rats had an allergic bronchiolitis with mucous cell hyperplasia (increase in the number of mucus-producing secretory cells lining the pulmonary airways) and an allergic alveolitis with marked increases in eosinophils in the bronchoalveolar lavage fluid (BALF). OVA-instilled and air-exposed rats had 538% more eosinophils in the BALF, 104% more stored mucosubstances in the bronchiolar epithelium, and a 6-fold increase in mucin-specific gene expression in bronchiolar airways than saline-instilled/air-exposed controls. Using this specific exposure regime of daily allergen and CAPs exposure we observed a marked particle-induced suppression, rather than enhancement, of the pulmonary inflammatory and epithelial responses to the inhaled allergen. Exposures to FCAPs (100% inhibition) or UFCAPs (by 66%), but not CCAPs, caused a marked attenuation of the OVA-induced allergic mucous cell hyperplasia. FCAPs also inhibited OVA-induced alveolitis (42% reduction in eosinophils in bronchoalveolar lavage fluid), and both FCAPS (65%) and UFCAPs (82%) blocked mucin-specific gene expression in bronchiolar epithelium (Figures 1 – 4).
Conclusion: These results indicate that fine (or ultrafine) ambient airborne particles may significantly interfere with allergen-induced airway responses during co-exposure of these airborne agents.
Study 2 Exposures: In January 2002, we conducted our second inhalation toxicology study at the same site in Claremont, CA. The experimental design was similar to that conducted in October 2001. Like in the first study, male, Brown Norway rats (10-12 wks of age) were exposed to filtered air, CCAPs, FCAPs or UFCAPs particles, 5 h/day (11am - 4pm), for three consecutive days. Immediately prior to each daily inhalation exposure, the rats were again intranasally challenged with saline alone or a 0.5% solution of OVA in saline.
Results: However in this winter study, rats were exposed to average mass concentrations of 86, 103, and 25μg/m3 for CCAPs, FCAPs, and UFCAPs, respectively (Table 2). These average mass concentrations were markedly lower than those to which rats were exposed in the first Claremont study in October 2001 (see above). In contrast to the results of the October study, no FCAPs-, UFCAPs- or CCAPs-related effects on OVA-induced allergic alveolitis, mucous cell metaplasia or mucin-specific gene expression were observed in these rats exposed to the much lower concentrations of CAPs.
Conclusions: These results, in relation to the first study, suggest that these lower mass concentrations were not sufficient to modulate the pulmonary responses induced by the allergen challenge. Differences in the chemical makeup of the CAPs in October and January may also have contributed, in part, to the marked differences in the pulmonary responses observed between the studies in Year 1.
Year 2: Studies in Los Angeles (PM Source Site)
Study 1 Exposures: In October 2002, we conducted our third inhalation toxicology study of CAPs-exposed Brown Norway Rats with and without OVA airway instillations. In this study the mobile research laboratory was parked in Los Angeles, CA near the main campus of the University of Southern California and highways with heavy motor vehicle traffic (i.e., our source site with locally generated particulate air pollution). We used the same experimental design and exposure regime as described above for the first and second studies in Claremont, CA. In this Los Angeles study, rats were exposed to average mass concentrations of 310, 324, and 31μg/m3 for CCAPs, FCAPs, and UFCAPs, respectively (Table 3).
Results: Similar to the particle-induced inhibition that we observed in the first study Claremont exposure study, we detected a trend for FCAPs-induced suppression of allergic responses. Sensitization and challenge with OVA induced significant accumulations of eosinophils (58-fold increase) and neutrophils (23-fold) in BALF (Figures 5, 6). In animals exposed to FCAPs these responses were not statistically significant with only 2.2-fold and 10-fold increases for eosinophils and neutrophils, respectively, when compared to saline-challenged rats. However, these inflammatory responses were not significantly less than air-exposed, OVA-challenged animals. Exposure to CCAPs, but not UFCAPs, also resulted in modest decreases of inflammatory cell recruitment. OVA induced a 160% increase in intraepithelial mucosubstances (p =0.06) that was inhibited by FCAPs by approximately 30% (Figure 7). Taken together, when compared to the inhibitory profile of CAPs we observed in Claremont, the present study showed a more modest effect for suppression of allergic responses. Average mass concentrations were approximately 60% of what was generated in Claremont 2001, but were 3-fold greater than in Claremont 2002 when no CAPs-related effects were documented.
Conclusion: Thus, our results describing a partial inhibition by CAPs may represent an intermediate point on a dose response curve.
Study 2 Exposures: In January 2003, we used Brown Norway rats and repeated the dosing and exposure protocol at the same site to assess the effects of the seasonal particle mixture and to compare to the January exposure in Claremont. In this second Los Angeles study, rats were exposed to average mass concentrations of 905, 1026, and 27μg/m3 for CCAPs, FCAPs, and UFCAPs, respectively (Table 4). During these exposures, the weather pattern was unseasonably warm and PM concentrations unusually high for January in Los Angeles. For CCAPs and FCAPs, concentrations were 3-fold greater than the October exposure at this site, and 10-fold greater than the January exposure in Claremont.
Results: Despite higher concentrations of CAPs, we did not detect particle-related changes in BALF cellularity in either saline-challenged or OVA-challenged rats. Similar to previous findings however, OVA-induced increases in intraepithelial mucosubstances were less in animals exposed to CAPs. Specifically, OVA-induced mucus storage was increased by only 45% and 59% when exposed to UFCAPs or FCAPs, respectively, compared to a 144% increase in air-exposed animals (Figure 8). The inhibitory effects of CAPs in the current study were less pronounced than we observed with lower particle concentrations at the receptor site in Claremont (FCAPS = 515 μg/m3), where allergic responses were blocked by 100%.
Conclusions: The degree of CAPs-induced inhibition of airway epithelial remodeling and inflammatory cell recruitment is not described with a simple dose-response relationship, and is more likely determined by contributions of specific particle components, their individual concentrations, and their potential interactions. Furthermore these results provide support for our hypothesis that transported, or aged particles, are more toxic than newly generated particles near source sites.
Year 3: Studies in Los Angeles (Source Site)
Study 1 Exposures: In the third year of the project we employed the same challenge and exposure protocol at the Los Angeles site but used younger Brown Norway rats (6-7 weeks old). In September 2003, rats were exposed to average mass concentrations of 587, 674, and 147μg/m3 for CCAPs, FCAPs, and UFCAPs, respectively (Table 5). CCAPs and FCAPs were similar in concentration to the September exposure at the receptor site in Claremont, CA in 2001, when the most pronounced inhibition occurred. In addition, UFCAPs was 5 to 6-fold greater than any of the previous exposures.
Results: As was observed previously, particles had no effect on BALF parameters or mucous cell hyperplasia in normal, non-allergic animals. Furthermore, significant CAPS-induced changes were not evident in OVA-challenged rats as we had seen with exposures with similar CAPs concentrations. Despite the lack of statistically significant differences, an apparent trend was nevertheless evident for the inhibitory effects by CAPs on OVA-induced BALF cellularity (e.g., eosinophils, Figure 9) and stored mucosubstances in pulmonary epithelium (Figure 10). During these exposures we also observed for the first time an effect of particles on nasal epithelium in non-allergic rats. CCAPs caused a 47% increase in stored mucosubstances in nasal respiratory epithelium, but had no effect on OVA-induced allergic rhinitis (Figure 11). Conversely, exposure to FCAPs inhibited by approximately 50% the OVA-induced increase in nasal intraepithelial mucosubstances.
Conclusion: It is notable that the higher concentrations of UFCAPs had no greater or lesser effects in normal and allergic rats than seen in previous exposures where lower concentrations were generated.
Study 2 Exposures: In February 2004, we repeated the protocols using the younger Brown Norway rats (6-7 weeks). In this study rats were exposed to average mass concentrations of 254, 505, and 114μg/m3 for CCAPs, FCAPs, and UFCAPs, respectively (Table 6).
Results: Similar to the previous September exposure with younger animals, a distinct trend for particle-induced inhibition of allergic alveolitis emerged, however the comparisons failed to reach statistical significance. Also, particles had no effect on allergic mucous cell metaplasia during this exposure.
Discussion: Summary and Conclusions
Our studies were designed to address four specific hypotheses. The first, that particles would enhance airway injury associated allergic airway disease, was not supported by our data. To the contrary, we found that particle exposure attenuated allergic bronchiolitis and alveolitis, and allergen-induced mucous cell metaplasia and the allergen-induced overexpression of mucin-specific gene (MUC5AC) in bronchiolar epithelium. This particle-induced attenuation of allergic airway disease was greatest with exposures to transported (“age”) FCAPs and UFCAPS in the October 2001 study in Claremont, CA (receptor site). In successive exposures, this inhibitory phenomenon was observed to varying degrees with changing variables of particle concentrations, exposure sites, and in younger animals.
The sum of our data supports our other hypotheses, that a) the magnitude of PM-induced effects is dependent on particle size, and b) PM in transported (“aged”) air pollution is more “toxic” on airways than that in locally generated air pollution, and c) that PM-induced airway “toxicity” is most severe during periods of intense photochemical activity, were supported by our experimental results. We consistently found that FCAPs, and with less frequency UFCAPs, were associated with particle-induced attenuation of allergic airway responses. Furthermore the effects of FCAPs were most pronounced during relatively warm weather (i.e. photochemical pollution) at the Claremont receptor site. By comparison the source-site exposures in Los Angeles during both summer and winter months produced less robust inhibitory effects of particles, but nonetheless demonstrated a trend for decreased allergic responses.
Although we predicted that exposures to CAPs would exacerbate the allergic airway responses, the inhibitory phenomenon we have described is not without precedent. Depression of inflammatory and immune cell function might be caused by oxidative stress that is measurable in pulmonary tissues of rats after FCAPs inhalation. We and others have demonstrated that exposure of alveolar macrophages to ambient particles in vitro induces dose-dependent cytotoxic responses, including oxidative stress, mitochondrial damage, and cytoskeletal derangement (Goldsmith et al. 1998; Moller et al. 2002; Becker et al. 2003; Kleinman et al. 2003; Li et al. 2003). Particle uptake by macrophages, lymphocytes, epithelial cells and dendritic cells may be counterproductive for normal immune responses to the presence of an airway allergen. Furthermore, ambient particles are known to induce a variety of cytokines from alveolar macrophages (e.g., IFN-γ, TNFα, IL-8,) that can interfere with allergic pathways to elicit eosinophil recruitment, lymphocyte activation, or IgE production.
Taken together, our results are reminiscent of data from allergic rodent models that examined the effects of airway endotoxin during allergen challenge. In these studies, endotoxin exposures at the time of allergen challenge inhibited allergic eosinophilic inflammation and airway hyperreactivity (Gerhold et al. 2002; Tulic et al. 2002). Attenuation of these allergic responses was associated with the production of IFNγ, IL-10 and IL-12 (Tulic et al. 2001; Gerhold et al. 2002), cytokines known to oppose Th2pathways of allergic inflammation. The paradigm of endotoxin-induced down regulation of allergic pathways is consistent with the “hygiene hypothesis”, where bacterial or fungal stimuli promote development of Th1-lymphocytes over allergy-promoting Th2 lymphocytes during the postnatal development of the immune system (Matricardi et al. 2002; Yazdanbakhsh et al. 2002). Just as bacterial stimuli present in low hygienic environments (e.g., rural, agricultural) can minimize the development of an allergy-prone immune phenotype in growing children, inhalation of a Th1-stimulus such as bacterial endotoxin opposes IgE production, eosinophilic inflammation, and airway hyperreactivity that are initiated by Th2 cytokines in subjects with allergic airway disease (Matricardi et al. 2002). Similar Th1/Th2cytokine dynamics may have occurred during CAPs inhalation and allergen challenge in the present series of experiments. Others have shown that treatment of ovalbumin-sensitized Brown Norway rats with IFNγ, a Th1 cytokine, blocks allergic inflammation and Th2 cytokine production (Huang et al. 1999). Inhalation of FCAPs and UFCAPs may have induced an early Th1 stimulus that interfered with allergic cytokine pathways that led to marked attenuation of more down stream responses such as eosinophilic inflammation and the development of mucous cell metaplasia. Further studies are needed to specifically investigate the Th1/Th2 cytokine responses in the lungs at various times after exposure to inhaled CAPs.
Interestingly, there are several reported epidemiology studies that have demonstrated that residents of the former East Germany who lived in communities with high levels of industrial air pollution had lower prevalence rates of asthma and other allergic diseases compared to residents of the former West Germany who lived in communities with lower levels of air pollution. Among East-German children, lower prevalence rates of asthma and positive skin-prick tests for allergery were observed compared to
West-German children who live in communities with less air pollution (Klein et al. 1992; von Mutius et al., 1994; Trepka et al., 1996). Similarly, East-German adults have been reported to have lower specific IgE levels and lower prevalence rates of asthma, wheezing, positive methacholine-challenge tests, allergic rhinitis, and positive skin-prick tests compared to those of West-German adults (Nicolai et al., 1997; Heinrich et al., 1998). It has yet to be determined what specific factors related to the air pollution (gaseous or particulate components), or related to life style, may account for these regional differences in the prevalence of asthma and allergies.
The results of our inhalation studies do lend support to the hypothesis that exposure to particulate air pollution may attenuate the development of allergic airway diseases. Since the rats in our study did not have allergic airway disease prior to exposure to the airborne CAPs, our studies were not designed to test the hypothesis that exposure to CAPs exacerbates pre-existing allergic airway disease. However, we have recently demonstrated that acute exposure of BN rats to FCAPs in a Detroit community may exacerbate pre-existing ovalbumin-induced allergic airway disease in BN rats under the right experimental and exposure conditions (Harkema et al., 2004). Therefore, it appears that the development and severity of this experimentally induced allergic airway disease is highly dependent on the time at which the laboratory rodents are exposed to both the allergen and the airborne particles.
Figure 4. Light photomicrographs of pulmonary tissue sections taken from rats exposed to filtered air and saline (A); filtered air and ovalbumin (OVA; sensitized and challenged) (B); coarse concentrated air particles (CCAPs) and OVA (C); or fine concentrated air particles (FCAPs) and OVA (D). No allergic alveolitis/bronchiolitis is present in A. Marked allergic alveolitis/bronchiolitis is present in B and C, but minimal pulmonary lesions are present in D. All tissues are stained with hematoxylin and eosin. Bar = 200 microns. Ba = bronchiolar airway; ap = alveolar parenchyma.
References:
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Gerhold, K, Blumchen, K, Bock, A, Seib, C, Stock, P, Kallinich, T, et al. 2002. Endotoxins prevent murine IgE production, T(H)2 immune responses, and development of airway eosinophilia but not airway hyperreactivity. J Allergy Clin Immunol 110: 110-116.
Goldsmith, CA, Imrich, A, Danaee, H, Ning, YY and Kobzik, L. 1998. Analysis of air pollution particulate-mediated oxidant stress in alveolar macrophages. J Toxicol Environ Health A 54: 529-545.
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Huang, TJ, MacAry, PA, Wilke, T, Kemeny, DM and Chung, KF. 1999. Inhibitory effects of endogenous and exogenous interferon-gamma on bronchial hyperresponsiveness, allergic inflammation and T-helper 2 cytokines in Brown-Norway rats. Immunology 98: 280-288.
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Matricardi, PM, Bouygue, GR and Tripodi, S. 2002. Inner-city asthma and the hygiene hypothesis. Ann Allergy Asthma Immunol 89: 69-74.
Moller, W, Hofer, T, Ziesenis, A, Karg, E and Heyder, J. 2002. Ultrafine particles cause cytoskeletal dysfunctions in macrophages. Toxicol Appl Pharmacol 182: 197-207.
Nicolai T, Bellach B, Mutius EV, Thefeld W, and Hoffmeister H. 1997. Increased prevalence of sensitization againsr aeroallergens in adults in West compared with East Germany. Clin Exp Allergy 27: 886-892.
Trepka MJ, Heinrich J, and Widhmann HE. 1996. The epidemiology of atopic diseases in Germany: An East-West comparison. Rev Envionm Health 11: 119-131.
Tulic, MK, Knight, DA, Holt, PG and Sly, PD. 2001. Lipopolysaccharide inhibits the late-phase response to allergen by altering nitric oxide synthase activity and interleukin-10. Am J Respir Cell Mol Biol 24: 640-646.
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Journal Articles:
No journal articles submitted with this report: View all 2 publications for this projectSupplemental Keywords:
RFA, Health, Scientific Discipline, Air, ENVIRONMENTAL MANAGEMENT, Geographic Area, particulate matter, Toxicology, air toxics, Health Risk Assessment, Chemistry, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Allergens/Asthma, Disease & Cumulative Effects, Environmental Monitoring, Children's Health, genetic susceptability, tropospheric ozone, Molecular Biology/Genetics, Biology, West Coast, Risk Assessment, asthma, health effects, particle size, sensitive populations, particulates, urban air, minority population, exposure and effects, stratospheric ozone, airway epithelial cells, fine particles, human health effects, air pollutants, cytokines, exposure, human airway epithelial calls, particulate emissions, airway disease, allergic airway disease, children, air pollution, particles, pariculate matter, human exposure, inhalation, particle pollutants, particulate exposure, airborne pollutants, immunology, urban air pollution, environmentally caused disease, PM, environmental effects, human health, Los Angeles Basin (LAB), allergen, disease, respiratory, ultrafine particles, air quality, environmental hazard exposures, toxics, exposure assessmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.