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Asthma is a known risk factor for acute ozone-associated respiratory disease. Ozone (O3) causes an immediate decrease in lung function and increased airway inflammation. The role of atopy and asthma in modulation of O3-induced inflammation has not been determined.
To determine if atopic status modulates O3 response phenotypes in humans.
Fifty volunteers (25 normal volunteers, 14 atopic non-asthmatics, 11 atopic asthmatics not requiring maintenance therapy) underwent a 0.4 ppm O3 exposure protocol. Ozone response was determined by changes in lung function and induced sputum composition, including airway inflammatory cell concentration, cell surface markers, cytokine and hyaluronic acid concentration.
All cohorts experienced similar decreases in lung function post O3. Atopics and atopic asthmatics had increased sputum neutrophils and IL-8 after O3 exposure; levels did not significantly change in normal volunteers. Following O3 exposure, atopic asthmatics had significantly increased sputum IL-6 and IL-1 β, and airway macrophage TLR4, FceRI, and CD23 expression; levels in normal volunteers and atopic non-asthmatics showed no significant change. Atopic asthmatics had significantly decreased IL-10 at baseline compared to normal volunteers: IL-10 did not significantly change in any group with O3. All groups had similar levels of hyaluronic acid at baseline, with increased levels after O3 exposure in atopics and atopic asthmatics.
Atopic asthmatics have increased airway inflammatory responses to O3. Elevated TLR4 expression suggests a potential pathway through which O3 generates the inflammatory response in allergic asthmatics but not in atopics without asthma.
These observations suggest that mild atopic asthma confers increased risk for exacerbation of O3-induced lung disease through promoting an enhanced innate immune inflammatory response to O3.
Ozone (O3) is a commonly encountered environmental pollutant. Asthma morbidity (ER visits, hospitalizations, rescue medication use) is clearly associated with exposure to increased levels of ambient air O3 1 Asthmatics are thought to have increased susceptibility to O3 due to aggravation of underlying allergic airways inflammation. Ozone exposure studies have shown that O3 induces an airway granulocyte response, characterized by sputum neutrophilia in healthy volunteers 1–3 and sputum neutrophilia and eosinophilia in atopic asthmatics 4. Ozone challenge has also been found to enhance responses to subsequent inhaled allergen challenge in mild atopic asthmatics 5–7. The ability of O3 to enhance airway inflammation and enhance responsiveness to subsequent inhaled allergen likely play important roles in asthma exacerbation. However, it is unclear if non-asthmatic persons with atopy also have enhanced susceptibility to O3 as has been shown in atopic asthmatics. Khatri and colleagues found associations between air quality and airway inflammation in both asthmatics and non-asthmatics with allergies 8, suggesting that atopic status itself may contribute to O3-induced inflammatory response in the airway.
Our group has also observed that response to inhaled lipopolysaccharide (LPS) yields airway inflammatory responses similar to those seen after O3 exposure. Like O3 exposure, airway LPS challenge induces airway neutrophilia, enhances response to inhaled allergen in allergic subjects 9–11, and induces airway pro-inflammatory cytokines1. We and others have also found that O3 and LPS induce selective increases in specific macrophage and monocyte populations in the airway 12 and that O3 induces influx of monocytes and macrophages with increased expression of CD11b and CD14, 13a co-receptor for LPS. Building on these observations, our group has recently found correlations in airway inflammatory responses in healthy individuals who underwent both O3 and LPS exposures (Hernandez, in press). The similarities between responses of normal volunteers and atopic asthmatics to O3 and LPS suggest that atopy modifies response to a number of innate stimuli and that common molecular mechanisms account for response to both O3 and LPS.
Consistent with this idea, mechanistic studies in mice suggest that at least some O3 responses are mediated through the primary LPs receptor, TLR4 14–16. As O3 is a reactive oxygen molecule, it is unlikely that a discrete receptor exists which modulates response to this agent, or that O3 would act directly on airway monocytes or macrophages to induce airway inflammation. Indeed, in vitro studies of airway macrophages or THP-1 cells exposed to O3 do not reveal an O3-induced release of inflammatory mediators 17–19. It seems more likely that O3 exerts a biological effect via interaction with components of airway lining fluid to produce secondary signaling molecules or danger signals which cause inflammatory responses. Ozone challenge of airway epithelial cells in culture has been shown to result in NF-κB activation and production of a variety of mediators 20–22. Pro-inflammatory mediators such as IL-8 are among those associated with O3-induced inflammation in asthmatics 23 and reduced levels of the anti-inflammatory cytokine IL-10 have been reported in asthmatics at baseline 23–25. Hyaluronic acid (HA) is an endogenous pro-inflammatory ligand for CD44 and TLR4 26–28 and may be an important mediator in asthma. Hyaluronic acid has also been recently identified as a candidate host danger signal which mediates O3-induced inflammatory responses 29, 30.
At present, it is unclear if O3 modifies levels of TLR4 in humans, and if atopic status itself modifies the O3-induced airway inflammatory response. The present study tests the hypothesis that atopic status alone modifies lower airway inflammatory response to a 0.4 ppm O3 exposure through examination of airway inflammatory cells and airway epithelial cell lining fluid from induced sputum.
These protocols were reviewed and approved by the University of North Carolina Committee on the Rights of Human Subjects (Institutional Review Board). All subjects underwent a physical examination, routine blood panel with CBC and differential cell count, allergy skin testing, and methacholine challenge. Atopy was demonstrated by positive immediate skin test response to one of the following allergen mixes: two species of house dust mite (Dermatophagoides farinae and Dermatophagoides Pteronyssinus), cockroach, tree mix, grass mix, weed mix, mold mix 1, mold mix 2, rat, mouse, guinea pig, rabbit, cat or dog. A positive methacholine challenge was determined as having a drop of 20% from post-saline FEV1 at a level of 10 mg/ml or less of methacholine. This study was an analysis of lung function data and samples obtained from a total of 50 subjects that were stratified into three groups based on the results of their allergy skin testing and methacholine challenge. Allergic asthmatics (AA, N=11) had positive skin prick testing and positive methacholine challenge and were required to have mild intermittent asthma, as defined by National Heart, Lung, and Blood Institute (NHLBI) 2007 guidelines (not on inhaled corticosteroid or leukotriene receptor antagonist therapy). Normal volunteers (NV, N=25) had negative skin prick testing and negative methacholine challenge, and atopic volunteers (N=14) had positive skin prick testing and negative methacholine challenge. All AA volunteers were newly recruited into this study protocol while other subjects were derived from previously completed studies 12, 31. Female subjects had to have a negative urine pregnancy test prior to challenge and all volunteers were required to be free of chronic cardiovascular illness, and be free of acute respiratory illness within 4 weeks of O3 challenge. All subjects had FEV1 and FVC ≥ 80% predicted normal for height and age 32, and FEV1/FVC ≥ 0.75 and were non-smokers with no smoking history. All subjects were screened for their ability to provide an adequate induced-sputum sample during their training session. Subjects provided an induced sputum sample during the screening visit and at 4–6 h after the O3 exposure. Sputum induction and processing methods are found in the online repository.
Spirometry testing was preformed according to ATS/ERS recommendations using the Viasys VMax 229 series spirometers. All subjects were seated, and at least 3 maneuvers were obtained, with the best of the three reported.
O3 Inhalation Challenge: The O3 exposures were conducted in an O3 exposure chamber at the US-EPA Human Studies Facility on the campus of the University of North Carolina, Chapel Hill, NC. Each subject was exposed to O3 (0.4 ppm) for 2 hours while performing four 15 minute sessions of intermittent moderate exercise (minute ventilation or VEmin = 30–40 L/min) on a treadmill, separated by 15 minutes of seated rest. Spirometry, breath sounds, and vital signs were assessed before and immediately after exposure. Sputum was obtained 4–6 hours post exposure and processed as previously described 12, 31, 33, 34. Sputum was assessed for total and differential cell counts, flow cytometric assessment of CD11b, CD14, CD86, TLR4, FceRI, CD23, and HLA-DR on macrophages and monocytes 12, 35. Sputum supernatants were also assessed for cytokine concentration.
Expression of selected cell surface molecules on sputum leukocytes was quantified via multicolor flow cytometry (FCM) using a BD LSR-II flow cytometer (BD Immunocytometry Systems; San Jose, CA). These included molecules associated with antigen presentation and specific immunity (CD86/B7.2, HLA-DR/MHC-II,), as well as innate immune function (CD11b/CR3, CD14/LPS receptor). First, sputum leukocytes were differentiated from cellular debris, bacteria, yeast, squamous and bronchial epithelial cells by gating on CD45 (pan leukocyte marker) positive cells. Up-or down-regulation of specific surface molecules was quantified as a change in the mean fluorescent intensity (MFI) of the gated population. Fluorochrome-labeled antibodies were obtained from BD Biosciences (CD11b-PE-CY5, CD45-APC-Cy7, HLA-DR-PerCP, Beckman-Coulter (CD14-APC, CD86-PE). Appropriate, nonspecific, labeled isotypic control antibodies were also obtained from these sources.
Cytokines from sputum supernatants were measured using multi-plex technology Meso ScaleDiscovery/MSD, Gaithersburg, MD). Each sample was analyzed with the HumanMIP-1 alpha Ultra Sensitive Kit (LOT#: K0031370) and the Human TH1/TH2 10-Plex Ultra Sensitive Kit (LOT#: K0031431). All supernatant samples were diluted 1:4 and had a final DTT concentration of < 1mM where no deleterious effects have been observed using the MSD platform.
Hyaluronic acid (HA) levels from induced sputum supernatants were performed with a commercially available enzyme-linked immunosorbent assay (Echelon, San Jose, CA) with minor modifications. The lowest concentration of available standard was 50 ng/ml. This was diluted 1:1 to make a 25 ng/ml standard, and the 25 ng/ml standard was diluted 1:1 to make a 12.5 ng/ml standard. In addition, standards were run with and without 0.1% DTT without notable differences in standard concentrations. Concentrations of HA from sputum supernatants were compared to standards containing DTT. The standard curve generated was log linear from 12.5 ng/ml to 1600 ng/ml.
The primary hypothesis to be tested was that atopic asthmatics or atopic non-asthmatics had increased susceptibility to ozone compared to normal volunteers, with the primary endpoint being sputum neutrophils. Changes in cell counts/mg sputum, differential cell count percentage (sputum), mean fluorescence intensity (MFI) measures (flow cytometry), cytokine values, and HA levels pre- and post-O3 exposures within each cohort were assessed. To determine if a particular cohort responded to O3, non-parametric paired Wilcoxon signed rank test was employed. Differences between either atopic asthmatics or atopic non-asthmatics and normal volunteers were analyzed using non-parametric t tests (Mann-Whitney Test). To confirm that baseline values between all three groups were not different, non-parametric one way analysis of variance (Kruskall Wallis Test) was employed. Significance was set at a p= 0.05.
Demographic data for the 50 volunteers, ranging in age from 19 to 39 years, recruited for challenge to 0.4 ppm of O3 are presented in Table 1. Fourteen were atopic, though they could not exhibit symptoms of atopy at the time of exposure. Eleven were atopic asthmatics, none of whom were on maintenance inhaled corticosteroid therapy or other scheduled drug.
The effect of O3 on lung function in cohorts of normal volunteers (n=25), atopics (n=13), and atopic asthmatics (n=11) was compared. Ozone caused similar significant decreases in mean FEV1 (p<0.001 all cohorts) and FVC (p<0.001 normal volunteers, p=0.003 atopics and atopic asthmatics) immediately after challenge in all cohorts (Figure 1) compared to their baseline values.
The effect of O3 on airway neutrophil, macrophage, and eosinophil numbers was a primary endpoint for this study. Paired data (pre and post O3 exposure) was available for 25 normal volunteers, 13 atopics, and 10 atopic asthmatics. Figure 2A shows that the atopic and the atopic asthmatic cohorts had significantly increased sputum neutrophil numbers (cells/mg sputum, p=0.045 atopics, p=0.04 atopic asthmatics) 4 hours after challenge. Although sputum neutrophils increased in normal volunteers, the increase in mean neutrophil numbers did not reach statistical significance, which may have been influenced by more variable baseline neutrophil numbers. Although macrophage numbers (cells/mg sputum) (Figure 2B) decreased in all cohorts, this was significant only among the atopic asthmatic volunteers (p=0.01). Figure 2C shows that eosinophil numbers at baseline were elevated in the airways of atopic (p=0.0002) and atopic asthmatic volunteers (p=0.0002) compared to very low values in normal volunteers. After O3 exposure, eosinophil numbers did not significantly change in the atopics or the atopic asthmatics and remained very low in normal volunteers.
We also examined sputum supernatants for a number of immunoregulatory cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, macrophage inflammatory protein 1 alpha, TNF alpha and Interferon gamma). Sputum supernatants were available for 24 normal volunteers, 12 atopics, and 10 atopic asthmatics. Four cytokines (IL-1β, IL-8, IL-6, and IL-5) were significantly altered by O3 exposure and this occurred only in the atopic and atopic asthmatic cohorts. Atopic asthmatics had significant increases in IL-1β (p=0.03), IL-8 (p=0.01), and IL-6 (p=0.01) (Figures 3A–C), while atopics had increased IL-8 (p=0.02) (Figure 3B) and IL-5 (p=0.03) following O3 exposure (data not shown). Two atopic asthmatic subjects had higher baseline and post O3 levels of IL- β and IL-8 compared to the rest of the cohort. Analysis of the data excluding these two subjects showed that the change in IL-8 and IL-β was significant (p=0.04) and close to significant (p=0.08) for these cytokines, respectively. We further analyzed the IL-1β data as % change from baseline; using this metric we found a significant difference when comparing NV vs. AA (p=0.009) (data not shown). When comparing baseline values, atopic asthmatics had increased IL-1β compared to normal volunteers (p=0.02) or atopics (p=0.05) (Figure 3A). No group experienced significant increases in IL-10 after O3 exposure. However, atopic asthmatics had significantly decreased IL-10 at baseline compared to normal volunteers (p=0.007) (Figure 3D).
Pre and post O3 exposure sputum supernatant samples from 10 subjects in each cohort were available for analysis. There were no differences in HA levels at baseline or after O3 exposure among the cohorts (Figure 3E). Ozone exposure caused increased HA levels in atopics (p=0.004) and atopic asthmatics (p=0.002) compared to their respective baseline values.
Macrophages from induced sputum were isolated as discussed in the methods, and changes in cell surface markers are presented in Figure 4 (n=13 normal volunteers, 7 atopics, and 6 atopic asthmatics). Macrophages in the atopic asthmatic cohort had increased expression of CD23 (p=0.003), FceRI (p=0.03), and TLR4 (p=0.03) after O3 exposure (Figure 4A–C). All cohorts showed increased CD11b after O3, but this increase attained significance only in the atopic (p=0.02) and atopic asthmatic (p=0.03) cohort (Figure 4D). There were no other significant changes in cell surface markers (CD80, CD86, TLR2, CD14, and HLA-DR) following O3 in any cohort (data not shown).
There are numerous epidemiologic studies linking increased environmental O3 levels with asthma exacerbations. Asthmatic susceptibility to ambient O3 has been purported to be due to enhanced underlying allergic airways inflammation 4, 36, however, not all studies have observed modified allergic airways inflammation following exposure in atopic individuals 4, 37.
In this study we examined whether atopic status alone or mild intermittent atopic asthma influenced the airways inflammatory response to inhaled O3. We found that all cohorts experienced similar decreases in lung function (FEV1 and FVC) immediately after O3 exposure, with no difference among cohorts. Atopy or atopic asthma did not appear to influence this immediate effect on lung function thought to be a reflex-mediated phenomenon triggered in part by eicosanoids 38, 39. We did find that although all cohorts had increased sputum neutrophils compared to their baselines after O3 exposure, these differences were significant only in atopic and atopic asthmatic subjects. We note however that the atopic and atopic asthmatic cohorts had less variability in their baseline PMNs/mg than the normal volunteers. We can speculate that atopic status may have influenced the variability of baseline neutrophilia when compared to normal volunteers. The potent neutrophil chemoattractant IL-8 was significantly increased only in the atopic and atopic asthmatic cohorts following O3 exposure with no significant differences between them. Thus, some markers of neutrophilia appear to be influenced by atopic status at the early time point sampled. However, the present study is limited in that we cannot comment on more chronic inflammatory changes such as reduced lung function or sputum neutrophilia that may selectively persist in some cohorts 24 hours after O3 exposure, t he time frame with which O3-induced asthma exacerbations are associated.
Although 0.4 ppm O3 did not significantly increase sputum eosinophils in this mild asthmatic cohort, we did find that O3 exposure upregulated cell surface expression of FceRI and CD23, the high and low affinity IgE receptors respectively, in the macrophages of atopic asthmatics. This finding suggests that O3-induced airway macrophages from atopic asthmatics have enhanced the ability to participate in the antigen uptake process40 following O3 exposure. In the past our group has shown that macrophages of allergic asthmatics have enhanced particle uptake capability compared to normal volunteers 35.
The lack of change in sputum eosinophils may be influenced in great part by the timepoint sampled (4 hours post O3), as previous studies by our group showed increased eosionphils in atopic asthmatics 18 hours post exposure 4. Of notable interest were the elevated baseline sputum eosinophils in the atopic non-asthmatic and the atopic asthmatic cohorts. This observation has been documented by two other groups 41, 42 in bronchial biopsy specimens, where baseline numbers appear to reflect atopic status alone. What is unclear is if and how these elevated baseline eosinophil numbers influence eosinophil trafficking at later timepoints and/or eosinophil activation with consequent exacerbation of disease.
In addition to O3’s enhancing allergic airways inflammation, we found that mild atopic asthmatics had increased cell surface expression of TLR4 on mature macrophages after O3 exposure. This finding corroborates murine studies of O3 challenge, where O3 exposure alone has been shown to upregulate TLR4 on murine alveolar macrophages 43. Mechanistic studies in animals suggest that at least some responses to O3 are mediated through TLR4 and consequent elaboration of innate immune cytokines such as IL-1 β 30. Hollingsworth and colleagues have showed that compared to wild type mice, TLR4 deficient mice had reduced airway hyper-responsiveness following subacute O3 exposure 15, and that inflammatory cytokine expression was altered in a TLR4 dependent manner 30. The role of TLR4 in O3-induced inflammation is further highlighted by our HA observations. Hyaluronan has recently been identified as an endogenous ligand of TLR426–28 and has been found to mediate O3-induced airway hyper-responsiveness29, 30 and pro-inflammatory cytokine production.30 Our finding of significantly increased HA levels in atopic and atopic asthmatic subjects but not in normals, makes the HA-TLR4 link an interesting mechanism to explore in future O3 studies.
We hypothesize that atopic asthmatics expressed more surface TLR4 on airway macrophages due to a combination of factors: a). increased potential ligands stimulating increased TLR4 expression, and/or b). the baseline cytokine environment in the atopic asthmatic cohort with increased IL-1β and decreased IL-10. Our HA assay was limited in that we could not distinguish between low and high molecular weight forms; the low molecular weight form serving as a putative TLR4 ligand. Atopic asthmatics may have had increased endogenous TLR4 ligands that were not assayed for in the present study. The baseline cytokine environment may also influence susceptibility to augmenting TLR4 expression, as others have shown that IL-10 can downregulate TLR4 expression 44, and that IL-1β overexpression is capable of provoking the release of endogenous TLR4 ligands 45, enabling TLR4-mediated production of cytokines such as IL-1β, IL-6, and IL-8.
In addition to the elevated IL-1β at baseline in the atopic asthmatic cohort, we found that IL-1β and IL-6 were elevated after O3 exposure in atopic asthmatics. This suggests an important role for IL-1β and possibly activation of the NLRP3 inflammasome which is central in release of IL-1β from macrophages. Administration of (human) IL-1 receptor antagonist before and after O3 exposure has been shown to prevent the development of airway hyper responsiveness and blunt increases in pro-inflammatory cytokines, as well as decrease neutrophilia in bronchoalveolar lavage (BAL) fluid in a mouse model of O3-induced lung injury 46. Similar to our study, IL-1β levels have been reported to be greater in BAL fluid and sputum of asthmatics compared to normal volunteers47, with airway macrophages from asthmatics also having increased expression of IL-1β48. Most notably, IL-1β has been shown to be elevated in BAL fluid from persons with symptomatic asthma vs. those with asymptomatic asthma 49. Our findings highlight the heterogeneity of IL-1β values at baseline and following O3 exposure in very mild asthmatics. Interestingly, Hastie et al. recently analyzed induced sputum from asthmatics stratified by granulocyte populations and found that those asthmatics who had >40% sputum neutrophils, independent of eosinophil level, had increased IL-1β compared to sputa with <40% neutrophils50. It is plausible then that in asthmatics with neutrophil-driven inflammatory responses, such as those evoked by O3 exposure or endotoxin inhalation challenge, IL-1β may play a significant role in the disease state. However, the importance of IL-1β in response to O3 exposure will need to be confirmed in a follow up study with a larger sample size. We hypothesize the increased TLR4 expression correlates with increased pro-inflammatory cytokine production, as has been shown in TLR4 transgenic animals 51. Taken together, our data on TLR4 and IL-1β lead us to suggest that innate immune inflammatory pathways involving activation of TLR4 and subsequent release of IL-1β may play an important role in driving O3-induced asthma exacerbations. Release of an endogenous TLR4 ligand may activate the NLRP3 inflammasome, with consequent production and release of IL-1β, documented in other models of sterile inflammation 52.
Another possible explanation for increased inflammatory cytokine production and increased TLR4 and IgE receptor surface expression after O3 exposure in atopic asthmatics may be the decreased level of IL-10 at baseline, with no increase after O3 exposure. Interleukin-10 is a potent anti-inflammatory cytokine, suppressing the production of a multitude of pro-inflammatory cytokines by activated macrophages, such as TNF-α, IL-β, IL-6, macrophage inflammatory protein-α, and IL-853. Decreased levels of IL-10 have been reported in the BAL24 and induced sputum25 of asthmatics compared to non-asthmatics. In the present study we found that baseline IL-10 levels in sputum of allergic asthmatics were significantly decreased compared to normal volunteers (p=0.007), and there was a similar trend for difference with atopic non-asthmatics (p=0.09). Studies of IL-10 knockout mice indicate that IL-10 appears to be protective against O3-induced neutrophilic inflammation and NFkB activation54. Therefore, suppressed baseline IL-10 in our atopic asthmatic cohort may have contributed to increased pro-inflammatory cytokine production after O3 exposure.
In conclusion, we report that atopic asthmatics exposed to O3 exhibit an elevated response of pro-inflammatory cytokines (IL-1β, IL-6, IL-8), TLR4 and IgE receptor expression in the airways. We suggest this airways milieu may confer increased reactivity to subsequently inhaled allergen and innate immune ligands. Low levels of IL-10 in the airways of atopic asthmatics may underlie this reactivity to O3. We propose that these findings have important health implications, as clarifying the mechanisms underlying susceptibility to environmentally-induced asthma and modulators of these inflammatory pathways, such as microRNAs 55 may allow for the development of more targeted traditional or alternative therapies to prevent asthma exacerbations.
The authors gratefully acknowledge the skillful assistance of Martha Almond, Aline Kala, Margaret Herbst, Carole Robinette, Heather Wells, Nathaniel Bailey, Fernando Dimeo, Danuta Sujkowski, Evan Trudeau, Nolan Sweeny, Wenli Zhang, and Tatiana Quintero-Matthews from the UNC Center for Environmental Medicine, Asthma and Lung Biology; Maryann Bassett, Tracy Montilla and Deborah Levin of the Environmental Public Health Division of the US EPA and Wes Gladwell of the Laboratory of Respiratory Biology of the NIEHS in the completion of this project.
This project was supported in part by grants R01ES012706 and P30ES010126 from the National Institute of Environmental Health Sciences, U19AI077437 from the National Institute for Allergy and Infectious Diseases, P01AT002620 from the National Center for Complementary and Alternative Medicine, and KL2RR025746, M01RR00046 and UL1RR025747 from the National Center of Research Resources of the National Institutes of Health, as well as CR 83346301 from the US Environmental Protection Agency.
Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through cooperative agreement CR 83346301 with the Center for Environmental Medicine and Lung Biology at the University of North Carolina at Chapel Hill, it has not been subjected to the Agency's required peer and policy review and therefore does not necessarily and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred.
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Disclosure of potential conflict of interest: B. Harris has received research support from the National Institute of Environmental Health Sciences, the US Environmental Protection Agency, and Purdue Pharmaceuticals–Quintiles. P. A. Bromberg has received research support from the US Environmental Protection Agency and the National Institutes of Health. D. B. Peden has consulted for GlaxoSmithKline and Funxional Therapeutics and has received research support from the National Institute of Environmental Health Sciences; the National Institute for Allergy and Infectious Diseases; the National Center for Complementary and Alternative Medicine; the National Heart, Lung, and Blood Institute; the US Environmental Protection Agency; the National Center for Research Resources. The rest of the authors have declared that they have no conflict of interest.