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Final Report: Asthma in Children: A Community-based Intervention Project
EPA Grant Number: R826708C001Subproject: this is subproject number 001 , established and managed by the Center Director under grant R826708
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: CECEHDPR - University of Southern California
Center Director: Gong, Henry
Title: Asthma in Children: A Community-based Intervention Project
Investigators: Gong, Henry , Diaz-Sanchez, David , Jones, Craig , McConnell, Rob
Institution: University of California - Los Angeles
EPA Project Officer: Fields, Nigel
Project Period: January 1, 1998 through January 1, 2002
Project Amount: Refer to main center abstract for funding details.
RFA: Centers for Children's Environmental Health and Disease Prevention Research (1998)
Research Category: Children's Health , Health Effects
Description:
Objective:The specific aims are:
Aim #1: Determine the mechanisms by which secondhand smoke (SHS) alters the in vivo IgE antibody response in the human upper airway.
Aim #2: Determine the mechanisms by which SHS alters IgE-independent inflammatory responses in the human upper airway.
Aim #3: Determine the effects of genetic background and age on the allergic antibody response induced by chronic exposure to SHS by employing a rat model.
Summary/Accomplishments (Outputs/Outcomes):Children Have Increased Susceptibility to the Inflammatory Effects of Oxidant Pollutants
Previous studies have categorically shown the potential of the model pollutant diesel exhaust particles (DEP) to induce and exacerbate inflammation in vivo in the human upper respiratory tract (Diaz-Sanchez, 2000). Given the similarity between DEP and SHS, the widespread exposure of children to passive smoking, and the immature immune system of children, we studied whether SHS could also modulate the immune system to enhance inflammation (Diaz-Sanchez, et al., 2002). Nasal lavages performed 24 hours after SHS exposure revealed a significant increase in levels of pro-inflammatory cytokines. The magnitude of this response was significantly higher in children than in adults, thus mean levels of tumor necrosis factor-alpha (TNF-α), granulocyte macrophage colony stimulating factor (GM-CSF), and interleukin 1-beta (IL-1β) were 228 percent, 321 percent, and 122 percent, respectively higher in children than adults.
Young Mice are More Susceptible to Adjuvant Effects of Oxidant Pollutants
We studied whether SHS could initiate a Th2 response and thus induce primary allergic sensitization (Rumold, et al., 2001). Mice aged 8 weeks old were exposed for 10 consecutive days to 1 percent nebuliz ed ovalbumin protein (OA) for 20 minutes/day either in the presence or absence of 1-hour daily smoke from five cigarettes. The ability of these animals to mount total and allergen-specific immunoglobulin E (IgE) response was then determined. In mice both IgE and IgG1 are “allergic antibodies” capable of binding FcεRI and inducing anaphylaxis. When mice were exposed to aerosolized OA alone, no OA-specific-IgE was observed at any of the time points studied. In contrast, in the group exposed to SHS plus OA for 10 days, antigen-specific IgE was apparent 12 days (Day 12) after the initial exposure (mean = 318 ± 108 U/mL) and persisted until Day 25.
Similar to IgE levels, OA-IgG1 could not be detected at any time in sera from C57BL/6 mice who had received aerosolized OA alone. However, by Day 12 OA-IgG1 could be detected in mice receiving both SHS and OA and remained elevated up to 30 days after initial exposure. Thirty days after the initial challenge (20 days after its completion), mice were re-exposed to OA. Broncho-alveolar lavage (BAL) performed 24 hours later revealed an influx of eosinophils in the group initially challenged with both environmental tobacco smoke (ETS) and OA but not in those exposed to either SHS alone or OA alone. Additionally, compared to mice challenged with OA alone, OA/ETS exposed mice had increased levels of IL-5 (73 vs.18 pg/mL), GM-CSF (107 vs. 7 pg/mL) and IL-2 (55.3 vs. 0 pg/mL) in their BAL fluid whilst interferon-gamma (IFN-γ) levels were significantly inhibited (7 vs. 77 pg/mL).
Induction of allergic antibody production accompanied by an increase in eosinophil numbers and a Th2 milieu are characteristic and hallmarks of murine allergic airway disease (a model for human asthma). Using this murine model we have shown that age is a significant factor in allergic sensitization by SHS. The experiment outlined above was repeated in both mature (8 week old) and younger sexually immature mice (2-3 weeks old). In young mice, antibody, cellular and cytokine responses to SHS were more exaggerated. We have obtained similar results using DEP inhalation exposure. We have also repeated the SHS experiments reducing daily exposure to SHS to that of only one cigarette/day. At this level allergen-specific IgE and IgG1 and changes in cytokine levels could still be observed in the young but were completely absent from the older mice.
These studies illustrate that the young may be at increased risk to the adverse effects of oxidant particulate pollutants.
Both SHS and DEP Elicit the Same Allergic Responses in the Human Upper Airways
We have previously shown that DEP is a potent adjuvant in the human upper airways. We examined whether this property was unique to DEP or was common to other oxidant particulate pollutants. We therefore tested the adjuvant effects of SHS. Nineteen ragweed-allergic subjects performed nasal lavages and 15 minutes later underwent controlled chamber exposures to 2 hours SHS (from five cigarettes) or clean air prior to nasal challenge with ragweed allergen (Amb a I) or placebo (300 µL saline).
Nasal washes were performed at different times after challenge. Subsequent visits to perform the other arms of the study were spaced at least 6 weeks apart. SHS promoted the production of allergen-specific IgE, the hallmark of allergic disease in nasal lavage fluid. At the peak of the response, ( 4 days after challenge) ragweed-specific IgE in the washes was on average 16.6- fold higher following ragweed plus SHS exposure compared to clean air/ragweed challenge. In addition, SHS interacted with allergen to promote a critical shift of cytokines measurable in nasal washes. Combined challenge with DEP plus ragweed resulted in the formation of a Th2 -cytokine milieu.
Following clean air/ragweed challenge, only low and inconsistent changes in cytokine levels occurred. In contrast, challenge with both SHS and allergen resulted in significantly increased levels of IL-4, IL-5, and IL-13. The levels of these cytokines were significantly greater at this time than following challenge with allergen alone (e.g., IL-5 mean = 4.8 pg/mL vs. 0.3 pg/mL, p < 0.01). In contrast, IFN- γ levels were not significantly changed by SHS plus allergen exposure. This induction of a Th2-cytokine nasal milieu is characteristic of an active allergic response.
Mast cell/basophil degranulation to allergen could also be enhanced by SHS. We measured histamine levels in nasal lavage fluid obtained 10 minutes following challenge with ragweed allergen. Baseline levels of histamine were virtually identical in all challenge days. Following clean air/ragweed challenge there was a 7.7 -fold rise in mean histamine from baseline values (4.02 vs. 0.52 nM). In contrast, this was significantly less than the 25.1- fold increase observed following SHS/ragweed challenge.
These studies are unique in demonstrating the adjuvant potential of SHS. Moreover, the ability to enhance the effect of allergen on IgE, Th2 cytokines, and histamine is identical to that observed previously for DEP. We hypothesize that adjuvancy is a common feature of oxidant pollutants. Our proposed studies will use DEP challenge or exposure models, but we believe the results will be equally applicable to SHS and other oxidant pollutants.
Antioxidants Block DEP-Induced Oxidative Stress in Mouse Lungs and Thereby Block Enhanced IgE Responses
If pollutants do induce allergic responses through the generation of oxidative stress, we would predict that their effects should be blocked by antioxidants that block all possible generation of reactive oxygen species (ROS). As shown before, SHS will induce allergic antibody production to OA in the absence of any prior sensitization (3). Similar results can also be obtained using aerosolized DEP. We studied whether antioxidants could block this response (4). We immunized BALB/c mice i.p. with either vehicle control or 25 mg/kg N-acetyl cysteine, a thiol antioxidant. The mice were then exposed to (1) aerosolized OA (1% for 20 minutes each day for 10 days), (2) aerosolized DEP ( 1 hour for 10 consecutive days), or (3) aerosolized DEP followed immediately by the aerosolized OA for 10 days. DEP was re-suspended in saline and aerosolized to achieve concentrations of 2000 μg/m3.
No OA-specific IgE and IgG1 could be detected in sera from mice exposed to OA alone, but control mice exposed to DEP plus OA produced significant levels of both allergic antibodies. In contrast, N-acetylcysteine treatment completely blocked production of both OA-IgE and OA-IgG1 in DEP plus OA exposed animals. Studies using SHS inhalation have shown similar results. Oxidative stress leading to covalent modification of cellular lipids and proteins was measured in lungs obtained at Day 12 by assaying levels of carbonyl proteins and lipid hydroperoxides. There was 6-fold increase in carbonyl protein content and a 2.9-fold increase in lipid peroxide levels in mice exposed to DEP + OA compared to OV only.
Induction of Phase II Enzymes Inhibits Enhancement of IgE Production by DEP-Extracts In Vitro
The studies above support our hypothesis that mechanisms that can regulate oxidative stress can also modulate DEP-induced allergic responses. Phase II enzymes are important detoxification agents that can moderate oxidative stress. We tested the hypothesis that induction of enzymes is important in regulating DEP adjuvant effects by using an in vitro IgE model. Stimulation with IL-4 and anti-C40 will induce IgE production by human peripheral blood mononuclear cells (PBMC). We had previously shown that co-stimulation of the cells with chemicals extracted by methanol from diesel particles will significantly enhance IgE production. Typically, after 14 days, cells co-cultured with this DEP-extract have IgE levels 200-600 percent higher than those cultured with IL-4/anti-CD40 alone. Sulforaphane is a potent inducer of Phase II enzymes and functions to activate Nrf2. GSTM1 expression is known to be high in lymphocytes.
As expected, addition of sulforaphane to PBMC cells resulted in increased gene expression of GSTM1 and NQO1 observed after 24 hrs. The addition of sulforaphane to the cell culture resulted in a dose-dependent inhibition of DEP-induced IgE potentiation. At concentrations of sulforaphane of 6 μM and higher, IgE levels from cells stimulated with IL4/α-CD40 and IL4/α-CD40/DEP-extract were indistinguishable. These results illustrate the principle that the pro-allergenic effects of diesel can be mediated by oxidative stress and can be alleviated by induction of Phase II enzymes.
Polymorphisms in Phase II Enzyme Genes Regulate Susceptibility to DEP and SHS
Although there is considerable variation in the magnitude of responses of subjects to nasal challenge with DEP, the DEP response is consistent in individuals. Indeed we have identified “poor responders” and tested them several years later, and DEP will once again elicit only modest responses.
Variations in activity of a number of human antioxidant enzymes have been identified and have been established to arise from polymorphisms in the coding genes (Nakajima and Aoyama, 2000; Siegel, et al., 1999; Fryer, et al., 2000; To-Figueras, et al., 1997; London, et al., 1995). Given the potential role of Phase II enzymes, we tested whether variation in key Phase II enzyme genes dictate differences in susceptibility to the adjuvant effects of DEP.
We used ragweed-sensitive subjects in our well-established human nasal model of secondary allergic responses. We performed a crossover single blind study. Subjects were challenged with either an “active” dose of allergen or both 0.3 mg DEP plus allergen. After at least a month, the subjects were recalled and those previously exposed to allergen alone received both DEP plus allergen and vice versa. Cells from buccal scrapes were then sent blinded to Dr. Gilliland's group who genotyped them to determine which variants of the GSTM1, GSTP1, and GSTT1 genes they carried.
Even with a small sample size of 19, significant associations were observed between genotype and nasal IgE response enhancement by DEP. Individuals with forms of the GSTM1 and GSTP1 genes which result in reduced or absent anti oxidant responses showed heightened allergic responses by DEP adjuvant effects. For example, compared with subjects with GSTM1 present genotype, subjects with GSTM1 null had a significantly larger increase in IgE (146 vs. 13.5 U/mL, p < 0.01) and in histamine levels (13.9 vs. 6.1 nM, p = 0.03) following DEP plus allergen challenge. In contrast, GSTT1 did not seem to regulate whether allergen-induced IgE responses could be elevated by DEP. It is important to note that while GSTM1 is highly expressed in lymphoid cell lines, GSTT1 is not, but is expressed predominantly in erythrocytes. Identical results were obtained when the experiment was repeated using SHS exposure instead of DEP.
Conclusions:- The adjuvant and inflammatory effects of oxidant pollutants are greater in the young.
- Oxidant particulate pollutants have the same adjuvant effects and probably share the same metabolic pathways and mechanisms regardless of their combustion source.
- The ability of anti oxidants to block immune-modulatory effects of these pollutants suggests a central role for oxidative stress in regulating effects.
- A subgroup of individuals with increased susceptibility to the adjuvant effects of pollutants can be identified.
- Individuals in this group have an increased prevalence of genotypes for key Phase II enzyme genes (GSTM1 and GSTP1), which result in the absence or reduced function of these antioxidant enzymes.
Taken as a whole, these results suggest that oxidant pollution (regardless of its sources) is likely to have an adjuvant effect on airway responses to allergen and that this effect will be enhanced in children and in individuals with poor anti oxidant defenses. A key to anti oxidant defense may be the ability to express Phase II enzymes.
References:
Diaz-Sanchez D. Pollution and the immune response: atopic disease—are we too dirty or too clean? Immunology 2000;101(1):1-10.
Diaz-Sanchez D, Rumold R, Gong H. Controlled second-hand smoke exposure enhances ragweed-induced IgE, Th2 cytokines and histamine production in allergic subjects. Journal of Allergy and Clinical Immunology 2002;109(S1):S90.
Rumold R, Jyrala M, Diaz-Sanchez D. Secondhand smoke induces allergic sensitization in mice. Journal of Immunology 2001;167(8):4765-4770.
Nakajima T, Aoyama T. Polymorphism of drug-metabolizing enzymes in relation to individual susceptibility to industrial chemicals. Industrial Health 2000;38(1):143-152.
Siegel D, McGuinness SM, Winski SL, Ross D. Genotype-phenotype relationships in studies of a polymorphism in NAD(P)H:quinone oxidoreductase 1. Pharmacogenetics 1999;9:113-121.
Fryer AA, Bianco A, Hepple M, Jones PW, Strange RC, Spiteri MA. Polymorphism at the glutathione S-transferase GSTP1 locus. A new marker for bronchial hyperresponsiveness and asthma. American Journal of Respiratory and Critical Care Medicine 2000;161:1437-1442.
To-Figueras J, Gene M, Gomez-Catalan J, Galan MC, Fuentes M, Ramon JM, Rodamilans M, Huguet E, Corbella J. Glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) polymorphisms and lung cancer risk among Northwestern Mediterraneans. Carcinogenesis 1997;18(8):1529-1533.
London SJ, Daly AK, Cooper J, Navidi WC, Carpenter CL, Idle JR. Polymorphism of glutathione S-transferase M1 and lung cancer risk among African-Americans and Caucasians in Los Angeles County, California. Journal of the National Cancer Institute 1995;87(16):1246-1253.
Journal Articles on this Report: 1 Displayed | Download in RIS Format
| Other subproject views: | All 11 publications | 9 publications in selected types | All 9 journal articles |
| Other center views: | All 80 publications | 52 publications in selected types | All 50 journal articles |
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Whitekus MJ, Li N, Zhang M, Wang M, Horwitz MA, Nelson SK, Horwitz LD, Brechun N, Diaz-Sanchez D, Nel AE. Thiol antioxidants inhibit the adjuvant effects of aerosolized diesel exhaust particles in a murine model for ovalbumin sensitization. The Journal of Immunology 2002;168(5):2560-2567. |
R826708 (2000) R826708 (2001) R826708 (2002) R826708 (Final) R826708C001 (Final) R826708C002 (Final) R827352 (Final) R827352C002 (Final) R831861 (2004) R831861 (2005) |
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children’s health, respiratory health, allergic airway disease, secondhand smoke, air pollution, diesel exhaust particles, integrated pest management, community-based participatory research, susceptibility, genetics, gene expression, inflammation,
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Air, Scientific Discipline, Health, RFA, Susceptibility/Sensitive Population/Genetic Susceptibility, Biology, indoor air, Risk Assessments, genetic susceptability, Health Risk Assessment, Children's Health, Atmospheric Sciences, Environmental Chemistry, Allergens/Asthma, adolescents, exposure assessment, asthma indices, allergen, health effects, indoor air quality, dust , air quality, dust mite, indoor environment, childhood respiratory disease, dust mites, harmful environmental agents, sensitive population, toxics, cockroaches, community-based intervention, respiratory, sensitive populations, biological response, air pollution, children, exposure, children's vulnerablity, minority population, school based study, asthma triggers, Breathmobile, asthma, human exposure
Relevant Websites:
Progress and Final Reports:
2000 Progress Report
2001 Progress Report
2002 Progress Report
Original Abstract
Main Center Abstract and Reports:
R826708 CECEHDPR - University of Southern California
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R826708C001 Asthma in Children: A Community-based Intervention Project
R826708C002 Children's Exposure to Environmental Tobacco Smoke: Changes in Allergic Response
R826708C003 Respiratory Disease and Prevention Center