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2001 Progress Report: Mechanisms of Age-dependent Ozone Induced Airway Dysfunction
EPA Grant Number: R827447Title: Mechanisms of Age-dependent Ozone Induced Airway Dysfunction
Investigators: Shore, Stephanie , Johnston, Richard , Murthy, G.G. Krishna , Want, Matt
Current Investigators: Shore, Stephanie , Johnston, Richard , Laporte, Johanne , Murthy, G.G. Krishna , Want, Matt
Institution: Harvard School of Public Health
Current Institution: Harvard University
EPA Project Officer: Deener, Kacee
Project Period: July 1, 1999 through June 30, 2002 (Extended to July 31, 2003)
Project Period Covered by this Report: July 1, 2001 through June 30, 2002
Project Amount: $852,937
RFA: Children's Vulnerability to Toxic Substances in the Environment (1999)
Research Category: Children's Health , Health Effects
Description:
Objective:The objective of this research project is to examine the hypothesis that intelenkin-6 (IL-6) protects against ozone (O3)-induced airway hyper-responsiveness (AHR) and inflammation, and mediates pulmonary adaptation to O3 in mice. Acute exposure to O3 causes damage to lung and airway epithelial cells, airway inflammation, and decreases lung function and AHR in human subjects. The mechanistic basis for O3-induced AHR appears to be inflammation arising from oxidant injury to the lungs and airways. However, the precise aspect of the inflammatory cascade required for AHR is still not firmly established. Ozone may be a particularly important respiratory hazard for children because they spend more time outdoors where O3 levels are higher, and because they are more physically active, and hence have a higher minute ventilation (VE), and consequently a higher delivered dose of O3. In addition, animal studies suggest that O3 may be more toxic to the lungs of immature rather than mature animals.
The questions that this research project seeks to address are: (1) Is O3 induced AHR greater in juvenile than adult animals? Why? (2) What is the mechanism of O3-induced AHR; and (3) Is the mechanistic basis for O3-induced AHR the same or different in juvenile and adult animals? In particular, we propose to test the following hypotheses:
· Age-related differences in ventilation, and in the effect of O3 on the ventilatory pattern, contribute to age-related differences in O3-induced AHR.
· Tumor necrosis factor alpha (TNFa) formation induced by O3 is important for O3-induced AHR.
· O3-induced TNFa formation is different in juvenile and adult animals.
Progress Summary:Role of IL-6 in Ozone-Induced AHR and Inflammation. In many species, IL-6 is released into bronchoalveolar lavage (BAL) fluid upon acute exposure to O3. IL-6 is a pleiotropic cytokine that has been reported to have both pro- and anti-inflammatory effects. In the lungs, it has been demonstrated that transgenic overexpression of IL-6 results in a decrease in AHR. Overexpression of IL-6 also protects against the acute lung injury induced by chronic hyperoxia, and mice genetically deficient in IL-6 have a reduced acute phase response to tissue injury. Taken together, these results suggest that O3-induction of IL-6 may serve to attenuate both the AHR and the airway injury induced by O3.
IL-6 also has been reported to play a role in the adaptation to the inflammatory effects of O3 that occurs with repeated exposure. McKinney, et al. reported that in rats, exogenous IL-6 markedly attenuated the polymorphonuclear neutrophil (PMN) migration into the lungs that was caused by a subsequent O3 exposure. IL-6 administration also caused a slight attenuation of O3-induced leak of protein into BAL fluid. The authors also demonstrated that administration of an anti-IL-6 receptor antibody prior to an initial O3 exposure prevented the attenuation in PMN migration but not in protein leak, which was observed with subsequent O3 exposures. The investigators did not examine O3-induced AHR in these studies.
We measured the effect of O3 on AHR in wildtype mice and mice genetically deficient in IL-6 (IL-6 -/- mice). We examined O3-induced AHR and inflammation in wildtype and IL-6 -/- mice following an initial O3 exposure, and following reexposure to O3, 4 days later. As indices of inflammation, we used BAL protein and PMN, as well as BAL IL-6, eotaxin, macrophage inflammatory protein-2 (MIP-2), and KC. Because IL-6 has been reported to increase subunit Tumor Necrosis Factor-Receptor 1 (sTNFR1) in blood, and because we previously have reported that tumor necrosis factor (TNF) is required for O3-induced AHR, we also analyzed the soluble TNF receptors, sTNFR1, and sTNFR2, in BAL fluid.
Whole body plethysmography was used to assess AHR. The outcome indicator for these experiments, Penh, has been shown by others to correlate with measurements of pulmonary resistance. There was no significant difference in baseline airway responsiveness to methacholine between wildtype and IL-6 deficient mice prior to O3 exposure. Three hours after cessation of the first O3 exposure (0.5 ppm for 3 hours), there was a marked leftward shift of the methacholine dose response curve in both wildtype and IL-6 deficient mice, indicative of AHR. However, there was no difference in the magnitude of the change in responsiveness between the 2 groups of mice.
Marked adaptation in the response to O3 occurred upon reexposure of wildtype mice to O3 (0.5 ppm for 3 hours) 4 days after the first exposure. In contrast to the AHR that was observed 3 hours after the first O3 exposure, AHR was not different from preexposure values 3 hours following cessation of the second O3 exposure. Similar results were obtained in IL-6 deficient mice.
We also examined the effect of repeated O3 exposure on BAL KC, IL-6, MIP-2, protein, sTNFR1, sTNFR2 levels, as well as BAL neutrophils in wildtype and IL-6 deficient mice. Three hours after cessation of an initial O3 exposure, there was an increase in all of these indices of inflammation in both wildtype and IL-6 -/- mice, compared to air exposed mice, but with the exception of BAL IL-6, which was undetectable in the IL-6 deficient mice, values were not different between wildtype and IL-6 deficient mice. A second exposure 4 days later resulted in further increases in all of these inflammatory indices, except IL-6 itself, which was lower on the second exposure than on the first. On the second O3 exposure, BAL neutrophils and BAL KC were significantly lower in IL-6 deficient mice than in the wildtype mice. The latter results are consistent with reports by others that the chemokine KC, which has chemotactic activity towards neutrophils contributes to the neutrophilia that is induced by O3. These results also indicate that IL-6 does not modulate the inflammation induced by an initial challenge, but are consistent with the hypothesis than IL-6 promotes O3-induced neutrophilia on subsequent challenges, perhaps through effects on KC expression.
Our results indicate that O3 increases the expression of soluble TNF receptors in BAL fluid. This is a novel observation that has not previously been reported in any species. Our results also indicate that IL-6 contributes to the expression of these receptors under basal conditions, because both sTNFRI and sTNFRII were lower in IL-6 deficient than in wildtype mice.
Effect of Chronic Ozone Exposure on Airway Inflammation in Mature and Immature Mice. In our previous progress report, we noted that following acute exposure to O3 (2 ppm for 3 h), immature mice have greater lung injury, as indicated by increases in BAL protein, likely as a result of a higher inhaled dose of O3. To more accurately reflect the types of exposures that occur in humans, we have now repeated the study using a more chronic exposure to lower concentrations of O3. Mice aged 2 weeks or 8 weeks were exposed to O3 (0, 0.2, 0.3, or 0.5 ppm) for 48 hours. O3-induced pulmonary inflammation and injury were assessed by measuring BAL cytokines protein and inflammatory cells, as described above.
In contrast to our previous results, which indicated greater BAL protein following acute exposure to high dose O3 in immature compared to mature mice, we observed that following more chronic exposure to lower concentrations of O3, immature mice actually had less protein in BAL fluid compared to adults. In fact, in 2-week-old mice, BAL protein was not significantly different from values obtained in air exposed control mice at any of the concentrations of O3 studied. In contrast, in 8-week-old mice, BAL protein was significantly greater than in air exposed mice at all O3 concentrations examined. We do not yet know the reason for the difference in age-related responses to O3 between acute and chronic O3 exposure. However, it is possible that the younger mice are more able to induce the expression of antioxidant enzymes in response to O3. Because such a process would be expected to take time, it might come into play during chronic, but not acute, exposures. We also noted substantial differences in BAL neutrophils induced by O3 between 2-and 8-week-old mice. In the 2-week-old mice, O3 exposure did not result in a significant increase in neutrophils at any of the concentrations studied, whereas in adult mice, even the lowest concentration of O3 (0.2 ppm) increased BAL neutrophils. Surprisingly, eosinophils did increase in the 2-week-old mice even at the lowest concentration of O3 studied, whereas they were undetectable in 8-week-old mice. We do not yet know the mechanistic basis for this difference, but are in the process of examining BAL eotaxin in the two groups, as eotaxin is an important chemoattractant for eosinophils. However, the results suggest that the immature animals may be predisposed to enhanced airway eosinophilia. Given that airway eosinophils are a major component of the inflammation of asthma, this may result in more severe episodes of asthma during periods of high O3.
Future Activities:In the coming year, we will complete analysis of BAL fluid and lung homogenates of 2- and 8-week-old mice exposed to O3 for 48 hours. Our current results indicate substantive differences in the response of these two groups, and this analysis will help to address the mechanistic basis for this difference. We will be focusing on TNF and TNF receptors, but also will analyze for eotaxin because of the differences noted in BAL eosinophils between the two groups. One explanation for the reduced inflammatory response to chronic O3 exposure in the 2-week-old mice is an enhanced ability to induce expression of antioxidants following O3 exposure. Because recent data from other laboratories indicate that heme oxygenase likely is to be an important antioxidant in the setting of O3 exposure, we will perform Western blot tests for heme oxygenase in homogenates of lungs from 2- and 8-week-old mice exposed to increasing concentrations of O3 (0.1 - 0.5 ppm).
Journal Articles on this Report: 1 Displayed | Download in RIS Format
| Other project views: | All 5 publications | 4 publications in selected types | All 4 journal articles |
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Shore SA, Johnston RA, Schwartzman IN, Chism D, Krishna Murthy GG. Ozone-induced airway hyperresponsiveness is reduced in immature mice. Journal of Applied Physiology 2002;92(3):1019-1028. |
R827447 (2000) R827447 (2001) R827447 (Final) |
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ozone, O3, exposure, health effects, age, biology, sensitive populations, mammalian. , Air, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Susceptibility/Sensitive Population/Genetic Susceptibility, Molecular Biology/Genetics, Risk Assessments, genetic susceptability, Health Risk Assessment, Physical Processes, air toxics, Children's Health, Biochemistry, tropospheric ozone, exposure and effects, environmental hazard exposures, acute exposure, enzyme systems, inhalation, respiratory problems, ozone, ozone induced airway dysfunction, assessment of exposure, age-related differences, lung injury, toxics, sensitive populations, ozone induced inflammation, air pollution, airway disease, children, stratospheric ozone, age dependent response, biomedical research, exposure, children's vulnerablity, dose response model, asthma
Relevant Websites:
http://www.hsph.harvard.edu/facres/shr.html
Progress and Final Reports:
1999 Progress Report
2000 Progress Report
Original Abstract
Final Report