Grantee Research Project Results
Final Report: Effect of Inhaled Acid-Coated Particles on Alveolar Macrophage Function: Genetic Susceptibility
EPA Grant Number: R825815Title: Effect of Inhaled Acid-Coated Particles on Alveolar Macrophage Function: Genetic Susceptibility
Investigators: Kleeberger, Steven R. , Jakab, George J.
Institution: The Johns Hopkins University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1997 through September 30, 2000
Project Amount: $561,577
RFA: Issues in Human Health Risk Assessment (1997) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The primary objectives of this project were to define the mode of inheritance of susceptibility to the pulmonary response to inhaled acid-coated particles (ACP) in the mouse, and to identify and map the susceptibility genes. The three specific aims designed to address these objectives were to: (1) characterize the kinetics of alveolar macrophage (AM) dysfunction and inflammation induced by inhaled ACPs in susceptible C57BL/6J (B6) and resistant C3H/HeJ (C3) inbred mice; (2) determine the mode of inheritance of susceptibility to ACP inhalation; and (3) determine the chromosomal location(s) of quantitative trait loci (QTL) that control susceptibility to inhaled ACPs.Summary/Accomplishments (Outputs/Outcomes):
Incomplete combustion of fossil fuel results in the production of sulfur oxides (e.g., SO2) and carbonaceous particles. This often results in the production of acid sulfates depending on atmospheric conditions such as high relative humidity. Considerable attention has been focused on the adverse respiratory health effects caused by inhalation of these products. Epidemiological studies in a number of industrialized countries throughout the world have reported a significant association of acute and chronic lung health effects in the general population with increases in these products. In addition to decrements in lung function, these exposures also may enhance respiratory illness (e.g., chronic cough, bronchitis, pneumonia). High particle and SO2 concentrations also have been attributed to a number of significant acute mortality episodes. Interestingly, although the epidemiology of the health effects of combustion pollutants has become clear, the physiologic and/or toxicological mechanisms have not been well characterized.It has become increasingly clear that some subpopulations are at increased risk to the effects of particulate exposure. These populations include children, the elderly, and patients with pre-existing chronic heart and lung diseases or compromised immune systems. Genetic background also may be an important contributing susceptibility factor. To determine whether genetic background contributes to susceptibility to toxic effects of inhaled particles, we studied the interstrain (genetic) and intrastrain (environmental) variance of lung responses to ACP aerosol in nine strains of inbred mice. A flow-past nose-only inhalation system was used to expose mice to ACPs produced by the co-generation of a carbon black aerosol-SO2 mixture at high humidity. Three days after a single 4-hour exposure to ACPs or filtered air, mice were assessed for pulmonary inflammation. To determine the effect of ACPs on AM function, pulmonary AMs were isolated from exposed animals and Fc receptor-mediated phagocytosis was evaluated. This phenotype was chosen because Fc receptor-mediated phagocytic function of AMs represents an important "first line" in immune host defense. The integrated activity of the phagocytic and immune system provides pulmonary defense against bacteria and other environmental pathogens. Phagocytosis of the invading pathogen is one of the events coordinated with intracellular killing (oxidative bust) to maintain sterility of the respiratory tract. Phagocytic function has been used as an indicator of pulmonary toxicity and, particularly, AM integrity in a number of airway toxicity models. A disruption of any of the steps involved in intracellular killing, including phagocytosis, compromises host defense. Increased risk of lower airway infection after particle exposure has been reported in several epidemiological studies, and is presumably mediated through effects on lung defense and AM function.
There was slight but significant inflammation in only two of the inbred strains of mice that were tested in this study, and the response resolved within 24 hours. Fc receptor-mediated phagocytosis was suppressed in eight of nine strains after ACP exposure. Full recovery occurred after 2 weeks. The significant interstrain variation in AM response to particle challenge indicated that genetic background has an important role in susceptibility. The effects of ACPs on AM function and inflammation were not correlated between strains. This model has important implications concerning interindividual variation in particle-induced compromise of host defense, and it provided the basis for determining the location of susceptibility genes for particle exposure.
The search for susceptibility loci was a two-step process. The first step was to apply methods of segregation analysis to establish inheritance patterns. The segregation analyses were done using susceptible B6 and resistant C3 strains of mice. Analysis of AM dysfunction in back-cross and intercross (F2) populations derived from B6 and C3 mice indicated that there are two major unlinked genes responsible for the differential responsiveness to AM phagocytic dysfunction after ACP exposure. These experiments suggested that the number of genes responsible for determining susceptibility to particle-induced AM dysfunction was amenable to linkage analysis. That is, if many genes (e.g., greater than six) contributed equally to determine susceptibility, then linkage analyses may not have been sensitive enough to identify all loci. However, two to five loci is a manageable number for linkage analyses. Therefore, we initiated the second step of the search for susceptibility loci, which was to complete a scan of the mouse genome using linkage analysis methods with an F2 cohort. These analyses identified statistically significant and suggestive QTLs on segments of chromosomes 17 and 11, respectively. The chromosome 17 QTL was then examined for candidate genes by comparative mapping between murine and human genomes, and a number of candidate genes were identified. These include tumor necrosis factor alpha (Tnf) and heat shock proteins Hsp70-1 and Hsp70-3. Each has been suggested to have important roles in cell and tissue responses to environmental stresses. Additional candidates include a number of histocompatibility loci and mast cell proteases Mcpt6 and Mcpt7. These genes have been found to be important in immune responses in the lung, and it is conceivable that they may have a role in responses to particulate challenge. The minor QTL on chromosome 11 also contains a number of potentially interesting candidate genes, including inducible nitric oxide synthase (Nos2), as well as a number of small inducible CC-chemokines. A functional role for each has been described in the pathogenesis of asthma, fibrosis, and other lung diseases.
Another important outcome of these studies was recognition that the major and minor QTLs for ACP susceptibility overlap directly with QTLS that we described previously for responsiveness to ozone (O3). Using an approach similar to that used for the ACP studies, we found that QTLs on chromosomes 17 and 11 accounted for a significant portion of the genetic variability in the inflammatory response to 0.3 ppm O3 exposure (0.3 ppm, 48 hours) in O3-susceptible B6 and resistant C3 mice.
We believe these observations have important implications in understanding the determinants of susceptibility to air pollutant mixtures and the epidemiologic associations of pollutant mixtures with respiratory morbidity and mortality. Particulates usually occur as mixtures with other pollutants, including sulfates and oxidants such as O3. Indeed, a number of epidemiological studies that monitor mixtures have found that respiratory symptoms (coughing, wheezing, lung function decrements) and other respiratory health outcomes (acute asthma exacerbation, lower respiratory tract infection) are associated with multiple pollutants. It is not clear whether the health effects are due to additive or synergistic properties of the pollutants. Results of our studies suggest that there may be specific genes within the major and minor QTLs that confer susceptibility to distinct response phenotypes induced by two prevalent air pollutants?sulfur-associated particles and O3. Fine-mapping studies will narrow the chromosomal length of the susceptibility QTLs to less than 1 cM. Subsequent physical mapping approaches will ultimately lead to the identification of the susceptibility genes and strain-specific polymorphisms.
We also have begun to investigate the genetic factors that control lung responses to residual oil fly ash (ROFA). ROFA is a particle pollutant derived as a combustion product of fuel oil. ROFA contains soluble sulfates and substantial levels (10% in mass) of soluble transition metals, including iron (Fe), vanadium (V), and nickel (Ni). These three principal ROFA-associated metals have well-documented human health effects in respiratory airways. In laboratory rodents, intratracheal instillation of ROFA induces airway hyperreactivity and acute pulmonary injury characterized by neutrophilic inflammation, pulmonary hyperpermeability and edema, pulmonary fibrosis, and increased susceptibility to microbial infections. Although high contents of transition metals are thought to account for the adverse pulmonary effects by generation of oxygen-based free radicals in the lung, the precise mechanisms through which ROFA induces lung injury and inflammation are not resolved.
Therefore, we initiated a study to investigate the role of genetic background in susceptibility to the pulmonary toxic effects of ROFA challenge in mice, and whether the genetic factors were similar to those identified for susceptibility to ACP. To address this objective, ROFA was instilled intratracheally in seven inbred strains of mice, and intra- and interstrain variation in the lung permeability and inflammatory responses were determined. ROFA induced dose- and time-dependent effects on lung hyperpermeability and inflammation in inbred mice. Among seven inbred strains, significant interstrain variation also was observed in the ROFA-induced lung hyperpermeability and inflammation. The greatest difference in lung hyperpermeability responses was between C3H/HeOuJ (OuJ) and C3 mice. This observation is particularly interesting as these two strains differ only at a polymorphism in the coding region of a gene for Toll-like receptor 4 (Tlr4), which plays an important role in innate immunity and responsiveness to bacterial endotoxin. We also have demonstrated (American Journal of Respiratory Cell and Molecular Biology 2000;22(5):620-627) that Tlr4 is an important determinant of the hyperpermeability response to O3 in inbred mice. Interestingly, we did not observe a difference in ACP-induced AM dysfunction between OuJ and C3 mice.
A marked upregulation of lung Tlr4 message level was observed in OuJ mice, but not in C3 mice following ROFA challenge. Differential expression of basal as well as induced lung Tlr4 protein also was detected between two strains of mice. We also examined possible involvement of Tlr4 signaling pathway in the variation of lung permeability response between OuJ and C3 mice. First, we determined the expression of an adaptor protein, MyD88, which is considered one of the most upstream components of Tlr4-mediated signaling cascade to interact directly with the cytoplasmic Toll/IL-1R homology domain of Tlr4. ROFA challenge markedly enhanced MyD88 protein levels in OuJ mice, but there was only a slight increase of MyD88 proteins in the lungs of C3 mice exposed to ROFA. We also evaluated expression of IRAK-1, the downstream serine/threonine kinase of MyD88, as well as TRAF6, a protein that may interact with activated IRAK-1. Expression of both was higher in OuJ than in C3 mice after ROFA challenge. Further, transcription factor NF- B was differentially activated in lungs of OuJ mice compared to C3 mice in response to ROFA. These data are consistent with a major role for Tlr4 signaling in response to ROFA, and may have implications for understanding the mechanisms through which this particle exerts its pulmonary effects. The effect of ROFA on the innate immune system is consistent with the hypothesis that one of the toxic outcomes of particulate exposure is compromise of host defense mechanisms.
These are the first demonstrations of genetic loci that are important determinants of responsiveness to particle-induced morbidity. Identification of susceptibility genes, and their regulation, will lead to a better mechanistic understanding of the causative effects of the pollutants and pollutant mixtures on respiratory health. Gene characterization also will provide a means for identifying susceptible individuals and developing strategies for intervention to prevent lung injury induced by exposure to environmental pollutants.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 5 publications | 2 publications in selected types | All 2 journal articles |
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Kleeberger SR, Jedlicka AE, Reddy SPM, Walters D, Clarke RW, Zhang L-Y. Genetic mechanisms of susceptibility to ROFA-induced lung injury. American Journal of Respiratory and Critical Care Medicine 2000;161:A912. |
R825815 (Final) |
not available |
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Ohtsuka Y, Eleff SM, Brunson K, Clarke RW, Mitzner W, Kleeberger SR. Mode of inheritance in susceptibility to inhaled acid-coated particles in mice. American Journal of Respiratory and Critical Care Medicine 1998;157(3):A696. |
R825815 (Final) |
not available |
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Ohtsuka Y, Brunson KJ, Jedlicka AE, Mitzner W, Clarke RW, Zhang L-Y, Eleff SM, Kleeberger SR. Genetic linkage analysis of susceptibility to particle exposure in mice. American Journal of Respiratory Cell and Molecular Biology 2000;22(5):574-581. |
R825815 (Final) |
not available |
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Ohtsuka Y, Clarke RW, Mitzner W, Brunson K, Jakab GJ, Kleeberger SR. Interstrain variation in murine susceptibility to inhaled acid-coated particles. American Journal of Physiology. Lung Cellular and Molecular Physiology 2000;278(3):L469-L476. |
R825815 (Final) |
not available |
Supplemental Keywords:
air, ambient air, indoor air, health effects, human health, sensitive populations, animal, mammalian, population, stressor, genetic predisposition, genetic polymorphisms, particulates, sulfates, toll-like receptor 4, Tlr4, intracellular signaling proteins, innate immunity, ozone, mixtures, linkage analyses., RFA, Health, Scientific Discipline, Toxicology, Genetics, Environmental Chemistry, Chemistry, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Children's Health, genetic susceptability, breeding studies, pulmonary immunotoxicity, asthma, sensitive populations, inhaled pollutants, Quantitative Trait Loci, airway disease, exposure, genetic predisposition, air pollution, acid coated particles, children, acidic particulate matter, human exposure, carbon black aerosol, inhalation, lung inflamation, inter-individual variation, human susceptibility, alveolar macrophage function, mortality, urban environment, inhaled particles, genetic susceptibility, air contaminant exposure, environmental hazard exposuresProgress 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.