Abstract
Inter-individual variation in human responses to air pollutants suggests that some subpopulations are at increased risk, and it is increasingly clear that genetic background is an important susceptibility factor.
Genetically standardised animal models provide useful investigative tools. Linkage analyses using inbred mice identified chromosomal segments (quantitative trait loci (QTL)), with genes controlling susceptibility to the lung inflammatory (chromosome 17), injury (chromosome 11), and hyperpermeability (chromosome 4) responses to ozone (O3) exposure. An immune dysfunction response induced by exposure to sulphate-associated particles is linked to the identical chromosome 17 and 11 QTLs described for O3 susceptibility, thus similar genetic mechanisms may be controlling pulmonary responses to these pollutants. Candidate genes within the QTLs on chromosomes 4 and 17 include the toll-like receptor 4 and the pro-inflammatory cytokine, tumour necrosis factor‐α, respectively. Functional analyses strongly support a role for these candidate genes in determining susceptibility to O3 and particulates. Because striking linkage homology exists between the human and mouse genomes, candidate susceptibility genes identified in the mouse are likely to aid research aimed at understanding human genetic factors that contribute to differential susceptibility.
To date, no studies have examined the interaction between age and genetic background in the development of air pollution-induced lung disease. However, investigations have suggested an influence of age on genetic susceptibility to lung cancer and other diseases, which indicate that an interaction between age and genetic background may be important in air pollution disease pathogenesis.
The author was supported by grants from the National Institutes of Health (HL57142, ES09606, and P30 ES00002) and the Environmental Protection Agency (R826724).
Air pollution continues to be an important public health and economic concern in industrialised cities throughout the world. Numerous epidemiological associations of adverse health outcomes with air pollution episodes support these concerns. Inter-individual variation in human responses to air pollutants indicates that not all individuals exposed to pollutants respond similarly. That is, some subpopulations are at increased risk to the detrimental effects of pollutant exposure. The United States Clean Air Act specifies that primary National Ambient Air Quality Standards for criteria pollutants be set low enough to protect the health of all susceptible groups within the population. Exceptions include those requiring life-support systems, intensive care unit patients, and newborn infants in nurseries. However, identification of those factors that determine susceptibility continues to be elusive.
Broadly, inter-individual variation in biological responses to environmental stimuli is a consequence of internal and external factors. External factors include physical forces (e.g. temperature, altitude), socioeconomic status, and previous exposure. Internal factors include sex, age, diet, and predisposing disease (e.g. asthma). Genetic background has become increasingly recognised as another important internal or host factor in environmental disease predisposition. In human populations, gene polymorphisms have been associated with susceptibility to environmental agents including pesticides and infectious agents 1–3.
It is also clear that multiple internal and external factors contribute to individual responses to air pollutants. A number of populations have been identified that are particularly susceptible to the toxic effects of airborne oxidants and particulates, including the elderly and individuals with cardiopulmonary disease 4, 5. Evidence also exists for genetic determinants of susceptibility to airborne pollutants, including ozone (O3) and particulates (see below). The primary objective of this manuscript is to discuss briefly studies that have identified a genetic basis for susceptibility to air pollution in animal models and human subjects, and the approaches used to identify susceptibility genes.
Research strategies employed to identify candidate genes
Two broad research strategies have been utilised to identify genes (quantitative trait loci (QTLs)) that determine susceptibility. The first is a genome scan or positional cloning (formerly known as reverse genetics). This strategy attempts to associate expression of genes or markers (e.g. microsatellite markers, see below) with phenotype(s) in segregant populations. A genome-wide screen is designed to identify linkage to any chromosomal intervals within the entire genome that may contain genes that are polymorphic between two strains of mice and may account for the differential response phenotype under study. That is, no a priori hypothesis about the role of a specific gene or genes is tested. This is ideally suited for genetically well-controlled models, particularly inbred mice, but is applicable to human populations as well. The second research strategy is the candidate gene approach (formerly “forward genetics”), in which genes are chosen a priori as likely mechanisms that determine the phenotype of interest. With the candidate gene approach, linkage is then assessed between the phenotype of interest and markers flanking the candidate genes or the candidate genes themselves. This strategy may implicate certain genes in the expressed phenotype. However, without a genome scan, the candidate gene approach may exclude other important loci that determine a quantitative trait, as well as the interaction between them. These strategies, alone or in combination, have been utilised in inbred mice to identify genetic mechanisms of a number of diseases. These include models of Huntingtons disease 6, 7, Duchenne muscular dystrophy 8, amyotrophic lateral sclerosis 9, insulin-dependent diabetes mellitus 10, Alzheimer's disease 11, von Willebrand disease 12, chronic granulomatous disease 13, and Niemann-Pick C1 disease 14. The considerable resources and energy applied to mapping of the mouse genome as an integral component of the Human Genome Project underlie the importance of this animal model for human diseases 15. One of the unifying concepts for genetic studies in mice is the linkage relationship of homologous loci from human and mouse. Specifically, highly significant homologies in gene order and chromosomal structure have been maintained between mice and humans since their divergence 16. Therefore, identification of the chromosomal location of a susceptibility gene in the mouse provides the basis for potentially localising a homologous gene in the human 17, 18.
Candidate genes for susceptibility to air pollutants
Identification of a QTL is a multi-step process that may be broadly grouped into three research objectives. The first objective is to determine whether the response phenotype is genetically determined and quantitative. After determining that the quantitative phenotype has a genetic basis, then the susceptibility QTL(s) are sought. Finally, significant informative QTLs are searched for candidate genes that may explain differential susceptibility/responsiveness in the model. It is beyond the scope of this manuscript to explain all of the details of linkage mapping; therefore, the reader is referred to excellent reviews of this topic by Broman 19, Moore and Nagle 20, and Silver 21. Selected investigations that have led to the identification of susceptibility QTLs for O3- and particle-induced pulmonary inflammation and injury are described below. This discussion is not exhaustive, and it is meant only to illustrate the approach to identify the genetic basis of susceptibility to air pollutants as well as candidate genes that may be important determinants of susceptibility.
Genetic determinants of susceptibility to ozone-induced lung inflammation and injury
O3 exposure induces multiple pulmonary and extrapulmonary responses in humans and animal models. O3 elicits inflammation, hyperreactivity and epithelial damage of the airways, as well as altered ventilation and decrements in pulmonary function 4, 22. O3 has also been demonstrated to either suppress or enhance immune responsiveness 23. Significant inter-strain variation in the magnitude of these responses has also been demonstrated in rats and mice, and has thus provided strong evidence of a genetic component to O3 responsiveness 24–26.
The inter-strain variation in susceptibility among inbred mice led the author's laboratory to conduct studies to identify the chromosomal location of the O3 susceptibility genes using susceptible C57BL/6J (B6) and resistant C3H/HeJ (C3) strains. A genome-wide search for linkage of the inflammation (polymorphonuclear leukocytes) phenotype was performed with informative simple sequence length polymorphisms (SSLPs) distributed at ∼10-centi-Morgan (cM) intervals throughout the genome (see 27 for more detail regarding choice of markers). The number and spacing of markers yielded complete coverage of the mouse genome with 95% confidence. Linkage was carried out with individual intercross animals derived from B6 and C3 progenitors (B6C3F2). The phenotyped F2 progeny were genotyped for each of the SSLPs, and linkage of susceptibility to O3 was evaluated using Map Manager QT and MAPMAKER-QTL software packages. Interval mapping by simple linear regression in the entire F2 cohort determined the presence of a susceptibility locus on chromosome 17 in the interval ∼16–22 cM (table 1⇓; 27). An additional QTL was detected on chromosome 11 between D11Mit20 and D11Mit12. Within the chromosome 17 QTL, there are a number of candidate genes, including the pro-inflammatory cytokine, tumour necrosis factor (TNF)‐α. Because TNF‐α may be postulated to have a role in the inflammatory response to oxidant-related lung injury, preliminary evaluations of this candidate gene were made for determination of differential O3‐induced inflammation in B6 and C3 mice. Pretreatment of susceptible B6 mice with a monoclonal antibody to TNF‐α significantly attenuated the inflammatory response to O3 relative to control B6 mice, thus providing support of TNF‐α as a candidate susceptibility gene in this model 27.
To further understand the mechanisms of O3‐induced lung injury, a genome-wide linkage analysis for susceptibility QTLs was performed to explain inter-strain differences in hyperpermeability induced by 72‐h exposure to 0.3 parts per million (ppm) O3. Because there is an apparent dissociation between inflammatory cell infiltration and lung hyperpermeability induced by O3 31, it was hypothesised that different loci control the hyperpermeability response. To determine the susceptibility QTLs, a genome screen was performed using recombinant inbred (RI) strains of mice derived from B6 and C3 progenitors (see 21 for explanation of the use of RIs for QTL mapping). A significant QTL was identified on chromosome 4, and suggestive QTLs were identified on chromosomes 3 and 11 (table 1⇑; 28). The chromosome 4 QTL contains a candidate gene, toll-like receptor 4 (TLR4) that has recently been implicated in innate immunity and endotoxin susceptibility 32–34. As a “proof of concept” that TLR4 has an important functional role in susceptibility, the hyperpermeability responses to O3 in C3H/HeOuJ (OuJ) and C3 mice were compared. These strains differ only at a polymorphism in the coding region of the TLR4 gene and the polymorphism confers resistance to endotoxin-induced injury in the C3 mouse compared to wild type OuJ. Significantly greater protein concentrations were found in OuJ mice compared with C3 mice after exposure to O3 28. Furthermore, reverse transcriptase polymerase chain reaction analysis demonstrated that TLR4 message levels in the lungs of C3 mice were markedly downregulated, while levels increased in the OuJ strain after O3 exposure 28. Together, results indicate that a QTL on chromosome 4 explains a significant portion of the genetic variance in O3‐induced hyperpermeability, and support a role for TLR4 as a strong candidate susceptibility gene. This is the first demonstration that innate immune mechanisms modulated by TLR4 are involved in the pulmonary response to oxidant exposure.
Prows et al. 26 performed a linkage analysis of susceptibility to death induced by exposure to high concentrations of O3. Using susceptible A/J and resistant B6 mice, these investigators identified a significant QTL on chromosome 11, and suggestive QTLs on chromosomes 13 and 17 (table 1⇑). Interestingly, the QTLs on chromosomes 11 and 17 are similar to those described by Kleeberger et al. 27 for susceptibility to inflammation induced by exposure to 0.3 ppm O3.
Evidence also exists for genetic determinants of susceptibility to O3 in human subjects. A number of laboratories have reported inter-individual variation in pulmonary function responses to O3 in otherwise normal, healthy human subjects 35–37. Inter-individual variation in the inflammatory response to O3 has also been described 38–42. A second line of evidence is the demonstration that specific gene polymorphisms associate with response phenotypes in exposed human subjects. Bergamaschi et al. 30 found that polymorphisms in genes for quinone-metabolising enzymes may have an important role in the pulmonary function and epithelial permeability responses to O3 in nonsmoking exercising subjects.
Genetic determinants of susceptibility to particle-induced lung inflammation and injury
Considerable attention has been focused on the adverse respiratory effects caused by inhalation of particles. Epidemiological studies have reported significant association of acute and chronic respiratory and nonrespiratory health effects with increases in particulate exposure throughout the industrialised world 4, 43, 44. Susceptible subpopulations include the aged (≥65 yrs) and patients with cardiopulmonary disease, such as chronic heart disease, chronic obstructive pulmonary disease, and asthma 4, 45–47. To determine whether genetic background is an important determinant of pulmonary responses to particulates, the inter-strain variance of lung responses to acid sulphate-coated particles (ACP) in inbred strains of mice was studied 48. Although the 4‐h challenge to ACP did not elicit a detectable inflammatory response, significant inter-strain differences were found in Fc receptor-mediated phagocytosis of alveolar macrophages (an indicator of innate immune defence). A genome scan similar to that described for O3 susceptibility (see above) was then performed with susceptible B6 and resistant C3 mice 29. Interestingly, linkage analyses identified a significant QTL on chromosome 17 and a suggestive QTL on chromosome 11 that nearly overlapped similar QTLs identified for O3 susceptibility. The common linkages suggest that similar genetic mechanisms may control pulmonary responses to O3‐induced inflammation and macrophage phagocytic dysfunction induced by ACP; however, further genetic analyses are required to confirm this hypothesis.
Contribution of age and genetic susceptibility to airborne pollutants
To date, few studies have examined the influence of age on genetic susceptibility to pulmonary diseases, and none have examined the interaction of these host factors on pollutant susceptibility. Gauderman and Morrison 49 performed a segregation analysis suggesting that the effect of genotype on lung cancer varies by age, such that age-specific relative risks are greatest in the young and decline thereafter. Though no specific genes were identified, this study strongly suggests that age and genetic background may be important co-determinants of pulmonary disease. Both factors should be considered in future proposals/studies to understand susceptibility to air pollutant-induced lung disease.
Conclusion
Air pollution-induced morbidity and mortality continue to be important public health concerns worldwide, and identification of susceptible subpopulations is of critical importance. Numerous factors may contribute to inter-individual susceptibility to the detrimental effects of air pollution, including age and genetic background. Linkage analyses with inbred mice and association studies with human subjects have led to identification of candidate susceptibility genes for pulmonary responses to ozone and sulphate-associated particles. The role of age as an interacting factor with genetic background has not been thoroughly examined in air pollution-induced lung disease, but likely has an important role in genetic susceptibility, and future studies should be designed to investigate the interaction of these two factors. An understanding of the biology of candidate genes will lead to an understanding of the genetic basis for differential responses to pollutant exposures. Furthermore, characterisation of a polymorphism in a pollutant susceptibility gene(s) may thus provide the means to identify individuals who are genetically susceptible to the development of injury.
- Received April 12, 2002.
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