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* From the Departments of Environmental Health, Pediatrics, & Medicine, University of Cincinnati, Cincinnati, OH; and the Divisions of Pulmonary Biology, Developmental Biology, Information Services, and Pulmonary Medicine, Children’s Hospital Medical Center, Cincinnati, OH.
Correspondence to: George D. Leikauf, PhD, Director, Molecular Toxicology Division, Department of Environmental Health, University of Cincinnati, PO Box 670056, Cincinnati, OH 45267-0056; e-mail: leikaugd{at}uc.edu
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Abstract |
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 TOP Abstract Results and DiscussionReferences |
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. Thus,
the functional genomic approaches of large gene set expression
(complementary DNA microarray) and genome-wide analyses continue to
provide novel insights into the genetic susceptibility of lung injury. In the past, numerous investigations have cataloged several relationships between environmental causes and respiratory diseases, such as silica and pulmonary fibrosis, coal dust and COPD, or asbestos and mesothelioma. The scientific strategy underlying this approach was to identify the hazards, understand the mechanisms, and evaluate the dose-response relationships so that human exposure could be limited and the disease could be prevented. Although individual susceptibility or resistance (eg, the "healthy worker" effect) has been suspected to influence these diseases, few scientists would evoke genetic causal factors and, until recently, avoided the systematic investigation of genetic risk factors, especially in acute cases of disease.
Gene-environmental interactions are critical to lung disease because each phenotype is only expressed when induced by an exposure (ie, penetrance depends on environmental exposures). Numerous environmental agents can induce common outcomes, thus common biological pathways may be controlling generalized responses to injury and repair. The relationships of individual exposure with individual risk are therefore very complex. An excellent example is cigarette smoking, which is a major cause of respiratory disease. Yet, not everyone who smokes cigarettes (even more than two packs per day) develops COPD, cardiovascular disease, or tobacco-related cancers.1 This is remarkable in that the amount of particulate matter that is deposited in the lung from 40 cigarettes exceeds 150 mg/d (assuming a 40% deposition of approximately 10 mg per cigarette). This dose is > 1,000 times greater than that resulting from exposure to ambient particulate matter (assuming a 30% deposition for Steubenville, OH, of particulate matter that is < 2.5 µm of 30 µg/m3 x 16.2 m3/d [based on the resting daily ventilation proposed by the National Council on Radiation Protection and Measurements]). However, these low-level ambient particulate matter exposures may be sufficient to produce acute cardiopulmonary mortality (especially in persons with COPD).2 Clearly, individuals vary in their resistance to the adverse pulmonary effects of a high-dose, single-day exposure to ambient particulate matter (ie, lifetime cigarette smoking), while others are extremely sensitive to a low-dose, single-day exposure to ambient particulate matter.
Currently, our understanding of the genetics of respiratory disease
that is induced by environmental exposures is very limited.
Environmental factors typically are viewed among geneticists as
confounders to the assessment of disease causality and the mechanisms
of heredity. For example, the initial studies of inheritance in lung
biology have focused mainly on diseases with strong genetic risk
factors (eg, cystic fibrosis or
1-antitrypsin deficiency in patients with
COPD). One motivation for this strategy was that predictions of
outcomes among offspring are more reliable in diseases with a single,
strong genetic component (ie, diseases following simple
mendelian inheritance patterns produced by single allelic variant [or
a few allelic variants]). Predictions of heritability are useful in
genetic counseling to inform parents and children of their individual
risks and in early diagnosis that is useful in monitoring patients
(eg, those with cystic fibrosis).3
4
Although
single-gene diseases are easy to study, these conditions are relatively
rare (eg, 1 cystic fibrosis patient per 2,500 white births)
compared to other respiratory diseases (eg, acute lung
injury, asthma, and COPD, which affect > 25 million people).
Common lung diseases, however, are likely to be controlled by multiple genes with varying influences on the disease process.5 This makes genetic analyses and predictions of inheritance difficult. Nonetheless, this task is becoming easier with the recent exposition of human genomic information that has changed our concept of many diseases. For complex diseases, like acute lung injury, gene-environmental interactions are likely to be major factors in controlling individual susceptibility, and additional genomic information should be applicable to this condition.
Acute lung injury is characterized by compromised gas exchange following macrophage activation, surfactant dysfunction, and epithelial destruction.6 Activated macrophages release a myriad of cytokines, reactive oxygen and nitrogen species, and proteolytic enzymes that, in turn, disrupt endothelial function. Together, these events lead to the key clinical manifestations of this condition, pulmonary edema, cellular infiltration, atelectasis, and, finally, complete respiratory failure.7 We reasoned that because the mortality rate varies between individuals who are initially classified with equivalent severity, persons could vary innately in their ability to withstand the progression of lung injury during mounting severe stress.
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Results and Discussion |
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TOPAbstractResults and DiscussionReferences |
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To determine the loci that may control resistance to acute lung injury, several inbred strains were screened, and then a quantitative trait locus (QTL) analysis was performed with the offspring generated from a sensitive mouse strain, A/J: (A), and a resistant mouse strain, C57BL/6-J: (B6), mouse strain following exposure to ozone. The genomic DNA was isolated from the liver, and polymerase chain reaction was performed for simple sequence length polymorphisms (SSLPs). Primers were chosen based on polymorphisms between the A and B6 strains (Research Genetics; Huntsville, AL). Using 65 SSLPs distributed at intervals of 20 to 30 centimorgan across the mouse genome, QTL analysis derived six regions of interest on chromosomes 1, 3, 7, 11, 12, and 13. These chromosomes then were analyzed with an additional 50 SSLPs to obtain a denser map, and the results then were analyzed by a software package (MapMaker/QTL, version 1.1b; MIT Center for Genome Research; Cambridge, MA).15 Significant linkage was found on chromosome 11 (proposed as "acute lung injury quantitative trait loci-1" [Aliq1]) and 13 (Aliq2) with log of odds (lod) scores of 4.1 (located at D11Mit263) and 3.3 (D13Mit59), respectively. The first value is above the level of significance of 3.3 for backcross analysis, as recommended by Lander and Kruglyak,16 and explains 13% of the genetic variance. (Note that an lod score of 3.3 is essentially a p value of 0.0005.) The second QTL, Aliq2, also had a lod score (3.3) that was significant for linkage and explained 10% of the genetic variance. In addition, this analysis determined suggestive linkages (lod scores, 1.7 to 2.3) on chromosomes 7 and 12.
To further assess the genetic determinants of this response, we used recombinant inbred (RI) mice. RI mice are derived by inbreeding the F2 progeny from two different strains of mice. By extending the inbreeding of the initial progeny for 20 generations, the initial segregation of each allele becomes fixed, and each RI mouse strain contains a unique combination of genetic material derived from the progenitor strains. This provides a tool for genetic mapping. The AXB and BXA RI strains are particularly useful because the progenitors differ in susceptibility to > 30 infectious or chronic diseases,17 18 and 31 live RI strains (and DNA of 41 strains) generated from these crosses are commercially available (Jackson Laboratory; Bar Harbor, ME). In addition, strain distribution patterns (SDPs) of > 700 SSLPs and other loci have been genotyped for this RI set.18 19 20 21 22 Following ozone exposure, we found that the mean survival times of the A and B6 progenitor strains were separated by 7 SDs. If resistance was controlled by a single dominant gene, then each RI strain should demonstrate a discrete sensitive or resistant phenotype. However, the RI strains demonstrated a continuous distribution, which is consistent with susceptibility to acute lung injury from oxidative damage being controlled by more than one gene, or modified by at least one other gene. Moreover, at least one RI strain had a survival time lower than the A progenitor strain. This also suggests that this may be an oligogenic or polygenic trait. The SDP for survival time in ozone was compared with all other SDPs that were available in the AXB, BXA RI set (MapManager).23 This analysis yielded another genetic region with significant linkage (lod score, 3.5) to survival time on mouse chromosome 17, which we proposed as Aliq3.
Subsequently, the F2 generation of mice that was
derived from the B6 and A strains was examined.13
These
data support the idea that acute lung injury induced by severe
oxidative stress is a complex oligogenetic trait in the mouse. The
primary finding strengthened our previous finding that a major QTL,
Aliq1, maps to mouse chromosome 11 (lod score, 6.8) in a
highly conserved region (having major areas of synteny with human
chromosome 17q21-q2224
25
26
). This region contains several
candidate genes, including nerve growth factor receptor, retinoic acid
receptor-, and granulocyte-colony stimulating factor. This QTL also
contains a gene cluster that houses at least 12 small inducible
cytokines, including chemokines that are similar to those induced in
the mouse lung by ozone exposure.27
28
In addition, the
F2 analysis provided evidence that was supportive
of the existence of modifier genes on chromosome 17 (Aliq3),
which had been identified by the RI analysis, and the suggestive QTLs
on chromosome 5 or 6. The QTL interval of Aliq1 on
chromosome 11 can be narrowed further using congenic strains and a
positional candidate gene approach.
Next, we conducted studies with additional agonists of acute lung injury. We have studied NiSO4 because it is an occupational contaminant, a component of cigarette smoke and ambient particulate matter,29 30 31 and because it is a potent respiratory irritant.32 33 In our study, NiSO4 exposure with 0.2-µm mass median aerodynamic matter modeled acute lung injury in mice, producing perivascular distention, alveolar epithelial damage, hemorrhage, and neutrophil infiltration.11
To compare inbred mouse strains, eight different strains were exposed
continuously to NiSO4, PTFE, or
O3, and the survival time was
recorded.11
13
Regardless of the irritant, the strain
phenotype pattern was similar with the A mouse strain being the most
sensitive, the C3H/He (C3) strain of intermediate sensitivity, and the
B6 strain being resistant to lung injury. The phenotype of the A
and B6 offspring (B6AF1) resembled the resistant
B6 parental strain with strains exhibiting sensitivity in the following
order (A>C3>B6 = B6AF1). Pulmonary pathologic
condition was comparable for the A and B6 mice, indicating that each
strain succumbed to severe edema (ie, acute lung injury).
The NiSO4 concentrations that were studied were
below the current occupational limit for soluble nickel (100 µg
Ni/m3) with continuous exposure to 15 µg
Ni/m3 producing a 20% mortality rate (14-day
study) in the sensitive A strain. The interstrain sensitivity
to protein (B6 > C3 > A) or polymorphonuclear leukocytes
(A 
B6 > C3) recovered in lung lavage fluid differed from that
of survival time. Thus, these phenotypes were discordant, suggesting
that they are not causally linked and are controlled by arrays of
independent genes. As was noted for acute lung injury, offspring from a
cross of the respective sensitive and resistant strain
(B6C3F1) exhibited phenotypes (lavage protein or
polymorphonuclear leukocytes) resembling the resistant parental strain.
The agreement of the strain phenotype pattern with these irritants
suggests that each agent may be initiating acute lung injury by a
common mechanism, possibly through genes controlling macrophage
activation and oxidative stress. In addition, the response of offspring
suggests that sensitivity is inherited as a recessive trait.
Subsequently, we characterized gene expression in this model by analyzing 8,734 sequence-verified murine complementary DNAs using microarray.34 In this study, messenger RNA levels were assessed in the B6 mouse lung following exposure to particulate NiSO4 for 0 (control), 3, 8, 24, 48, or 96 h. Lung polyadenylated messenger RNA was isolated, reverse-transcribed, and fluorescent-labeled. Samples from exposed mice (Cy5-labeled) were competitively hybridized against samples from unexposed control mice (Cy3-labeled) to microarrays containing murine complementary DNAs. To evaluate temporal patterns of gene expression, genes were clustered according to similarities in expression with time. To evaluate the pathophysiology of changes in gene expression, genes were categorized according to function. Changes in the expression of five selected genes also were analyzed by conventional (ie, Northern blot and S1 nuclease protection) assays. The clustering of coregulated genes (displaying similar temporal expression patterns) revealed the increased expression of relatively few genes, including those associated with macrophage activation, oxidative stress, antiproteolytic function, and extracellular matrix repair. Most notable was a delayed decrease in transcripts encoding surfactant proteins (SPs) (including SP-A, SP-B, and SP-C), which was confirmed by S1 nuclease protection analysis.
To further evaluate the genetics of NiSO4-induced acute lung injury, we performed a genome-wide analysis with 307 mice generated from the backcross of resistant B6xA F1 with susceptible A strain mice.12 Significant linkage was limited to a single region on chromosome 6 (proposed as Aliq4). Suggestive linkages were identified on chromosomes 1 and 12. Mice containing a genotype of either A/A or A/B6 alleles in each of these three regions (combined with an additional putative modifying locus chromosome 16 and the opposing A/B6 or A/A on another possible modifying locus on chromosome 9) had phenotypes that differed from each other that were comparable to those of the A and B6 parental strains. This suggests that the interplay of genes in these five chromosomal regions together is sufficient to explain this phenotype. This was supported by a Sewell-Wright analysis that estimated that five genes could explain the variance in the phenotype.
Importantly, the region on chromosome 6 (Aliq4) with
significant linkage to acute lung injury when exposed to
NiSO4 previously had been identified as a region
with significant linkage in an F2 population of
mice that had been exposed to ozone.13
Several interesting
candidate genes exist in the QTL on chromosome 6, including aquaporin
1, SP-B, and transforming growth factor (TGF)-.
A polypeptide member of a protein family that includes the epidermal
growth factor (EGF), TGF- is a ligand for the EGF receptor and
shares many similar biological properties with EGF.35
The
precise physiologic role of TGF- in lung injury is not completely
understood, but TGF- and EGF both appear to play a role in
initiating and sustaining tissue repair. Experiments in animals and
humans demonstrate that treatment with TGF- and EGF enhances the
healing of a variety of wounds. The topical administration either of
recombinant TGF- or EGF in pigs and humans accelerated the rate of
epidermal regeneration in skin burns,36
37
increased the
tensile strength of injured rabbit corneas,38
and
accelerated healing in patients with corneal defects.39
Parental pretreatment with TGF- or EGF decreased gastric mucosal
damage that had been induced by strong irritants, including absolute
ethanol, acidified aspirin, or stress in rodents.40
41
42
Complementary studies using TGF- knockout mice and induced
overexpressing mice found, respectively, increased and decreased levels
of dextran-sulfate-induced colitis compared with wild-type
mice.43
44
The administration of TGF- in models of lung injury has had moderate
success. For example, Kheradmand et al45
using denuded rat
type II epithelial cells in vitro demonstrated that the
TGF- level increased and that a monoclonal antibody to TGF- in
the presence of serum decreased the rate of "wound closure." To
study the effects of TGF- on lung injury in vivo, we
developed transgenic mice overexpressing human TGF- in the distal
lung under the control of the SP-C promoter.46
47
Mice
from four TGF- transgenic lines were exposed to ultrafine PTFE
particles that induce rapid lung injury48
and are fatal
within 8 h of administration in wild-type mice.10
TGF- transgenic mice demonstrated increased survival rates, reduced
inflammation, and pulmonary edema compared with wild-type mice,
indicating that human TGF- expression in the lung could
protect against acute lung injury.
To further investigate whether TGF- protects against acute lung
injury, four transgenic mouse lines with varying amount of expression
of human TGF- directed to the lung were exposed to
NiSO4. Acute lung injury was characterized by
assessing pulmonary histology, the BAL fluid cell differential and
protein level, lung wet-to-dry weight ratios, and lung homogenate
cytokine levels. The length of survival of four separate TGF-
transgenic mouse lines was significantly longer than that of
nontransgenic control mice, and survival time correlated with the
levels of TGF- expression in the lung. The transgenic line
expressing the highest level of TGF- (line 28) and nontransgenic
controls were compared for differences in lung histology, BAL fluid
protein and cell differential, lung wet-to-dry weight ratios, and lung
homogenate proinflammatory cytokines at 24, 48, and 72 h after
initiating nickel exposure. Within 72 h of nickel exposure,
line-28 mice demonstrated reduced histologic changes of lung
inflammation and edema, decreased BAL fluid protein and neutrophil
levels, reduced lung wet-to-dry weight ratios, and reduced production
of interleukin-1ß, interleukin-6, and macrophage inflammatory
protein-2 levels compared with nontransgenic controls. In this
transgenic mouse model, TGF- protects against nickel-induced
acute lung injury, at least in part, by attenuating the inflammatory
response.
In summary, acute lung injury is a severe (ie, mortality
rate, > 40%) respiratory disease associated with numerous
precipitating factors. Despite extensive research since its initial
description > 30 years ago, questions remain about the basic
pathophysiologic mechanisms that are critical to diminished survival
and their relationship to therapeutic strategies. Histopathology
revealed surfactant disruption, epithelial perturbation, and sepsis,
either as initiating factors or as secondary complications, which in
turn increased the expression of cytokines that sequester and activate
inflammatory cells, most notably neutrophils. The concomitant release
of reactive oxygen and nitrogen species subsequently modulates
endothelial function. Together, these events orchestrate the principal
clinical manifestations of the syndrome, pulmonary edema and
atelectasis. To better understand the gene-environmental interactions
controlling this complex process, we examined the relative sensitivity
of inbred mouse strains to acute lung injury induced by ozone,
ultrafine PTFE, or fine particulate NiSO4
(ie, 0.2-µm median mass aerodynamic matter, 15 to 150
µg/m3). Measuring survival time, protein and
neutrophil levels in BAL fluid, lung wet-to-dry weight ratio, and
histology, we found that these responses varied among inbred mouse
strains, and susceptibility is heritable. To assess the molecular
progression of NiSO4-induced acute lung injury,
the temporal relationships of 8,734 genes and expressed sequence tags
were assessed by complementary DNA microarray analysis. The clustering
of coregulated genes (displaying similar temporal expression patterns)
revealed the altered expression of relatively few genes. Enhanced
expression occurred mainly in genes associated with oxidative stress,
antiproteolytic function, and the repair of the extracellular matrix.
Subsequent to these changes, SPs and Clara cell secretory protein
messenger RNA expression decreased. A genome-wide analysis of 307 mice
generated from the backcross of resistant B6xA F1
mice with the susceptible A strain mice identified significant linkage
to a region on chromosome 6 (proposed as Aliq4) and
suggestive linkages on chromosomes 1 and 12. The combining of these
QTLs with two additional possible modifying loci (ie,
chromosome 9 and 16) accounted for the difference in survival time that
was noted in the A and B6 parental strains. In addition, the QTL on
chromosome 6 is in a region that we previously identified by a
genome-wide analysis conducted for acute lung injury that was induced
by ozone exposure and that contains several relevant candidate genes,
including SP-B, and TGF-. These candidates, in turn, can be directed
to the lung epithelium in transgenic mice or can be abated in inducible
and constitutive gene-targeted mice. The initial results are
encouraging and suggest that several of these mice vary in their
susceptibility to oxidant-induced lung injury. Thus, these combined
approaches have led to new insights into the functional genomics of
lung injury and diseases.
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Footnotes |
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Abbreviations: EGF = epidermal growth factor; lod = log of odds; PTFE = polytetrafluoroethylene; QTL = quantitative trait locus; RI = recombinant inbred; SDP = strain distribution pattern; SP = surfactant protein; SSLP = simple sequence length polymorphism; TGF = transforming growth factor
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References |
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TOPAbstractResults and DiscussionReferences |
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. Am J Physiol Lung Cell Mol Physiol 277,L1045-L1050 to dextran sodium sulphate induced colitis. Gut 43,64-70 (TGF-). Am J Pathol 151,1075-1083[Abstract]
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