- Open Access
Quantitative trait analysis of the development of pulmonary tolerance to inhaled zinc oxide in mice
© Wesselkamper et al. 2005
Received: 16 March 2005
Accepted: 18 July 2005
Published: 18 July 2005
Individuals may develop tolerance to the induction of adverse pulmonary effects following repeated exposures to inhaled toxicants. Previously, we demonstrated that genetic background plays an important role in the development of pulmonary tolerance to inhaled zinc oxide (ZnO) in inbred mouse strains, as assessed by polymorphonuclear leukocytes (PMNs), macrophages, and total protein in bronchoalveolar lavage (BAL) phenotypes. The BALB/cByJ (CBy) and DBA/2J (D2) strains were identified as tolerant and non-tolerant, respectively. The present study was designed to identify candidate genes that control the development of pulmonary tolerance to inhaled ZnO.
Genome-wide linkage analyses were performed on a CByD2F2 mouse cohort phenotyped for BAL protein, PMNs, and macrophages following 5 consecutive days of exposure to 1.0 mg/m3 inhaled ZnO for 3 hours/day. A haplotype analysis was carried out to determine the contribution of each quantitative trait locus (QTL) and QTL combination to the overall BAL protein phenotype. Candidate genes were identified within each QTL interval using the positional candidate gene approach.
A significant quantitative trait locus (QTL) on chromosome 1, as well as suggestive QTLs on chromosomes 4 and 5, for the BAL protein phenotype, was established. Suggestive QTLs for the BAL PMN and macrophage phenotypes were also identified on chromosomes 1 and 5, respectively. Analysis of specific haplotypes supports the combined effect of three QTLs in the overall protein phenotype. Toll-like receptor 5 (Tlr5) was identified as an interesting candidate gene within the significant QTL for BAL protein on chromosome 1. Wild-derived Tlr5-mutant MOLF/Ei mice were tolerant to BAL protein following repeated ZnO exposure.
Genetic background is an important influence in the acquisition of pulmonary tolerance to BAL protein, PMNs, and macrophages following ZnO exposure. Promising candidate genes exist within the identified QTL intervals that would be good targets for additional studies, including Tlr5. The implications of tolerance to health risks in humans are numerous, and this study furthers the understanding of gene-environment interactions that are likely to be important factors from person-to-person in regulating the development of pulmonary tolerance to inhaled toxicants.
Individuals may develop tolerance to adverse health effects elicited from repeated exposure to inhaled toxicants in several different occupational and environmental situations. Pulmonary tolerance can be defined as the lung's ability to withstand the detrimental effects of a toxic compound following multiple exposures. There are several implications of tolerance to human health, having both advantages and disadvantages with respect to the development of harmful health effects. Clinical investigations of zinc oxide (ZnO)- , endotoxin- , and ozone- [3–6] induced adverse respiratory effects have demonstrated inter-individual variability in the capacity to develop pulmonary tolerance following inhalation exposure. Because these clinical studies are more tightly controlled than epidemiologic studies, they suggest that genetic background and gene-environment interactions contribute to the development of pulmonary tolerance in humans.
We initially characterized the pulmonary tolerance phenotype in an outbred mouse model by assessing levels of BAL protein and polymorphonuclear leukocytes (PMNs) following single (1X) and 5 daily repeated (5X) exposures to inhaled ZnO to begin the identification of the genes regulating the development of pulmonary tolerance to repeated toxicant exposure . Significant genetic variability in the development of pulmonary tolerance to ZnO, endotoxin, and ozone was established in several inbred strains of mice in a subsequent study . Of the strains tested, the BALB/cByJ (CBy) strain was tolerant and the DBA/2J (D2) strain was non-tolerant to BAL protein, PMNs, and macrophages following repeated ZnO exposure.
Because inbred mouse strains are virtually identical at all loci throughout their genome, and also share several chromosomal regions of conserved synteny with humans, they are an ideal animal model in which to investigate genotype-environment interactions and identify genes controlling pulmonary responses to inhaled toxicants where no a priori evidence for their location exists . Inbred strains of mice have been successfully utilized in quantitative trait locus (QTL) analyses to identify candidate genes that control susceptibility to adverse pulmonary responses induced by a variety of inhaled gases [10–13] and particulates [14, 15]. In the present study, a CByD2F2 mouse cohort was used to determine QTLs linked to the development of pulmonary tolerance to BAL protein, PMN, and macrophage phenotypes following repeated ZnO exposure.
Materials and methods
Inbred BALB/cByJ (CBy), DBA/2J (D2) and CByD2F1/J (F1) mice (6–7 weeks of age) were purchased from The Jackson Laboratory (Bar Harbor, ME). CByD2F1/J mice were crossed to produce F2 (intercross) offspring in our laboratory animal facility. All mice were acclimated for at least 1 week before exposure, housed in a positive pressure environment with a 12-hour light/dark cycle starting at 6:00 a.m., and provided with water and standard laboratory rodent chow (Purina, Indianapolis, IN) ad libitum except during exposure. All mice were handled in accordance with the standards established by the U.S. Animal Welfare Acts set forth in National Institutes of Health guidelines, and by the New York University School of Medicine Division of Laboratory Animal Resources.
Zinc oxide generation, characterization, and exposure
Mice were exposed to ZnO (1.0 ± 0.2 mg/m3, mean ± SD) in stainless steel cages placed inside a 0.07 m3 Plexiglas chamber. ZnO fumes were generated in a furnace as previously described [7, 16]. The ZnO particles had a mass median aerodynamic diameter of 0.3 μm and geometric standard deviation of 1.5. Samples of the chamber atmosphere were collected approximately every 40 minutes from the manifold of the exposure system with polytetrafluoroethylene filters (Type TX40HI20-WW, Pallflex Products Corp., Putnam, CT), and the ZnO concentration was determined gravimetrically using a microbalance (Model C-30, Cahn Instruments, Cerritos, CA).
For linkage analyses, all F2 offspring (n = 299, 138 males and 161 females) were exposed at 7–12 weeks of age in a total of seven 5X exposure regimens. Development of pulmonary tolerance was assessed 24 hours after the fifth exposure, and exposure group sizes ranged from 36 to 45 animals. All ZnO exposures of F2 mice also contained at least two CBy, D2, and F1 mice for control purposes.
Bronchoalveolar lavage (BAL)
Mice were euthanized by intraperitoneal injections of ketamine HCl (100 mg/kg, Vetalar, Fort Dodge Laboratories, Inc., Fort Dodge, IA) and sodium pentobarbital (175 mg/kg, Sleepaway, Fort Dodge Laboratories, Inc.), and the posterior abdominal aorta was severed. The lungs of each mouse were lavaged two times with 1.2 ml of Dulbecco's phosphate buffered saline without Ca2+ or Mg2+ (pH 7.2–7.4, 37°C, Invitrogen, Carlsbad, CA). The collected BAL was immediately placed on ice (4°C) following recovery. Measurement of BAL protein, total cell counts, and differential cell counts were performed as previously described .
DNA isolation and genotyping
Genomic DNA was isolated from kidney tissue of each phenotyped F2 animal and for CBy, D2, and F1 controls (Wizard Genomic DNA Purification Kit, Promega, Madison, WI). DNA concentration was determined using a Beckman DU-650 spectrophotometer, and each sample was diluted to 10 ng/μl for genotype analysis. PCRs were performed to genotype F2 offspring for SSLPs located throughout the mouse genome. Eighty-six unlabeled primer pairs for SSLPs that differed in length by at least 5% between the CBy and D2 progenitor strains were purchased from Research Genetics (ResGen/Invitrogen). PCR was performed in 20 μl reaction volumes in 96-well low profile plates (Fisherbrand, Fisher Scientific, Fairlawn, NJ) using a PTC-100 thermal cycler (MJ Research, Watertown, MA). The final concentration for each reaction was: 10 mM Tris-HCl (pH 8.3), 50 mM KCL, 2.5 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate (Promega), 1.1X Rediload (ResGen/Invitrogen), and 0.132 μM of each SSLP primer pair. This reaction mixture was added to 100 ng of genomic DNA and 0.45 U of Taq DNA polymerase (Roche Applied Science, Indianapolis, IN). Final reaction mixtures were initially denatured at 94°C for 3 min, followed by 36 amplification cycles (94°C for 30 seconds, 57°C for 45 seconds, and 72°C for 30 seconds + 1 second/cycle). A final extension step at 72°C for 7 min was followed by refrigeration (4°C). PCR products were differentiated on 3% agarose (Invitrogen) gels and all samples were visualized by ethidium bromide staining using a ChemiImager-4400 low light imaging system (Imgen Technologies, Alexandria, VA) and called by a single investigator. Any questionable calls in reading the genotype from the image were reviewed by a second investigator and if not resolved, that sample was rerun.
Estimation of loci
The number of independently segregating loci was calculated using the following formula by Wright : n = (P2 - F1)2/4(|σ2 F2 - σ2 F1|), where n is an estimate of the number of independent loci; P2 and F1 are the mean BAL protein responses following 5X ZnO exposure in CBy and CByD2F1 mice, respectively; σ2 F2 and σ2 F1 are the computed variances of the F2 and CByD2F1 mice, respectively.
A genome scan was performed to identify associations between genotypes and the BAL protein phenotype using a CByD2F2 mouse cohort. All phenotypic data were natural log normalized to generate a normal distribution to meet normality assumptions of the Map Manager QT computer program. Interval analyses were then performed by fitting a regression equation for the effect of a theoretical QTL at the position of each SSLP and at 1-centimorgan (cM) intervals between SSLPs using free, additive, recessive, and dominant regression models. The regressions and significance of each genotype/phenotype association (or likelihood χ2 statistic) were calculated by Map Manager . Permutation tests were performed on the phenotypic and genotypic data using Map Manager to generate empirical thresholds for significance following the methods of Churchill and Doerge [19, 20].
For the initial genome scan, the 15 most tolerant and 15 most non-tolerant F2 animals with respect to BAL protein, PMNs, and macrophages (i.e., the phenotypic extremes) were used for selective genotyping [9, 21]. Interval analyses were done as stated previously, and 10,000 permutations were performed to generate significant and suggestive likelihood χ2 statistic thresholds for the BAL protein phenotype. Following the identification of a suggestive QTL for BAL protein on chromosome 1, the entire F2 cohort was examined for additional QTLs. For the BAL protein phenotype, three additional SSLPs on chromosome 1 were analyzed, and a permutation test (10,000 permutations) was performed with only chromosome 1. This method was similar to that used in previous linkage studies with inhaled particles and gases that utilized Map Manager QT [10, 14, 22]. All likelihood χ2 statistic thresholds corresponded to those reported in the aforementioned linkage studies.
A haplotype analysis was carried out similar to that done previously by Prows and Leikauf to determine the contribution of each QTL and QTL combination to the overall BAL protein phenotype . This method quantifies any difference in mean BAL protein levels that are linked with a particular haplotype. Haplotypes for the following SSLPs were used for this analysis: D1Mit291 (101.5 cM), D4Mit254 (82.5 cM), and D5Mit193 (1.0 cM). Mean BAL protein concentrations for groups of F2 mice with the same haplotype at each QTL or QTL combinations were calculated and compared with the mean BAL protein of F2 mice with the other haplotypes to determine the contributions of these QTLs to the overall BAL protein phenotype.
Phenotypes of the CByD2F2 cohort
Genotyping of the entire F2 cohort
The combinatorial effect of the QTLs on chromosomes 1, 4, and 5 were also examined. For any combination of two QTLs, F2 mice that had a DD-DD haplotype for markers on chromosomes 1 and 5 (D1Mit291 and D5Mit193) had an average of 310 μg/ml more BAL protein than those that were CC or H (CC/H) for those markers. The greatest difference in mean BAL protein levels were found in F2 mice that had a DD-CC/H-DD haplotype for the three QTLs on chromosomes 1, 4, and 5, (i.e., D1Mit291, D4Mit254, and D5Mit193), respectively. These mice had an average of 345 μg/ml more BAL protein than those that were CC/H at the markers across the three chromosomes.
Identification of candidate genes
Positional candidate genes from linkage analysis of pulmonary tolerance to BAL protein following repeated ZnO exposure.
Dfy, Duffy blood group
Tlr5, toll-like receptor 5
Adprt1, ADP-ribosyltransferase 1
Tgfb2, transforming growth factor-β2
Traf5, TNF receptor-associated factor 5
Slc30a1, solute carrier family 30 (zinc transporter), member 1
Ptafr, platelet-activating factor receptor
Slc30a2, solute carrier family 30, member 2
1 (p35), 1 (p34-p36)
Pla2g2a, 2c, 2d-f, 5, phospholipase A2 groups
1 (p36.3-p36.2), 1 (p36)
Tnfrsf1b, 8, 9, TNF-receptor superfamily members
Tnfrsf4, TNF-receptor superfamily, member 4
Slc4a2, solute carrier family 4, member 2
Il6, interleukin 6
Slc30a3, solute carrier family 30 (zinc transporter), member 3
Positional candidate genes from linkage analysis of pulmonary tolerance to BAL PMNs and macrophages following repeated ZnO exposure.
Chr 1 (PMNs)
Ccl20, chemokine (C-C motif) ligand 20
Cxcr4 chemokine (C-X-C motif) receptor 4
Il10, interleukin 10
Chr 5 (Macrophages)
Cxcl1, chemokine (C-X-C motif) ligand 1
Cxcl2, chemokine (C-X-C motif) ligand 2
Cxcl5, chemokine (C-X-C motif) ligand 5
Cxcl9, chemokine (C-X-C motif) ligand 9
Cxcl10, chemokine (C-X-C motif) ligand 10
Spp1, secreted phosphoprotein 1
Development of pulmonary tolerance in MOLF/Ei mice
Clinical studies on the acquisition of tolerance to inhaled toxicants suggest that genetic background and gene-environment interactions contribute to the development of pulmonary tolerance in humans. We have previously determined that a genetic component exists in a mouse model of pulmonary tolerance to repeated ZnO exposure . In the present study, we performed linkage analyses on an F2 mouse population derived from tolerant CBy and non-tolerant D2 strains to further ascertain the contribution of genetic background to the development of pulmonary tolerance, and identify candidate genes that may be important regulators in the acquisition of tolerance.
Initial analysis using the most tolerant and non-tolerant F2 mice with respect to BAL protein generated a putative QTL (designated as the zinc-induced tolerance (ZIT 1) locus) on the distal end of chromosome 1. Further assessment of the entire F2 cohort demonstrated that the QTL on chromosome 1 attained an LRS value for significant linkage, and also identified two suggestive QTLs located on chromosomes 4 and 5. Using a variation of the Wright equation , a minimum of three loci were estimated to be independently segregating with the BAL protein phenotype following 5X ZnO exposure, thus in agreement with the results of the linkage analysis. There are several approaches that can be pursued to focus in on the QTL intervals that were identified. For instance, increasing the number of F2 mice used in the QTL analysis is one alternative. The major disadvantage of this, however, is that segregating QTLs contribute a great deal of phenotypic "noise," making it problematic when determining whether or not a given mouse has inherited a particular QTL . Thus, in order to separate the effects of multiple loci, congenic mouse strains for each QTL could be generated, which could then be used to breed multicongenic lines to examine the existence of any epistatic effects. Additionally, future studies could employ the use of a backcross (CByD2F1 × CBy) mouse population to expand the evaluation of the QTL effects to the overall BAL protein phenotype.
To measure the contribution of each individual QTL and each QTL combination to the overall BAL protein phenotype, the protein levels for F2 mice with each particular haplotype were compared. Mice with opposite allelic combinations for QTLs on chromosomes 1 and 5 had a difference of 310 μg/ml BAL protein, which accounts for approximately one-third of the total difference in mean BAL protein between the parental CBy and D2 strains. Additionally, the mean BAL protein level of F2 mice with a DD haplotype for QTLs on chromosomes 1, 4, and 5 was over half that of the non-tolerant D2 parental strain. These analyses suggest that although three QTLs were identified, the development of pulmonary tolerance to BAL protein is a decidedly complex phenotype that is regulated by a number of different genes, some of which were likely not identified by linkage analysis using an F2 cohort.
Candidate genes within the significant QTL on chromosome 1 (ZIT 1) that could play a role in controlling the development of tolerance to BAL protein following repeated ZnO exposure are presented in Table 1. The Duffy blood group (Dfy) has been shown to modulate the intensity of inflammation following endotoxin exposure , and has a role in enhancing inflammatory cell recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium . ADP-ribosyltransferase 1 (Adprt1) and tumor necrosis factor (TNF) receptor-associated factor (Traf5) are functionally associated with nuclear factor (NF)-κB, a key transcription factor in the regulation of the inflammatory process [28, 29]. Additionally, activation of ADPRT1 plays a role in endotoxin-induced BAL protein increases [30, 31]. Activated transforming growth factor-β has been shown to be a mediator of bleomycin- and endotoxin-induced lung permeability (i.e., BAL protein) in mice . Finally, solute carrier family 30 (zinc transporter), member 1 (Slc30a1) is a metal transporter on the plasma membrane that confers resistance to zinc and cadmium toxicity in vitro via an efflux mechanism [33, 34].
Candidate genes for BAL protein tolerance identified within the suggestive QTL intervals on chromosomes 4 and 5 are also presented in Table 1. Notable candidates include platelet-activating factor receptor (Ptafr) and phospholipase A2 (Pla2) that have been implicated as potential mediators of toxicant-induced BAL protein increases [35–37]. Interleukin (IL)-6 protein was increased following ZnO exposure in humans [1, 38], and was hypothesized to be an anti-inflammatory suppressor of ZnO-induced lung injury. Interestingly, increased lung IL-6 levels have been shown to mediate pulmonary tolerance to ozone in rats . Lastly, solute carrier family 30 (zinc transporter) members 2 and 3 (Slc30a2 and Slc30a3) have been identified as zinc transporters that protect against zinc-induced toxicity in both cell culture and animal models [40, 41]. The development of pulmonary tolerance to BAL protein in our model could be regulated by any number of the aforementioned candidate genes. These candidates will be investigated in future studies of transcriptional and protein regulation to determine their roles in the development of tolerance.
The suggestive QTLs for tolerance to BAL PMNs and macrophages (on chromosomes 1 and 5, respectively) were also examined for positional candidate genes (Table 2). Chemokine (C-C motif) ligand 20 (Ccl20), chemokine (C-X-C motif) receptor 4 (Cxcr4), and interleukin-10 (Il10) were identified as candidates for BAL PMN tolerance. The movement of PMNs into inflammatory tissues is regulated by chemotactic factors (e.g., chemokines) that signal through numerous chemokine receptors . PMNs are capable of producing several chemokines and proinflammatory cytokines, indicating that PMNs may be important in auto-direction of cell trafficking during inflammation. CXCR4 expression has been detected on human PMNs , and coordinated chemokine receptor gene expression may control the tissue-specific migration and activation status of PMNs into the lung. Human PMNs are also able to express CCL20 , which is able to recruit immature dendritic cells that play an important role in the initiation of the immune response as well as chronic inflammation [45–47]. Finally, IL-10 can be produced by T cells and is able to diminish PMN influx by inhibition of expression of proinflammatory chemokines , NF-κB (via I kappa kinase) , and TNF , as well as modulate cells and effector functions associated with an allergic response. With respect to the BAL macrophage phenotype, the epidermal growth factor receptor (EGFR) ligands amphiregulin (Areg) and betacellulin (Btc) were identified as candidate genes within the suggestive QTL on chromosome 5. Ligand-dependent activation of the EGFR by particles rich in metal content lead to activation of the MAP kinase signaling cascade and cytokine expression and secretion [51, 52]. Secreted phosphoprotein 1 (Spp1, also known as osteopontin) was also identified, and it can act as a chemoattractant for macrophages [53, 54]. Lastly, a host of chemokine (C-X-C motif) ligands were identified between 51 and 53 cM that could be involved in macrophage chemotaxis . Again, tolerance to BAL PMNs and macrophages may be under the influence of any number of these candidate genes.
In the present study, we identified toll-like receptor (Tlr5) as a candidate gene within the significant ZIT 1 QTL on chromosome 1 for tolerance to BAL protein. Toll-like receptors activate intra-cellular signaling that culminates in the induction of a multitude of effector genes . Tlr5 has been shown to be an important gene in the immune response to Gram-positive and Gram-negative bacterial flagellin [57, 58]. We utilized a wild-derived inbred mouse strain called MOLF/Ei which has non-conservative mutations in Tlr5 that are associated with a lower level of expression  to determine whether Tlr5 plays a role in the development of tolerance to BAL protein in our ZnO model. Because MOLF/Ei mice have a lower level of TLR5 mRNA expression compared to other strains , it was unclear whether we would observe tolerance after a single exposure to ZnO. Although the MOLF/Ei strain has no "wild-type" control strain per se, MOLF/Ei mice were tolerant to increased BAL protein following repeated ZnO exposure when compared to the non-tolerant D2 strain. These data support a role for Tlr5 in the development of tolerance to BAL protein. Interestingly, Kleeberger and colleagues identified Tlr4 as a candidate gene in a study of susceptibility to increased BAL protein in mice following a single exposure to 0.3 ppm ozone for 72 hours , which also suggests toll-like receptor signaling may be important in the regulation of inhaled toxicant-induced changes in BAL protein.
The mechanism through which Tlr5 signaling may regulate the development of tolerance to ZnO is unknown. While endotoxin  and bacterial flagellin  have been demonstrated as the primary ligands for Tlr4 and Tlr5, respectively, the ligand(s) that is responsible for toll-like receptor signaling following exposure to inhaled toxicants such as ozone and ZnO is unknown. Although there have been no studies on endogenous ligands for Tlr5, several endogenous ligands for Tlr4 have been identified. For example, fibronectin  and hyaluronic acid  are produced by lung cells during lung injury and are endogenous Tlr4 ligands. The downstream pathway through which Tlr5 may regulate the development of tolerance to ZnO-induced BAL protein is potentially via NF-κB, a transcription factor that is known to induce several cytokines involved in inhaled ZnO responsiveness such as IL-8 and IL-6 [1, 63]. Furthermore, NF-κB-dependent gene expression is decreased in Tlr5-mediated tolerance to flagellin in vitro . Thus, it is plausible that a mutation in Tlr5, a regulatory element upstream of NF-κB signaling, could modulate tolerance to BAL protein from repeated ZnO exposure. Finally, Tlr5 may function as a danger signal receptor in the development of ZnO tolerance, consistent with the "danger model" of innate immunity that explicates activation of the innate immune system by factors other than foreign antigens . Whatever the case, much work is needed in understanding how toxicants function through toll-like receptor signaling mechanisms in the lung to regulate adverse responses such as BAL protein.
In summary, linkage analysis of a large F2 mouse cohort identified significant linkage of a QTL (ZIT 1) on chromosome 1 associated with tolerance to BAL protein following repeated exposure to inhaled ZnO. Suggestive QTLs were also identified on chromosomes 4 and 5 for BAL protein, on chromosome 1 for BAL PMNs, and on chromosome 5 for BAL macrophages. Haplotype analysis suggested that the combinatorial effects of these three loci contributed to the overall phenotype, which agrees with the calculated number of segregating loci. Tlr5 was identified within the significant QTL for BAL protein on chromosome 1. Wild-derived Tlr5-mutant MOLF/Ei mice were determined to be tolerant to BAL protein following repeated ZnO exposure, suggesting a role for Tlr5 in the development of pulmonary tolerance to inhaled toxicants.
These data substantiate genetic background as an important influence in the acquisition of pulmonary tolerance following exposure to inhaled toxicants such as ZnO, and promising candidate genes exist within the identified QTL intervals that would be good targets for additional studies on the pathogenesis of tolerance.
The authors would like to thank the following people for their contributions to this study: Karen Galdanes, Margaret Krasinski, Kathy Baker, and Dr. Moon-shong Tang (New York University); Dr. George D. Leikauf (University of Cincinnati); Dr. Daniel R. Prows (Cincinnati Children's Hospital); Dr. Steven R. Kleeberger (National Institute of Environmental Health Sciences); Dr. Carrie L. Welch (Columbia University). This study was supported by USEPA Grant R-826244, USEPA Particulate Matter Center Grant R-827351, USEPA STAR Fellowship U-91578301, NIEHS Center Grant of Excellence ES-00260, and CDC/NIOSH (via Mt. Sinai School of Medicine, New York, NY) Grant T-42CCT210425-06-01.
- Fine JM, Gordon T, Chen LC, Kinney P, Falcone G, Beckett WS: Metal fume fever: characterization of clinical and plasma IL-6 responses in controlled human exposures to zinc oxide fume at and below the threshold limit value. J Occup Environ Med 1997, 39:722–726.View ArticlePubMedGoogle Scholar
- Kline JN, Cowden JD, Hunninghake GW, Schutte BC, Watt JL, Wohlford-Lenane CL, Powers LS, Jones MP, Schwartz DA: Variable airway responsiveness to inhaled lipopolysaccharide. Am J Respir Crit Care Med 1999, 160:297–303.View ArticlePubMedGoogle Scholar
- Christian DL, Chen LL, Scannell CH, Ferrando RE, Welch BS, Balmes JR: Ozone-induced inflammation is attenuated with multiday exposure. Am J Respir Crit Care Med 1998, 158:532–537.View ArticlePubMedGoogle Scholar
- Jorres RA, Holz O, Zachgo W, Timm P, Koschyk S, Muller B, Grimminger F, Seeger W, Kelly FJ, Dunster C, Frischer T, Lubec G, Waschewski M, Niendorf A, Magnussen H: The effect of repeated ozone exposures on inflammatory markers in bronchoalveolar lavage fluid and mucosal biopsies. Am J Respir Crit Care Med 2000, 161:1855–1861.View ArticlePubMedGoogle Scholar
- Horvath SM, Gliner JA, Folinsbee LJ: Adaptation to ozone: duration of effect. Am Rev Respir Dis 1981, 123:496–499.PubMedGoogle Scholar
- Hackney JD, Linn WS, Mohler JG, Collier CR: Adaptation to short-term respiratory effects of ozone in men exposed repeatedly. J Appl Physiol 1977, 43:82–85.PubMedGoogle Scholar
- Wesselkamper SC, Chen LC, Gordon T: Development of pulmonary tolerance in mice exposed to zinc oxide fumes. Toxicol Sci 2001, 60:144–151.View ArticlePubMedGoogle Scholar
- Wesselkamper SC, Chen LC, Kleeberger SR, Gordon T: Genetic variability in the development of pulmonary tolerance to inhaled pollutants in inbred mice. Am J Physiol Lung Cell Mol Physiol 2001, 281:L1200–9.PubMedGoogle Scholar
- Silver LM: Mouse genetics: concepts and applications. New York, Oxford University Press; 1995.Google Scholar
- Cho HY, Jedlicka AE, Reddy SP, Zhang LY, Kensler TW, Kleeberger SR: Linkage analysis of susceptibility to hyperoxia. Nrf2 is a candidate gene. Am J Respir Cell Mol Biol 2002, 26:42–51.View ArticlePubMedGoogle Scholar
- Kleeberger SR, Levitt RC, Zhang LY, Longphre M, Harkema J, Jedlicka A, Eleff SM, DiSilvestre D, Holroyd KJ: Linkage analysis of susceptibility to ozone-induced lung inflammation in inbred mice. Nat Genet 1997, 17:475–478.View ArticlePubMedGoogle Scholar
- Prows DR, Shertzer HG, Daly MJ, Sidman CL, Leikauf GD: Genetic analysis of ozone-induced acute lung injury in sensitive and resistant strains of mice. Nat Genet 1997, 17:471–474.View ArticlePubMedGoogle Scholar
- Kleeberger SR, Reddy SP, Zhang LY, Cho HY, Jedlicka AE: Toll-like receptor 4 mediates ozone-induced murine lung hyperpermeability via inducible nitric oxide synthase. Am J Physiol Lung Cell Mol Physiol 2001, 280:L326–33.PubMedGoogle Scholar
- Ohtsuka Y, Brunson KJ, Jedlicka AE, Mitzner W, Clarke RW, Zhang LY, Eleff SM, Kleeberger SR: Genetic linkage analysis of susceptibility to particle exposure in mice. Am J Respir Cell Mol Biol 2000, 22:574–581.View ArticlePubMedGoogle Scholar
- Prows DR, Leikauf GD: Quantitative trait analysis of nickel-induced acute lung injury in mice. Am J Respir Cell Mol Biol 2001, 24:740–746.View ArticlePubMedGoogle Scholar
- McCarthy JF, Yurek GJ, Elliott JF, Amdur MO: Generation and characterization of submicron aerosols of zinc oxide. Am Ind Hyg Assoc J 1982., 880–886: Google Scholar
- Wright S: The genetics of quantitative variability. In Quantitave Genetics. Edited by: Reeve ECH and Waddington CH. London, HMSO; 1952.Google Scholar
- Manly KF, Olson JM: Overview of QTL mapping software and introduction to map manager QT. Mamm Genome 1999, 10:327–334.View ArticlePubMedGoogle Scholar
- Churchill GA, Doerge RW: Empirical threshold values for quantitative trait mapping. Genetics 1994, 138:963–971.PubMedPubMed CentralGoogle Scholar
- Doerge RW, Churchill GA: Permutation tests for multiple loci affecting a quantitative character. Genetics 1996, 142:285–294.PubMedPubMed CentralGoogle Scholar
- Lander ES, Botstein D: Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 1989, 121:185–199.PubMedPubMed CentralGoogle Scholar
- Kleeberger SR, Reddy S, Zhang LY, Jedlicka AE: Genetic susceptibility to ozone-induced lung hyperpermeability: role of toll-like receptor 4. Am J Respir Cell Mol Biol 2000, 22:620–627.View ArticlePubMedGoogle Scholar
- Lander E, Kruglyak L: Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 1995, 11:241–247.View ArticlePubMedGoogle Scholar
- Collins FS: Positional cloning moves from perditional to traditional. Nat Genet 1995, 9:347–350.View ArticlePubMedGoogle Scholar
- Sebastiani G, Leveque G, Lariviere L, Laroche L, Skamene E, Gros P, Malo D: Cloning and characterization of the murine toll-like receptor 5 (Tlr5) gene: sequence and mRNA expression studies in Salmonella-susceptible MOLF/Ei mice. Genomics 2000, 64:230–240.View ArticlePubMedGoogle Scholar
- Dawson TC, Lentsch AB, Wang Z, Cowhig JE, Rot A, Maeda N, Peiper SC: Exaggerated response to endotoxin in mice lacking the Duffy antigen/receptor for chemokines (DARC). Blood 2000, 96:1681–1684.PubMedGoogle Scholar
- Lee JS, Frevert CW, Wurfel MM, Peiper SC, Wong VA, Ballman KK, Ruzinski JT, Rhim JS, Martin TR, Goodman RB: Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo. J Immunol 2003, 170:5244–5251.View ArticlePubMedPubMed CentralGoogle Scholar
- Hassa PO, Hottiger MO: A role of poly (ADP-ribose) polymerase in NF-kappaB transcriptional activation. Biol Chem 1999, 380:953–959.View ArticlePubMedGoogle Scholar
- Nakano H, Oshima H, Chung W, Williams-Abbott L, Ware CF, Yagita H, Okumura K: TRAF5, an activator of NF-kappaB and putative signal transducer for the lymphotoxin-beta receptor. J Biol Chem 1996, 271:14661–14664.View ArticlePubMedGoogle Scholar
- Albertini M, Clement MG, Lafortuna CL, Caniatti M, Magder S, Abdulmalek K, Hussain SN: Role of poly-(ADP-ribose) synthetase in lipopolysaccharide-induced vascular failure and acute lung injury in pigs. J Crit Care 2000, 15:73–83.View ArticlePubMedGoogle Scholar
- Liaudet L, Pacher P, Mabley JG, Virag L, Soriano FG, Hasko G, Szabo C: Activation of poly(ADP-Ribose) polymerase-1 is a central mechanism of lipopolysaccharide-induced acute lung inflammation. Am J Respir Crit Care Med 2002, 165:372–377.View ArticlePubMedGoogle Scholar
- Pittet JF, Griffiths MJ, Geiser T, Kaminski N, Dalton SL, Huang X, Brown LA, Gotwals PJ, Koteliansky VE, Matthay MA, Sheppard D: TGF-beta is a critical mediator of acute lung injury. J Clin Invest 2001, 107:1537–1544.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim AH, Sheline CT, Tian M, Higashi T, McMahon RJ, Cousins RJ, Choi DW: L-type Ca(2+) channel-mediated Zn(2+) toxicity and modulation by ZnT-1 in PC12 cells. Brain Res 2000, 886:99–107.View ArticlePubMedGoogle Scholar
- Palmiter RD, Findley SD: Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. Embo J 1995, 14:639–649.PubMedPubMed CentralGoogle Scholar
- Furue S, Kuwabara K, Mikawa K, Nishina K, Shiga M, Maekawa N, Ueno M, Chikazawa Y, Ono T, Hori Y, Matsukawa A, Yoshinaga M, Obara H: Crucial role of group IIA phospholipase A(2) in oleic acid-induced acute lung injury in rabbits. Am J Respir Crit Care Med 1999, 160:1292–1302.View ArticlePubMedGoogle Scholar
- Kaneko T, Ikeda H, Fu L, Nishiyama H, Okubo T: Platelet-activating factor mediates the ozone-induced increase in airway microvascular leakage in guinea pigs. Eur J Pharmacol 1995, 292:251–255.PubMedGoogle Scholar
- Longphre M, Zhang L, Harkema JR, Kleeberger SR: Ozone-induced pulmonary inflammation and epithelial proliferation are partially mediated by PAF. J Appl Physiol 1999, 86:341–349.PubMedGoogle Scholar
- Fine JM, Gordon T, Chen LC, Kinney P, Falcone G, Sparer J, Beckett WS: Characterization of clinical tolerance to inhaled zinc oxide in naive subjects and sheet metal workers. J Occup Environ Med 2000, 42:1085–1091.View ArticlePubMedGoogle Scholar
- McKinney WJ, Jaskot RH, Richards JH, Costa DL, Dreher KL: Cytokine mediation of ozone-induced pulmonary adaptation. Am J Respir Cell Mol Biol 1998, 18:696–705.View ArticlePubMedGoogle Scholar
- Palmiter RD, Cole TB, Findley SD: ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. Embo J 1996, 15:1784–1791.PubMedPubMed CentralGoogle Scholar
- Palmiter RD, Cole TB, Quaife CJ, Findley SD: ZnT-3, a putative transporter of zinc into synaptic vesicles. Proc Natl Acad Sci U S A 1996, 93:14934–14939.View ArticlePubMedPubMed CentralGoogle Scholar
- Baggiolini M: Chemokines and leukocyte traffic. Nature 1998, 392:565–568.View ArticlePubMedGoogle Scholar
- Yousefi S, Cooper PR, Potter SL, Mueck B, Jarai G: Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes. J Leukoc Biol 2001, 69:1045–1052.PubMedGoogle Scholar
- Scapini P, Laudanna C, Pinardi C, Allavena P, Mantovani A, Sozzani S, Cassatella MA: Neutrophils produce biologically active macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19. Eur J Immunol 2001, 31:1981–1988.View ArticlePubMedGoogle Scholar
- Dieu-Nosjean MC, Vicari A, Lebecque S, Caux C: Regulation of dendritic cell trafficking: a process that involves the participation of selective chemokines. J Leukoc Biol 1999, 66:252–262.PubMedGoogle Scholar
- Dieu-Nosjean MC, Massacrier C, Homey B, Vanbervliet B, Pin JJ, Vicari A, Lebecque S, Dezutter-Dambuyant C, Schmitt D, Zlotnik A, Caux C: Macrophage inflammatory protein 3alpha is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors. J Exp Med 2000, 192:705–718.View ArticlePubMedPubMed CentralGoogle Scholar
- Sallusto F, Lanzavecchia A: Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J Exp Med 1999, 189:611–614.View ArticlePubMedPubMed CentralGoogle Scholar
- Shanley TP, Vasi N, Denenberg A: Regulation of chemokine expression by IL-10 in lung inflammation. Cytokine 2000, 12:1054–1064.View ArticlePubMedGoogle Scholar
- Spight D, Zhao B, Haas M, Wert S, Denenberg A, Shanley TP: Immunoregulatory effects of regulated, lung-targeted expression of IL-10 in vivo. Am J Physiol Lung Cell Mol Physiol 2005, 288:L251–65.View ArticlePubMedGoogle Scholar
- Mulligan MS, Warner RL, Foreback JL, Shanley TP, Ward PA: Protective effects of IL-4, IL-10, IL-12, and IL-13 in IgG immune complex-induced lung injury: role of endogenous IL-12. J Immunol 1997, 159:3483–3489.PubMedGoogle Scholar
- Wu W, Samet JM, Ghio AJ, Devlin RB: Activation of the EGF receptor signaling pathway in airway epithelial cells exposed to Utah Valley PM. Am J Physiol Lung Cell Mol Physiol 2001, 281:L483–9.PubMedGoogle Scholar
- Blanchet S, Ramgolam K, Baulig A, Marano F, Baeza-Squiban A: Fine particulate matter induces amphiregulin secretion by bronchial epithelial cells. Am J Respir Cell Mol Biol 2004, 30:421–427.View ArticlePubMedGoogle Scholar
- O'Regan AW, Hayden JM, Body S, Liaw L, Mulligan N, Goetschkes M, Berman JS: Abnormal pulmonary granuloma formation in osteopontin-deficient mice. Am J Respir Crit Care Med 2001, 164:2243–2247.View ArticlePubMedGoogle Scholar
- Ophascharoensuk V, Giachelli CM, Gordon K, Hughes J, Pichler R, Brown P, Liaw L, Schmidt R, Shankland SJ, Alpers CE, Couser WG, Johnson RJ: Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis. Kidney Int 1999, 56:571–580.View ArticlePubMedGoogle Scholar
- Driscoll KE: Macrophage inflammatory proteins: biology and role in pulmonary inflammation. Exp Lung Res 1994, 20:473–490.View ArticlePubMedGoogle Scholar
- Kopp EB, Medzhitov R: The Toll-receptor family and control of innate immunity. Curr Opin Immunol 1999, 11:13–18.View ArticlePubMedGoogle Scholar
- Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A: The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001, 410:1099–1103.View ArticlePubMedGoogle Scholar
- Gewirtz AT, Navas TA, Lyons S, Godowski PJ, Madara JL: Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol 2001, 167:1882–1885.View ArticlePubMedGoogle Scholar
- Lien E, Means TK, Heine H, Yoshimura A, Kusumoto S, Fukase K, Fenton MJ, Oikawa M, Qureshi N, Monks B, Finberg RW, Ingalls RR, Golenbock DT: Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide. J Clin Invest 2000, 105:497–504.View ArticlePubMedPubMed CentralGoogle Scholar
- Donnelly MA, Steiner TS: Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5. J Biol Chem 2002, 277:40456–40461.View ArticlePubMedGoogle Scholar
- Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, Chow JC, Strauss JF: The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 2001, 276:10229–10233.View ArticlePubMedGoogle Scholar
- Taylor KR, Trowbridge JM, Rudisill JA, Termeer CC, Simon JC, Gallo RL: Hyaluronan fragments stimulate endothelial recognition of injury through TLR4. J Biol Chem 2004, 279:17079–17084.View ArticlePubMedGoogle Scholar
- Kuschner WG, D'Alessandro A, Wong H, Blanc PD: Early pulmonary cytokine responses to zinc oxide fume inhalation. Environ Res 1997, 75:7–11.View ArticlePubMedGoogle Scholar
- Mizel SB, Snipes JA: Gram-negative flagellin-induced self-tolerance is associated with a block in interleukin-1 receptor-associated kinase release from toll-like receptor 5. J Biol Chem 2002, 277:22414–22420.View ArticlePubMedGoogle Scholar
- Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301–305.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.