Published ahead of print on December 13, 2007, doi:10.1164/rccm.200703-373OC
American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 613-621, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200703-373OC
IRF-1 Gene Variations Influence IgE Regulation and Atopy
Michaela Schedel1,*,
Leonardo A. Pinto1,*,
Bianca Schaub1,
Philip Rosenstiel2,
Dmitry Cherkasov3,
Lisa Cameron1,
Norman Klopp4,
Thomas Illig4,
Christian Vogelberg5,
Stephan K. Weiland6, ,
Erika von Mutius1,
Michael Lohoff3,* and
Michael Kabesch1,*
1 University Children's Hospital, Ludwig Maximilian's University Munich, Munich, Germany; 2 Institute of Clinical Molecular Biology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; 3 Institute of Microbiology, University of Marburg, Marburg, Germany; 4 Institute of Epidemiology, GSF–Research Centre for Environment and Health, Neuherberg, Germany; 5 University Children's Hospital, Dresden, Germany; and 6 Institute of Epidemiology, Ulm University, Ulm, Germany
Correspondence and requests for reprints should be addressed to Michael Kabesch, M.D., University Children's Hospital, Ludwig Maximilian's University Munich, Lindwurmstrasse 4, D-80337 München, Germany. E-mail: michael.kabesch{at}med.uni-muenchen.de
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ABSTRACT
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Rationale: The development of atopic diseases is characterized by skewed immune responses to common allergens. Only recently, interferons have been identified to play a crucial role in these mechanisms.
Objectives: Because interferon regulatory factor (IRF)-1 is critical for interferon expression, we tested the hypotheses that genetic changes in this essential transcription factor may have consequences for the development of atopy.
Methods: The IRF-1 gene locus was resequenced in 80 human chromosomes. Association and haplotype analyses were performed in a cross-sectional study population of German children from Dresden (n = 1,940), and results were replicated in a second population sample from Munich (n = 1,159), both part of the ISAAC (International Study of Asthma and Allergy in Childhood) phase II. Promoter polymorphism effects were studied using electrophoretic mobility shift assay and colorimetric binding assays. Allele-specific IRF-1 gene expression was studied in vitro using luciferase reporter assays, whereas we assessed ex vivo expression of IRF-1 by real-time polymerase chain reaction and IFN- protein by Luminex technology (Bio-Rad, Hercules, CA). Statistical analyses were performed using SAS/Genetics (SAS 9.1.3; SAS Institute, Cary, NC).
Measurements and Main Results: By resequencing, 49 polymorphisms were identified within the IRF-1 gene. Four blocks containing 11 polymorphisms were significantly associated with atopy, total IgE levels, or specific IgE levels in both populations (P < 0.05). Two polymorphisms changed transcription factor binding of nuclear factor (NF)- B and EGR1 (early growth response 1) to the IRF-1 promoter, altered gene expression in vitro (P = 0.0004), and altered IRF-1 mRNA and IFN- protein expression ex vivo.
Conclusions: Our results suggest that functionally relevant IRF-1 polymorphisms influence atopy risk, potentially by altering transcription factor binding, IRF-1 gene expression, and IFN- regulation.
Key Words: asthma • genes • interferons • IRF-1 transcription factor • genetic polymorphism
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AT A GLANCE COMMENTARY
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Scientific Knowledge on the Subject
Interferon regulatory factor (IRF)-1 is critical for interferon expression. However, little is known concerning the influence of genetic changes in this essential transcription factor on the development of atopic diseases.
What This Study Adds to the Field
Our results suggest that functionally relevant IRF-1 polymorphisms influence atopy risk, potentially by altering transcription factor binding, IRF-1 gene expression, and IFN- regulation.
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Genetic predisposition and the exposure to (or absence of) certain environmental stimuli may determine the development of atopy by activating allergen-associated immune cells and the expression of specific cytokine patterns. Type I and II interferons, inducible by environmental stimuli, such as microbial exposure, are key players in the human immune system and may suppress atopy-associated skewed immunity (1). IFN- production is controlled by several transcription factors of which IFN regulatory factor (IRF)-1 is of special importance. IRF-1 expression is induced by viruses, lipopolysaccharide (LPS), and IL-1 (2). The induction of IRF-1 expression by either type I IFN (IFN- or IFN-β) or type II IFN (IFN-) is mediated by the transcription factor signal transducer and activator of transcription 1 (STAT1) (3). So far, it is not fully understood whether other transcription factors and pathways influence IRF-1 regulation. IRF-1 binds to the promoter region of genes critical for IFN expression and genes responsive to IFN (2). IRF-1 stimulates the production of IL-12, IL-18, and IL-23. In addition, IRF-1 binds to the IL-4 promoter and represses IL-4 transcription, thereby inhibiting T-helper 2 (Th2) responses (4). IRF-1 up-regulates the expression of IL-15, which induces the generation of IFN-–producing natural killer (NK) cells. As a consequence, mice deficient in IRF-1 (Irf1–/–) show a strongly increased susceptibility to intracellular infections (5). Overall, IRF-1 has a complex role influencing many aspects of T-cell immunology.
The gene coding for IRF-1 is located in a cytokine gene cluster on chromosome 5q31, which is considered to play an important role in the development of allergic disorders (Figure 1) (6). To investigate the presence and role of genetic variations in the IRF-1 gene in a white population, we resequenced the gene, performed association studies with allergy-related phenotypes in two independent populations of German children (n = 1,940 and n = 1,159), and studied associated promoter polymorphisms for their putative functional role in gene regulation.
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Figure 1. Localization of IRF-1 on chromosome 5q31 and description of intragenetic (A) and intergenetic (B) linkage disequilibrium (LD) patterns (r2 plots). (A) Position of the IRF-1 gene on chromosome 5q31 in relation to adjacent genes (top), gene structure showing exons (middle) and position of frequent IRF-1 single nucleotide polymorphisms (SNPs) (minor allele frequencies [MAF] > 0.1), and LD (r2 plot) in the resequenced population sample (n = 40). (B) LD (r2 plot) between IRF-1 tagging SNPs representing blocks 1, 2, 3, and 4 and neighboring SNPs in the genes coding for IL-3, CSF2, IL-5, IL-13, IL-4, and CD14. Shades in the LD plot from Haploview: white (r2 = 0), shades of gray (0 < r2 < 1) and black (r2 = 1).
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METHODS
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Additional details on methods are provided in the online supplement.
ISAAC Population
Between 1995 and 1996, a cross-sectional study was performed in Munich and in Dresden, Germany, as part of ISAAC (International Study of Asthma and Allergy in Childhood) phase II to assess the prevalence of asthma and allergies in 5,629 schoolchildren at the age of 9 to 11 years (7). Here, all children of German origin with DNA available (n = 3,099) were analyzed (Table E1 of the online supplement). Children whose parents reported a physician's diagnosis of asthma, recurrent asthmatic bronchitis, or spastic bronchitis in a self-administered questionnaire were classified as having asthma. The sensitivity to six common aeroallergens was assessed by skin prick test (SPT). A child was considered atopic if a wheal reaction of 3 mm or larger occurred to one or more allergens after subtraction of the negative control. Total serum IgE levels were measured using the Imulite System (DPC Biermann, Bad Nauheim, Germany), and specific IgE antibodies (Sx1; Pharmacia, Lund, Sweden) against inhalative allergens were assessed.
Mutation Screening and Genotyping
Using an ABI Prism 3730 sequencer (Applied Biosystems, Foster City, CA), 80 chromosomes from 40 unrelated, randomly selected adult volunteers were sequenced for 12,985 bp in and around the IRF-1 gene (exons, introns, 2,522 bp upstream and 2,820 bp downstream of the gene; except a 868-bp SINE [short interspersed nuclear element] of highly repetitive sequence 232 bp downstream of the gene) (8).
Genomic DNA was extracted from whole blood by a standard salting-out method (9). DNA samples were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom, Inc., San Diego, CA) (Tables E2–E3).
Functional Analyses of the Promoter Polymorphisms
Alterations in transcription factor binding to the proximal promoter of the IRF-1 gene were investigated by electrophoretic mobility shift assay (EMSA) and colorimetric nuclear factor (NF)-B assay. To study IRF-1 promoter polymorphism–dependent gene expression in vitro, Jurkat T cells were transfected with pGL3 plasmids under the control of the proximal (763 bp) IRF-1 promoter containing the respective wild-type or polymorphic alleles at positions A-1710C, G-1705A, and G-1595A (block 1), and analyzed in a luciferase reporter assay. Single nucleotide polymorphism (SNP)–dependent effects on IRF-1 gene expression were analyzed ex vivo using real-time polymerase chain reaction on peripheral and cord blood mononuclear cells (PBMCs and CBMCs), and protein expression of the downstream cytokine IFN- was measured in the supernatant of these cells using Luminex technology (Bio-Rad, Hercules, CA) (Tables E5 and E6).
Statistical Analyses
Polymorphisms with r2  0.8 were defined as a linkage disequilibrium (LD) block. Deviation from Hardy-Weinberg equilibrium was analyzed with  2 tests. Association between SNPs and dichotomous outcomes were evaluated using 2 tests in a dominant model of the rare allele. All tests were two-sided and the differences were considered significant when P values were less than 0.05. For IgE, t tests of log-transformed values were used in a dominant model. Haplotype frequencies were estimated with an expectation–maximization algorithm (10), and associations of common haplotypes (frequency > 0.03) with atopy and IgE levels were calculated with Haploview (Version 3.32; MIT, http://www.broad.mit.edu/mpg/haploview) (11). For haplotype analysis, the 90th percentile of total IgE distribution within the study population was used as a dichotomous outcome variable as previously described (12). Calculations were performed with the SAS software (version 9.1.3; SAS Institute, Cary, NC).
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RESULTS
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Mutation Screening and Polymorphism Identification in the IRF-1 Gene
Upon resequencing of 40 adult volunteers of German origin, a total of 73 sequence variants were found. Of these, 39 variants and one 16-bp deletion with minor allele frequencies (MAF) of at least 10% were identified in the region of the IRF-1 gene (Table 1) and considered common SNPs. In addition, nine infrequent polymorphisms (MAF 0.03–0.10) and 24 mutations (MAF < 3%) were identified but not studied further (Table E4). Two polymorphisms (C-2606T and 3513del) were previously not described in public SNP databases (dbSNP). Twenty-three polymorphisms were located in intronic regions, nine in the promoter region, three in the 5' untranslated region (5'UTR), one in the 3'UTR, and three SNPs in the 3' flanking region. One SNP (A3116G) found in exon 7 did not change the amino acid sequence.
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TABLE 1. DESCRIPTION OF IRF-1 POLYMORPHISMS AND THEIR RESPECTIVE POSITION WITHIN THE GENE, ALLELE FREQUENCIES, rs NUMBERS, LINKAGE DISEQUILIBRIUM, AND GENOTYPED TAGGING SINGLE NUCLEOTIDE POLYMORPHISMS
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Associations between IRF-1 SNPs, IgE Levels, and Atopy in Two Independent Populations
On the basis of resequencing results, the existence of polymorphisms in a white population was verified and LD analysis could be performed. Analyzing polymorphisms with MAF 0.10 (Figure 1A), five major LD blocks were identified with r2 0.8 (Table 1). No relevant LD between these SNPs in the IRF-1 gene and polymorphisms in the nearby genes IL-5, IL-4, and IL-13 were observed in our population (Figure 1B).
SNP block 1 (A-1710C, G-1705A, and G-1595A), block 2 (C-2606T, A-672C, and G5250T), and block 4 (C-1243G, C-1152T, C4174T) contained three SNPs each, whereas two SNPs were found in block 3 (A1244G and T8268G). SNP block 5 contained 26 polymorphisms in strong LD. An additional three polymorphisms could not be assigned to any LD block. Thus, three single SNPs and one tagging SNP per LD block were genotyped in a cross-sectional study population of 1,940 children from Dresden (Table 1). All SNPs showing associations in the first sample were also genotyped in a second population from Munich (n = 1,159) to minimize the risk of type I errors due to multiple testing.
In the Dresden population, four of the tested SNPs representing blocks 1, 2, 3, and 4 were associated with significant changes in total IgE levels (Table 2). In addition, polymorphism C4174T (representing block 4), which was associated with lower total serum IgE levels, also reduced the risk for atopic sensitization (measured by SPT). SNP A-672C (representing block 2) was not only associated with elevated total serum IgE levels but also with an increased risk for specific sensitization (Table 2) in the Dresden population at age 9 through 11 years. Interestingly, the tagging SNP T8460G representing the largest LD block of 26 polymorphisms was not associated with any one of the investigated phenotypes. Next, genotyping was replicated in the second ISAAC phase II population (Munich) for all four tagging SNPs that had shown associations with elevated total serum IgE levels in the Dresden population. In the Munich population, changes in total IgE levels were significant for SNP C4174T and showed trends similar to the previous observations in the Dresden sample for all other SNPs. The associations with atopic sensitization observed in Dresden with SNPs A-672C and C4174T were replicated and extended to SNPs A-1710C and A1244G. In addition, C4174T was strongly protective against the development of asthma in the presence of atopic sensitization (odds ratio [OR], 0.35; 95% confidence interval [CI], 0.19–0.65; P < 0.001), whereas A-1710C (OR, 1.96; 95% CI, 1.02–3.76; P = 0.040) and A1244G (OR, 1.84; 95% CI, 1.03–3.28; P = 0.036) increased the risk for atopic asthma (data not shown). Haplotype analyses with the IRF-1 region were also performed and confirmed previous observations (Table 3). H_a, the most common IRF-1 haplotype, was associated with a protective effect for atopy measured by RAST and SPT in Munich, whereas H_b increased the risk for strongly elevated total serum IgE (above 90th percentile) in Dresden and atopy in Munich. The same trends for atopy were observed in Dresden.
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TABLE 2. GEOMETRIC MEANS OF TOTAL SERUM IgE AND ODDS RATIOS FOR ASSOCIATIONS WITH ELEVATED SPECIFIC SERUM IgE TO INHALATIVE ALLERGENS (MEASURED BY Sx1, >0.35 IU/ml) AND A POSITIVE SKIN PRICK TEST (3 mm) COMPARING HOMOZYGOTE CARRIERS OF THE WILD-TYPE ALLELE WITH CARRIERS OF AT LEAST ONE MINOR ALLELE
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TABLE 3. HAPLOTYPE ASSOCIATIONS BETWEEN IRF-1 HAPLOTYPES, 90TH PERCENTILE OF TOTAL SERUM IgE (>457 IU/ml), SPECIFIC SERUM IgE (>0.35 IU/ml), AND ATOPY MEASURED BY SKIN PRICK TEST (3 mm)
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Functional Studies Reveal SNP-dependent Variations in the Binding of Transcription Factors in the Proximal Promoter of the IRF-1 Gene
Three of the 11 associated SNPs in blocks 1, 2, 3, and 4 were located in the previously defined proximal promoter (13) of the IRF-1 gene (SNP A-1710C, G-1705A, G-1595A) and two in the 5'UTR (C-1152T and C-1243G), potentially influencing IRF-1 gene expression through changes in transcription factor binding (Figures 2A–2C). Thus, these regions were analyzed for polymorphism-associated changes in transcription factor binding using EMSA with nuclear extracts from the Jurkat T-cell line either unstimulated or stimulated with phorbol myristate acetate (PMA)/ionomycin.
At the site harboring SNPs A-1710C and G-1705A, NF-B binding was significantly stronger in the probe carrying the polymorphic C allele at position –1710 (Figure 2B). In EMSA experiments, alterations in NF-B binding depend on SNP A-1710C, whereas G-1705A showed no significant effects in EMSA and colorimetric assays (Figures E3 and E6). Effects on NF-B binding were observable in the Jurkat T-cell line and the YT cell line containing T cells and NK cells (Figure E4b), whereas no allele-specific NF-B binding to the SNP locus was detectable in HEK293 cells (Figure E4a).
At position –1595, EMSA experiments identified an allele-specific DNA/protein complex inducible by PMA/ionomycin stimulation and present in nuclear extract of different cell lines, namely Jurkat T cells (Figure 2C) and HEK293 (Figure E5). Using supershift experiments, this complex, binding exclusively in the presence of the wild-type allele, was identified as EGR1 (early growth response 1). For the sites harboring C-1152T and C-1243G in the 5'UTR, no significant changes in transcription factor binding were observed (data not shown).
IRF-1 Promoter Polymorphisms (Block 1) Influence IFR-1 Gene Expression In Vitro and Ex Vivo
Next, the effect of IRF-1 promoter polymorphisms located within block 1 (A-1710C, G-1705A, and G-1595A) showing associations with atopy phenotypes and changes in transcription factor binding were investigated for their role in IRF-1 gene expression. Both A-1710C and G-1595A had shown alterations in transcription factor binding, whereas G-1705A had to be included in the analysis due to its almost complete linkage with and close vicinity to A-1710C. Thus, a 763-bp construct of the proximal IRF-1 promoter either carrying wild-type or polymorphic alleles at positions A-1710C, G-1705A, and G-1595A was cloned into a luciferase reporter vector and transiently transfected into Jurkat T cells. SNP-dependent effects were studied in vitro with and without ionomycin stimulation. Although the wild-type promoter construct showed a clear induction of activity after ionomycin stimulation (Figure 3A), induction of gene expression was almost completely lacking in the presence of the polymorphic alleles (P = 0.0004).
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Figure 3. Polymorphisms in the IRF-1 promoter (block 1) decrease IFR-1 gene expression in vitro (A) and ex vivo (B–E). (A) Jurkat T cells were transiently transfected with a 763-bp reporter construct of the proximal IRF-1 promoter carrying the wild-type or the polymorphic alleles at position A-1710C, G-1705A, and G-1595A. Eight hours after transfection, medium was exchanged by medium containing 100 ng/ml ionomycin or pure medium (unstimulated). At 18 hours after stimulation, cells were harvested (n = 6). Luciferase activity was normalized for transfection efficiency using the control plasmid pRL-TK (plasmid Renilla luciferase-thymidine kinase; Promega, Madison, WI) activity. The relative luciferase activity is presented in relative light units (RLU). (B–E) IRF-1 mRNA gene expression in peripheral blood mononuclear cells (PBMCs) (B and C) from adults (n = 13) and cord blood mononuclear cells (CBMCs) (D and E) from newborns (n = 19) was determined after stimulation with phytohemagglutinin (PHA) or lipid A (LpA). In adults, five individuals carried all wild-type alleles at positions A-1710C, G-1705A, and G-1595A, and eight were polymorphic in these locations (CBMCs: n = 8 wild-type, n = 11 polymorphic). Results are displayed as fold difference in stimulated as compared with unstimulated cells compared with the housekeeping gene 18SrRNA. Quantitative gene expression was assessed with real-time polymerase chain reaction (I-Cycler; BioRad, Hercules, CA). For B and C, the respective median IRF-1 mRNA values (interquartile ranges, 25–75%) are displayed. P values were calculated using the Mann-Whitney rank sum test. In E, mean IRF-1 mRNA values (±SEM) are depicted as samples and were normally distributed, and P values were calculated using a t test. Light gray bars represent the wild-type alleles at position A-1710C, G-1705A, and G-1595A, and dark gray bars represent the respective polymorphic alleles.
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To conduct ex vivo experiments, PBMCs and CBMCs of individuals either carrying all wild-type or all polymorphic alleles at positions A-1710C, G-1705A, and G-1595A were identified from a population of 83 unrelated adult volunteers and from 115 cord blood samples. At the same time, all samples included in the experiments were restricted to be wild-type for SNPs from LD block 4 to control for potential confounding by these inversely related SNPs. On the basis of this selection, 13 adult samples (5 wild-type, 8 polymorphic) and 19 cord blood samples (8 wild-type, 11 polymorphic) could be analyzed. IRF-1 gene expression was assessed in unstimulated cells and after lipid A (LpA) or phytohemagglutinin (PHA) stimulation. In adult and cord blood samples, IRF-1 mRNA expression was lower in homozygous carriers of the polymorphic alleles (–1710C/–1705A/–1595A) than in wild-type individuals, after LpA as well as PHA stimulation (Figures 3B–3E). These differences reached significance in cord blood samples after LpA stimulation and showed the same trends under all other conditions. Thus, the same naturally occurring promoter SNPs in block 1 associated with a reduced inducibility of luciferase activity in vitro were also associated with reduced IRF-1 gene expression ex vivo.
In the same samples, IRF-1 promoter SNP effects on IFN- cytokine secretion from supernatants of PBMCs and CBMCs before and after PHA or LpA stimulation were investigated. After PHA stimulation, median IFN- expression was slightly lower in polymorphic than in wild-type samples (PBMCs: 491 vs. 574 pg/ml, P = 0.83; and CBMCs: 41 vs. 136 pg/ml, P = 0.65). Also, the presence of polymorphic IRF-1 promoter alleles induced a significant decrease in IFN- levels after LpA stimulation in PBMCs (4.44 vs. 32.38 pg/ml, P = 0.01). In contrast, after LpA stimulation, no significant difference in IFN- expression was observed in cord blood of polymorphism carriers (21.8 vs. 5.4 pg/ml, P = 0.45).
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DISCUSSION
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Chromosome 5q31-33 has been a hot spot of allergy genetics in recent years, due to repeated linkage of the locus with allergy in whole genome linkage studies and the knowledge that many genes important for the immune system are clustered in this region. So far, the focus of 5q studies has been on Th2-related cytokines, but our results may indicate that there is more to the 5q locus than that. Our association studies and functional analyses suggest that genetic variations in IRF-1, potentially acting through changes in transcription factor binding, influence the development of atopy and the regulation of total IgE levels. Even though the IRF-1 gene is crucial for the regulation and expression of IFNs, involved in many important and diverse immune responses and diseases (14, 15), no systematic screening for polymorphisms in a white population has so far been reported, and previous association studies with atopic phenotypes were limited to the analysis of a few selected SNPs and tandem repeat markers in Asian populations (16–18). In our study, 49 polymorphisms (40 frequent and 9 infrequent) were identified by resequencing 80 chromosomes. On the basis of this information, LD patterns could be analyzed and eight tagging SNPs were selected for genotyping in a large and well-phenotyped white population. Thus, it is unlikely that frequent SNPs or significant associations were missed. Replication rather than correction for multiple testing was used to minimize the risk of a type I error, always present when multiple tests are performed. In addition, a number of different approaches to study functional implications of those SNPs in the IRF-1 promoter that most likely alter gene function were performed. Taking all the evidence together, it is very unlikely that the observed associations in two independent populations and the differences in functional analyses would all have occurred by chance.
Four blocks of SNPs genotyped by tagging SNPs A-1710C, A-672C, A1244G, and C4174T were associated with total or specific IgE regulation as well as the development of atopy measured by SPT in the two populations. Although in the Dresden population IRF-1 SNPs had a stronger influence on total IgE levels, the effect on atopy was much more pronounced in the Munich sample, where, in addition, associations with the development of atopic asthma were observed. Differences in IRF-1 effects between Dresden (in former East Germany) and Munich (in former West Germany) are thus present. Such differences in effects between populations of different environmental exposures have also been observed previously with genetic variants in other immunoregulatory genes strongly susceptible to environmental stimuli. Thus, mechanisms similar to those recently identified for a promoter SNP in the CD14 gene, where the amount of endotoxin exposure determines the role of the CD14 SNP in the development of atopy (either conferring a risk or even protection) (19), may also be suggested for IRF-1.
Functional studies of promoter SNPs as presented here give a first impression on how these complex interactions between IRF-1 polymorphisms, IgE regulation, and atopy may develop. Of the 11 polymorphisms represented by the four tagging SNPs showing significant associations, five SNPs in the proximal promoter and the 5'UTR of the IRF-1 gene were studied and two SNPs were identified to lead to significant and profound changes in transcription factor binding in the proximal promoter of the IRF-1 gene. The presence of the C allele at position –1710 significantly increases binding of NF-B in the proximal IRF-1 promoter and the polymorphic A allele at position –1595 abrogates EGR1 binding. In independent previous studies, cancer cell lines carrying polymorphic alleles at position –1710/–1705 and –1595 showed increased type I IFN expression (IFN- and IFN-β) (20). We studied the effects of these polymorphisms on gene expression using in vitro and ex vivo approaches and a variety of stimuli. First, an in vitro assay was used where the three promoter polymorphisms could be tested independent of other SNP influences in and around the IRF-1 gene, as the promoter was cloned into a luciferase reporter construct. Indeed, IRF-1 promoter SNPs significantly decreased gene expression after ionophore stimulation compared with the wild-type construct. Ex vivo experiments confirmed these observations: IRF-1 mRNA inducibility after mitogen (PHA) and LpA stimulation were considerably reduced in carriers of the polymorphic promoter alleles in PBMCs and CBMCs. Taken together, all these experiments point in the same direction: the IRF-1 promoter SNPs altering transcription factor binding decrease IRF-1 expression (in vitro and ex vivo) and could suggest that these SNPs diminish a T-cell–specific and broader Th1-derived IRF-1 signal which may be associated with increased atopic sensitization. However, differences in the strength of these polymorphism effects were also observed between PBMCs and CBMCs after LpA stimulation, a rather indirect Th1 stimulus compared with the more T-cell–directed PHA stimulus. Thus, it could be speculated that genetically altered IRF-1 expression may have other effects on IFN- regulation during the neonatal period than later in life when the diverse and complex interaction between different inflammatory cells is taken into account.
The combination of transcription factors altered by SNPs in the proximal promoter of IRF-1 is intriguing. NF-B is a key player in many inflammatory processes induced by microbial stimuli but is also necessary for IgE switching, expression, and regulation, potentially acting through a feedback loop (21). EGR1 has been shown to play a role in LPS-induced gene regulation, potentially also influencing Th2 suppression via suppressor of cytokine signaling (SOCS) pathways (22). Thus, both identified alterations in transcription factor binding may play a role in linking microbial exposure, IRF-1 regulation, and the development of atopy. However, other tagged SNPs located in the distal promoter region (C-2606T), in intronic regions (A-672C, A1244G, C4174T, and G5250T) or the 3'UTR (T8268G), may also potentially contribute to changes in IRF-1 function but have not yet been studied in detail. The way genetic changes in IRF-1 affect gene function may vary considerably. Although some variants may create new binding sites for transcription factors, other SNPs may act in the opposite way, and whereas a number of SNP blocks increase atopy risk, some SNPs (in block 4) may counterbalance these effects. Mutation screening also identified a previously described (17), multiallelic polymorphism in intron 7 (GT repeat) in our screening population (rs41498144). Considering the difficulties in measuring LD between biallelic SNPs and multiallelic variations, rs41498144 has been excluded from further LD analyses. However, when rs41498144 is modeled as a biallelic marker using 11–12 GT repeats as a frequent allele and 13–15 repeats as a minor allele, rs41498144 may be considered to be somewhat in LD with A1244G (D' = 0.77, r2 = 0.54). It cannot be excluded that the previously described association between this intronic GT repeat and atopic asthma may be due to LD within the IRF-1 gene. Neither we nor the authors of the original report (17) studied this variant further on a population or functional level.
Also, the possibility that associations observed with SNPs in the IRF-1 gene may have originated from extended LD with SNPs in other candidate genes in the cytokine cluster on chromosome 5q31 was addressed. Thus, LD between IRF-1 SNPs (A-1710C, C4174T, A-672C, A1244G) and SNPs in the proximal genes IL-5 (C-746T), IL-4 (C-589T), and IL-13 (C-1112T and G2044A) previously associated with atopy was studied (23–25). However, LD between SNPs in the IRF-1 gene and polymorphisms in IL-5, IL-4, and IL-13 was not relevant in our population and did not explain the observed associations (Figure 1B).
Thus, we concluded that genetic variants in the IRF-1 gene seem to influence the regulation of specific and total IgE levels and the development of atopy, putatively by altered transcription factor binding in the proximal promoter of the IRF-1 gene, changing IRF-1 expression and consecutive IFN regulation. Considering the importance of IRF-1 in diverse immune responses, polymorphisms in IRF-1 may influence a number of different diseases in which susceptibility to microbial exposure plays a role.
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Acknowledgments
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The authors thank Michael Kormann and Martin Depner for support and critical review of the manuscript.
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FOOTNOTES
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Supported by the German Ministry of Education and Research (BMBF)/National Genome Research Network (NGFN) research grant NGFN 01GS 0429, and the German Research Foundation as part of the Transregional Collaborative Research Program TR22, grants A15, A16, and Z3.
* These authors contributed equally to this article. 
 Deceased.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200703-373OC on December 13, 2007
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
Received in original form March 6, 2007;
accepted in final form December 13, 2007
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W. C. Moore
Update in Asthma 2008
Am. J. Respir. Crit. Care Med.,
May 15, 2009;
179(10):
869 - 874.
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ABSTRACT
AT A GLANCE COMMENTARY
Competition and supershift experiments using an unrelated oligo and unrelated antibody. PO = polymorphism; WT = wild-type.