Published ahead of print on February 12, 2004, doi:10.1164/rccm.200307-933OC
© 2004 American Thoracic Society Functional Polymorphisms in the Promoter Region of Macrophage Migration Inhibitory Factor and AtopyFirst Department of Medicine, Hokkaido University School of Medicine, Sapporo; Department of Respiratory and Allergy Medicine, Aichi Medical University, Aichi; and Department of Molecular Chemistry, Hokkaido University Graduate School of Medicine, Sapporo, Japan Correspondence and requests for reprints should be addressed to Nobuyuki Hizawa, M.D., First Department of Medicine, Hokkaido University School of Medicine, Kita-Ku N-15 W-7, Sapporo, Japan 060-8638. E-mail: nhizawa{at}med.hokudai.ac.jp
Macrophage migration inhibitory factor (MIF) is a pleiotrophic lymphocyte and macrophage cytokine; it is likely to play an important role in innate immunity. Genome-wide search for atopy susceptibility genes recently identified human chromosome 22q11, where the gene encoding MIF resides, as a region of interest for atopic traits. Both the –173G/C and –794 [CATT]5–8 repeat polymorphisms in the MIF promoter region are associated with altered levels of MIF gene transcription in vitro. We, therefore, hypothesized that these potentially functional polymorphisms may influence susceptibility to atopy and asthma. A case–control analysis examined the genetic influence of these promoter polymorphisms on the development of atopy and asthma in a Japanese population (n = 584). Evidence for significant association between the –173G/C and –794 [CATT]5–8 repeat polymorphisms and atopy was found; odds ratio for homozygotes of –173C allele was 3.67 (compared with homozygotes of –173G allele, 95% confidence interval = 1.43–9.46, p < 0.01), and odds ratio for noncarriers of the –794 [5-CATT] allele was 3.51 (compared with 5-CATT repeat homozygotes, 95% confidence interval = 1.82–6.78, p < 0.0005). No associations with asthma were detected. These results indicate that promoter polymorphisms in the MIF promoter region are risk factors for atopy and implicate MIF in the pathogenesis of atopy in a Japanese population.
Key Words: candidate gene • case–control analysis • specific IgE Macrophage migration inhibitory factor (MIF) was initially described as an immune activity isolated from the supernatants of T lymphocytes (1) and has been implicated in macrophage activation and in antigen-driven T cell responses (2, 3). A recent investigation indicated that MIF regulates innate immune responses by macrophages through modulation of expression of toll-like receptor 4 (4); toll-like receptor 4 is the principal receptor for bacterial endotoxin recognition. There is evidence that endotoxin exposure during early life is protective against development of atopy and asthma (5–7); it is hypothesized that bacterial signals, such as endotoxin, play a functional role in maturation of T helper cell type (Th) 1–type immune responses, suppressing the Th2 response (8). We previously demonstrated expression of MIF protein in serum and induced sputum of patients with asthma (9). Bronchoalveolar lavage fluid obtained from patients with asthma having atopy also contains significantly elevated levels of MIF, compared with volunteers not having atopy (10). Furthermore, increased levels of MIF protein are associated with atopic dermatitis (11). A recent linkage study has found that human chromosome 22q11, where the gene encoding MIF is located, shows evidence of linkage with atopy-related phenotypes (12). Polymorphisms with potential functional relevance have also been identified in the MIF promoter; a single nucleotide polymorphism at position –173 (G to C) (13) and a tetranucleotide CATT repeat beginning at nucleotide position –794 (14) have been found to be associated with altered levels of MIF gene transcription in vitro. Further evidence of the functional importance of these variants includes findings of significant association with several immune-mediated inflammatory diseases including juvenile idiopathic arthritis (13), sarcoidosis (15), and rheumatoid arthritis (14). Given the role of MIF in innate immune responses against microbial pathogens and regulation of inflammatory responses, we hypothesized that common allelic variations in these potentially functional polymorphisms may be involved in the genetic–environmental interaction underlying the pathophysiology of atopy and asthma. In a case–control association study using 584 unrelated Japanese subjects, we investigated whether the above two polymorphisms in the MIF promoter region contribute to the risk of development of atopy and asthma.
Complete details are provided in the online supplement.
Study Population
All subjects gave written informed consent for enrollment in the study and all associated procedures. The Ethics Committee of the School of Medicine, Hokkaido University, approved the study.
Identification of Polymorphisms
Statistical Analysis The association of the MIF promoter polymorphisms was measured by odds ratio with 95% confidence intervals, as estimates of relative risk for development of atopy and asthma. The –794 [CATT]5–8 genotypes were combined into three categories: 5, 5 genotype; 5, X genotypes; and X, X genotypes (allele X represents any allele other than five repeats of CATT). Odds ratios were adjusted for potentially confounding factors using a multivariate logistic regression model. We estimated haplotype frequencies for –173G/C and –794 [GATT]5–8 repeat polymorphisms using the estimated haplotype program and tested the statistical association between the MIF promoter haplotypes and atopy using the program Haplo.Score that provided a global test for association, as well as haplotype-specific tests (20).
Luciferase Reporter Gene Assay
Characteristics of the 235 subjects without atopy (155 subjects without asthma, 80 subjects with asthma) and 349 subjects with atopy (152 subjects without asthma, 197 subjects with asthma) are presented in Table 1. The mean age was highest for subjects with asthma not having atopy, and females predominated in this group. Subjects with atopy (both healthy control subjects and subjects with asthma) had higher total serum IgE levels than subjects without atopy (unpaired t-test, p < 0.001). In addition, subjects with asthma (both subjects with atopy and subjects without atopy) had higher total serum IgE levels than healthy control subjects (unpaired t-test, p < 0.001). Alleles of the two promoter polymorphisms were in Hardy–Weinberg equilibrium in subjects without atopy. We found that both the –173G/C and –794[CATT]5–8 repeat promoter polymorphisms were significantly associated with atopy (Table 2) ; odds ratio for CC homozygotes of the –173G/C polymorphism was 3.67 (compared with GG homozygotes, 95% confidence interval = 1.43–9.46, p < 0.01), and odds ratio for noncarriers of the 5-CATT allele of the –794 [CATT]5–8 repeat polymorphism was 3.51 (compared with 5-CATT homozygotes, 95% confidence interval = 1.82–6.78, p < 0.0005). In contrast, there were no significant differences in genotype distribution of these promoter polymorphisms between healthy control subjects and subjects with asthma (Table 2). Because we initially studied two markers (–173G/C and –794 CATT polymorphisms) in two phenotypes (atopy and asthma), we multiplied our significance levels by 4 (two markers x two phenotypes), although these statistical tests were not independent due to the linkage disequilibrium between the two polymorphisms. Using this correction, the association between the two promoter polymorphisms and atopy was significant at p = 0.05.
The –173G/C and –794 [CATT]5–8 promoter polymorphisms were in significant linkage disequilibrium, with the –173C allele strongly associated with the 7-CATT repeat allele (p < 0.000001). The three most frequent haplotypes common to both groups with and without atopy were G/5-CATT, G/6-CATT, and C/7-CATT. These three haplotypes constituted 89.9% of haplotypes in the group with atopy and 90.1% of haplotypes in the group without atopy (Table 3) . The haplotype composed of these two promoter polymorphisms was significantly associated with atopy, with a p value of 0.009 from 10,000 simulations of a global score test, as implemented in Haplo.Score (20). The haplotypes most strongly associated with atopy, as judged by the haplotype-specific scores, were G/5-CATT (p < 0.0001) and C/7-CATT (p = 0.0036), on the basis of 10,000 simulations; the G/5-CATT haplotype was associated with a lower risk of atopy and the C/7-CATT haplotype was associated with an increased risk of atopy (Table 3).
Transfection of the clone containing the C/7-CATT haplotype into A549 cells resulted in significantly reduced luciferase activity, relative to cells containing the other two common haplotypes (Figure 1) .
In the present case–control study using a Japanese population, we found significant association between atopy and two promoter polymorphisms of the MIF gene. The G/5-CATT haplotype was associated with reduced risk for development of atopy, and the C/7-CATT haplotype was associated with increased risk for development of atopy. In previous in vitro functional studies, levels of MIF expression significantly differed among –173G/C genotypes in a cell type–specific manner. Promoter sequence analysis indicates that the –173C allele creates a potential activator protein 4 transcription factor–binding site (13). With the CATT tetranucleotide polymorphism, the 5-CATT allele was shown to be associated with lower basal and stimulated MIF promoter activity in vitro than the 6-, 7-, or 8-repeat alleles (14). Using the A549 epithelial cell line, we characterized in vitro function of the three most common haplotypes. The C/7-CATT haplotype had lower promoter activity than the G/5-CATT and G/6-CATT haplotypes, suggesting functional importance of the MIF promoter haplotype in determining levels of MIF gene transcription. There is, however, no clear and simple discernable relationship between these polymorphisms and the differences observed in transcription levels of the three haplotype constructs, illustrating the complex nature of transcriptional regulation of the MIF gene. Thus, the physiologic relevance of the functional consequences of these promoter polymorphisms remains uncertain, and we cannot exclude the possibility that they act as markers of another important genetic abnormality without themselves being functionally relevant. Recognition of endotoxin and Gram-negative bacteria by the host requires cooperative interplay between the endotoxin-binding protein (lipopolysaccharide-binding protein) (21), CD14 (22), and toll-like receptor 4 (23). Microbial toxins are powerful inducers of MIF release by immune cells, and, by upregulating basal expression of toll-like receptor 4 in the macrophage, MIF promotes recognition of endotoxin-containing particles and Gram-negative bacteria by the innate immune system (4). Several epidemiologic studies suggest that lack of exposure to endotoxin in early childhood is a risk factor for development of atopic phenotypes (5–7). Furthermore, genetic variants in the genes encoding endotoxin-signaling molecules such as CD14 (24) and CARD15 (25) have been described and found to be associated with levels of total serum IgE or allergy, supporting the hypothesis that exposure to endotoxin modulates IgE regulation by activating innate immune systems. We speculate that individuals carrying the C/7-CATT haplotype have lower expression of MIF in response to inhaled endotoxin at the respiratory mucosa, lower expression of toll-like receptor 4, and, consequently, lower endotoxin-inducible expression of interleukin-12 and interleukin-18, resulting in enhanced Th2 differentiation. MIF is involved in antigen-specific immune responses; neutralizing anti-MIF antibodies inhibited T cell proliferation and interleukin-2 production in vitro, suppressing antigen-driven T cell activation and antibody production in vivo (26). MIF has recently been shown to be coded for by the same gene as glycosylation-inhibiting factor (27); glycosylation-inhibiting factor has been described as an immunosuppressive cytokine in a series of studies of regulation of antigen-specific IgE responses (28). Glycosylation-inhibiting factor is involved in antigen presentation involving B and T cell receptors and regulates generation of Th effectors from naive CD4 T cells (29), consequently regulating the balance of Th1/Th2-type immune responses. The importance of regulatory roles of MIF/glycosylation-inhibiting factor in antigen-specific immune responses is additional evidence that the MIF gene is a promising candidate for atopy or antigen-specific IgE responsiveness. It is important to note that the significant association between atopy and the two promoter polymorphisms could be due to type I error or population stratification (30). However, as we evaluated only two loci in an entirely Japanese population and as the control group was in Hardy–Weinberg equilibrium for the two polymorphisms, the usual problems associated with population stratification may be of limited importance in the present study. As for type I error, none of the reported p values were adjusted for multiple comparisons, because not all of the statistical tests were independent, due to the linkage disequilibrium between the two polymorphisms and the dependence between genotype and haplotype. In addition, given strong prior evidence for MIF as a candidate gene for atopy and evidence for functionality of MIF promoter polymorphisms, the present results appear to significantly support the hypothesis that individuals carrying certain genotypes of the present MIF promoter polymorphisms are at increased risk of developing atopy under certain additional environmental and genetic conditions. Nevertheless, we acknowledge that type I error and population stratification may have influenced the present findings and that these findings are preliminary and do not by themselves conclusively confirm an etiologic relationship. Additional evidence is needed from studies of other groups of individuals with and without atopy, especially in cohorts well characterized in terms of levels of endotoxin exposure in infancy. In conclusion, MIF is an excellent positional and biologically plausible candidate gene for atopy and may be involved in the endotoxin-signaling pathway, contributing to the development of atopy. However, given the great diversity of functions performed by MIF, further functional studies of genetic variation in the MIF promoter region are needed to clarify the pathophysiologic mechanisms by which these polymorphisms affect development of atopy.
The authors thank all the subjects of this study for their participation. They also thank A. Honda, Y. Obata, and K. Hosono for their excellent technical assistance and the Pharmaceutical Research Laboratory, Hitachi Chemical Co., Ltd., for measuring Ag-specific IgE levels (MAST).
Supported in part by a Grant-in-Aid for Scientific Research 13670585 from the Japan Society for the Promotion of Science. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Conflict of Interest Statement: N.H. has no declared conflict of interest; E.Y. has no declared conflict of interest; D.T. has no declared conflict of interest; J.N. has no declared conflict of interest; M.N. has no declared conflict of interest. Received in original form July 10, 2003; accepted in final form February 6, 2004
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ABSTRACT
METHODS
0.35 UA/ml, or multiple radioallergosorbent test 
2 test was used to compare qualitative risk factors (sex, smoking status) among the four groups (healthy control subjects without atopy, healthy control subjects with atopy, subjects with asthma not having atopy, and subjects with asthma having atopy). One-way analysis of variance was used to compare quantitative risk factors (age, serum IgE levels). We used the Hardy–Weinberg equilibrium program (
