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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

Objective

To study the effect of the innate cytokine macrophage migration inhibitory factor (MIF) on the susceptibility and severity of systemic lupus erythematosus (SLE) in a multinational population of 1,369 Caucasian and African American patients.

Methods

Two functional polymorphisms in the MIF gene, a −794 CATT5–8 microsatellite repeat (rs5844572) and a −173 G/C single-nucleotide polymorphism (rs755622), were assessed for association with SLE in 3,195 patients and healthy controls. We also measured MIF plasma levels in relation to genotypes and clinical phenotypes, and assessed Toll-like receptor 7 (TLR-7)–stimulated MIF production in vitro.

Results

Both Caucasians and African Americans with the high-expression MIF haplotype −794 CATT7/−173*C had a lower incidence of SLE (in Caucasians, odds ratio [OR] 0.63, 95% confidence interval [95% CI] 0.53–0.89, P = 0.001; in African Americans, OR 0.46, 95% CI 0.23–0.95, P = 0.012). In contrast, among patients with established SLE, reduced frequencies of low-expression MIF genotypes (−794 CATT5) were observed in those with nephritis, those with serositis, and those with central nervous system (CNS) involvement when compared to patients without end-organ involvement (P = 0.023, P = 0.005, and P = 0.04, respectively). Plasma MIF levels and TLR-7–stimulated MIF production in vitro reflected the underlying MIF genotype of the studied groups.

Conclusion

These findings suggest that MIF, which has both proinflammatory properties and macrophage and B cell survival functions, exerts a dual influence on the immunopathogenesis of SLE. High-expression MIF genotypes are associated with a reduced susceptibility to SLE and may contribute to an enhanced clearance of infectious pathogens. Once SLE develops, however, low-expression MIF genotypes may protect from ensuing inflammatory end-organ damage.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease with a multifactorial etiology (1). Several observations support the important role of both environmental factors and susceptibility genes in disease pathogenesis. The contribution of a genetic etiology to SLE is suggested by a disease concordance rate of ∼30% in monozygotic twins (2). In addition, there is a strong association between SLE incidence and particular HLA loci (3) and several non-HLA genes (4). Studies to date have not fully accounted for the total genetic contribution to SLE, however, and most studies have been limited to Caucasian patients.

The cytokine macrophage migration inhibitory factor (MIF) is recognized as playing a central role in the regulation of innate immunity and in the differentiation of the adaptive response. MIF counterregulates the immunosuppressive actions of glucocorticoids and promotes the production of tumor necrosis factor α (TNFα), leading to further MIF release and a re-entrant activation response that supports the maximal expression of proinflammatory mediators, matrix-degrading enzymes, and cyclooxygenases (5, 6). There also is evidence indicating that genetic deletion or neutralization of MIF has a protective effect in mouse models of spontaneous lupus (7, 8).

Both the circulating level and the tissue expression of MIF are elevated during inflammation, and recent data indicate that functional polymorphisms in the MIF gene influence the clinical expression of autoimmune or infectious diseases (9, 10). Two polymorphisms have been identified in the MIF gene promoter; the first is a microsatellite repeat at position −794 bp upstream of the MIF gene (−794 CATT5–8), in which the repeat length correlates with gene expression, and the second is a single-nucleotide polymorphism (SNP) at position −173 bp upstream of the MIF gene, in which the −173*C allele is associated with higher levels of circulating MIF (Figure 1). These 2 polymorphisms have been associated, in different populations, with increased severity of clinical manifestations or disease susceptibility in rheumatoid arthritis and juvenile idiopathic arthritis (11, 12), sarcoidosis (13), scleroderma (14), asthma (15), and inflammatory bowel disease (16).

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Figure 1. Diagram of the human macrophage migration inhibitory factor (MIF) gene showing its 3 exons, predicted transcription factor binding sites, and the −173 G/C single-nucleotide polymorphism (rs755622) and −794 CATT5–8 microsatellite repeat (rs5844572). The numerical prefixes refer to nucleotide distance (in basepairs) upstream from the transcription start site. The −173C allele, −794 CATT7, and −794 CATT8 repeats, as well as the −794 CATT7/−173C haplotype, are associated with higher expression of MIF.

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To determine the potential relationship between MIF gene polymorphisms and the systemic autoimmune disease SLE, we conducted a multicenter observational study of 1,369 patients with SLE, in whom MIF genotypes were analyzed in relation to disease incidence and clinical phenotype. In a subset of the patients, we examined the levels of circulating MIF and TNFα proteins for their correlation with MIF genotypes, and also analyzed MIF production by peripheral blood mononuclear cells of known genotype in response to Toll-like receptor 7 (TLR-7) agonism.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

Patients.

Our study included 1,369 patients with SLE and 1,826 healthy controls. Patients were included in the study if they fulfilled at least 4 of the 11 American College of Rheumatology revised criteria for SLE (17). Patient and control subjects were recruited locally from the US, Sweden, and Argentina. Participants in the US were included from 4 academic centers: Oklahoma University (Oklahoma City, OK), The Hospital for Special Surgery (New York, NY), Upstate Medical University (Syracuse, NY), and Yale University (New Haven, CT). Participants in Sweden were recruited from Karolinska University Hospital (Stockholm, Sweden). In Argentina, patients were included from Sanatorio Parque (Rosario, Argentina). Patients and controls were divided into 2 different ethnic groups: Caucasians and African Americans. Other ethnicities were excluded from the analysis because of their relatively small sample numbers. Ethnicity was determined by self-report and, in the University of Oklahoma cohort, was verified by admixture analysis.

The clinical manifestations studied included articular involvement, mucocutaneous lesions or photosensitivity, renal disease, serositis, hematopoietic abnormalities, and central nervous system (CNS) involvement. Patients were considered to have renal disease if they had one or more of the following features: active urinary sediment, significant proteinuria, or histopathologic evidence of lupus nephritis.

The relevant Institutional Review Boards approved the study. All subjects gave their signed informed consent to participate, and the Health Insurance Portability and Accountability Act Notification was completed by each subject when applicable.

Genotyping.

Genomic DNA was isolated from serum samples using the easy-DNA kit (Invitrogen). The two MIF promoter polymorphisms (the −794 CATT5–8 microsatellite repeat [rs5844572] and the −173 G/C SNP [rs755622]) were analyzed using the methods described previously by Wu et al (14). Of note, neither of these loci is represented in the high-density genotyping chips that have been used previously for genome-wide association studies in SLE. Briefly, MIF −173 G/C genotyping was performed using a predeveloped TaqMan assay for allelic discrimination (Applied Biosystems). The genotype of each sample was automatically attributed using fluorescence detection in an ABI Prism 7900HT instrument. Allelic discrimination was analyzed with SDS version 2.1.1 software (Applied Biosystems). MIF −794 CATT5–8 genotyping was carried out by polymerase chain reaction using a forward primer (5′-TGCAGGAACCAATACCCATAGG-3′) and a TET fluorescence–labeled reverse primer (TET-lab–5′-AATGGTAAACTCGGGGGAC-3′). Automated capillary electrophoresis on a DNA sequencer was performed on each sample, and the CATT alleles were identified using Genotyper version 3.7 software (Applied Biosystems).

Analysis of plasma MIF and TNFα levels by enzyme-linked immunosorbent assay (ELISA).

Plasma MIF levels were measured by sandwich ELISA using specific antibodies (18). Samples were run in triplicate. TNFα levels were measured using the Ready-Set-Go ELISA kit (eBioscience). Plasma MIF and TNFα levels were compared and culled from both Caucasian and African American patients with SLE and healthy control subjects, all of whom were identified as having either the high-expression MIF haplotype −794 CATT7/−173*C (7C) or the low-expression MIF haplotype −794 CATT5/−173*G (5G).

Peripheral blood leukocyte analysis.

Human peripheral blood mononuclear cells were isolated from heparinized blood using Ficoll-Hypaque gradient centrifugation. Cells were washed and resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, and the mononuclear cells were then purified by adherence (5 × 105 cells/well) for 2 hours. Cells were stimulated for 20 hours with the TLR-7 agonist polyU (0.25 mg/ml; Sigma-Aldrich), and the conditioned medium was then assayed by ELISA for MIF content.

Statistical analysis.

Given the population stratification of the MIF alleles under study (19), the study participants were subdivided by ethnic origin to allow appropriate comparison and analysis. Pearson's chi-square test was used to determine the homogeneity between the different Caucasian populations, and to analyze Hardy-Weinberg equilibrium. Allele and genotype frequencies were compared using chi-square test and, when appropriate, Fisher's exact test. Logistic regression was used to adjust for other covariates. Comparisons were made, first, between patients and controls and, then, between patients with and those without end-organ involvement, including patients with nephritis, those with serositis, and those with CNS involvement. Between these groups, the frequency of the high-expression genotypes (−794 CATT7–containing genotypes −794 CATT5/7, −794 CATT6/7, and −794 CATT7/7 and the −173*C-containing genotypes −173 C/C and −173 G/C) was compared with the frequency of the low-expression genotypes (those not containing −794 CATT7, or −794 CATT5/5, −794 CATT5/6, and −794 CATT6/6, and the −173 G/G genotype). The −794 CATT8 allele was found rarely (in 0.5% of all subjects) (19), and was therefore excluded from the analysis.

Studied haplotypes were reconstructed using the −173 G/C SNP and the −794 CATT5–8 repeat, from which 8 haplotype combinations are theoretically possible (designated 5G, 6G, 7G, 8G, 5C, 6C, 7C, and 8C). Haplotype frequencies and their inferred probabilities were calculated using HPlus version 2.5 software (obtained from the Quantitative Genetic Epidemiology Group of the Fred Hutchinson Cancer Research Center in Seattle, WA) (20). Both the 8G haplotype and the 8C haplotype were not included, because of their rare occurrence. The incidence of SLE associated with the different MIF haplotypes, in relation to the 5G haplotype as referent, was calculated using logistic regression. Age, race, sex, geographic location, MIF polymorphisms, and antinuclear antibody (ANA) status were used as covariates. We also determined the relationship between the MIF haplotypes and the incidence of different clinical manifestations, including nephritis (n = 299), serositis (n = 255), and CNS involvement (n = 101). African Americans were not included in the analyses of clinical manifestations because of the small number of clinical phenotypes reported for these subjects.

To address the issue of multiple testing, we used the permutation test in R (available at http://www.R-project.org/) (21). The permutation test was used in those instances in which the comparisons produced marginal P values.

To account for population stratification and its effect on genotype/haplotype differences, we used formulas established by Lee and Wang (22). The population stratification bias can be quantified by the confounding rate ratio (CRR). Through a mathematical calculation, Lee and Wang showed that the CRR is always bound above by “U” and below by “1/U,” in which “U” is the highest OR that can be attributed to the population stratification bias and “1/U” is the lowest OR. “U” takes into account the highest and lowest reported prevalence (“G”) of a disease in a population affected by stratification and the highest and lowest frequency (“B”) of the genotype/haplotype in question reported in the same population (22). If the observed OR is above “U” or lower than “1/U,” then these observed ORs cannot be explained simply by population stratification.

For calculation of power and sample size, we referred to data from a study by Barton et al (23), in which 438 patients with inflammatory arthritis and 343 controls were used to detect a 11.5% difference in the −173C-containing genotypes (P = 0.001), a 7.7% difference in the CATT7-containing genotypes (P = 0.001), and an 8.1% difference in the 7C haplotype (P = 0.0001) (23). We thus determined that 215 patients, 365 patients, and 149 patients, respectively, would be necessary to detect such differences in the respective genotypes and haplotype in the Caucasian population, with a 95% confidence interval (95% CI) and 80% power to detect such differences. Similarly, based on a study by Gao et al (24), we determined that 129 African American patients would be needed to detect a difference of 11.1% in −173C-containing genotypes, with a 95% CI and 80% power. No prior studies on differences in the CATT7-containing genotypes or haplotype have been conducted in African Americans.

Statistical analyses were performed using SPSS version 16. Two-tailed P values less than or equal to 0.05 were considered statistically significant. Differences in circulating MIF and TNFα levels between groups were compared using the Student's t-test.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

Patient demographics.

The demographic characteristics of the SLE patients and healthy controls are shown in Table 1. There was no difference in the mean age between patients and controls (mean ± SD 38.7 ± 12.6 years versus 39.4 ± 12.4 years; P = 0.447). The majority of subjects were female in both the patient group and control group (89% versus 86%; P = 0.108). The studied clinical manifestations of SLE included articular involvement (82%), mucocutaneous lesions or photosensitivity (81%), renal disease (60%), serositis (48%), hematopoietic abnormalities (46%), and CNS involvement (19%).

Table 1. Characteristics of the patients with systemic lupus erythematosus (SLE) and healthy controls*
 SLE patients (n = 1,369)Healthy controls (n = 1,826)
  • *

    Except where indicated otherwise, values are the number (%) of subjects. Missing data were not included in the calculation of the mean age, clinical manifestations, and frequency of positivity for antinuclear antibodies (ANAs) and anti–double-stranded DNA (anti-dsDNA). CNS = central nervous system.

  • Defined as a titer ≥1:40.

Age, mean ± SD years38.7 ± 12.639.4 ± 12.4
Female1,306 (89)1,569 (86)
Ethnicity
 Caucasian1,082 (74)1,439 (78)
 African American180 (12)180 (10)
Clinical manifestations
 Arthralgias/arthritis598 (82) 
 Mucocutaneous lesions/photosensitivity590 (81) 
 Renal disease475 (60) 
 Serositis380 (48) 
 Hematopoietic abnormalities256 (46) 
 CNS involvement/neuropsychiatric disorders153 (19) 
Antibodies
 ANA positive658 (93)128 (7)
 Anti-dsDNA positive164 (75) 

The analysis reported herein focused on the Caucasian and African American populations, since these comprised the majority of our cases. The genotype frequencies did not deviate from Hardy-Weinberg equilibrium for the polymorphisms studied in either of the ethnic groups. Significant differences in the genotype and allele distributions of −794 CATT between Caucasians and African Americans were noted, as previously reported, and their frequencies were similar to previously published ethnic-specific frequencies (19).

MIF promoter polymorphisms and SLE incidence.

To determine the potential association between MIF polymorphisms and the incidence of SLE, the frequencies of MIF alleles, genotypes, and haplotypes as well as the frequency of the high-expression genotypes (those containing −794 CATT7 or −173*C) and the high-expression haplotype 7C were calculated and compared between SLE patients and healthy controls. The frequencies of the −173 G/C SNP genotype in Caucasians and African Americans are shown in Table 2. The frequency of the high-expression −173*C-containing MIF genotype, which has been previously shown to be associated with elevated levels of circulating MIF (10, 25), was significantly lower in SLE patients than in controls in the African American cohort (49.7% versus 64.2%; P = 0.007) but not in the Caucasian cohort (32.7% versus 34.6%; P = 0.36). The P values on permutation testing were 0.0154 for African American patients and 0.578 for Caucasian patients compared with controls. This finding suggests that the high-expression −173*C-containing MIF genotype may be protective of SLE in African Americans.

Table 2. Frequencies of MIF −173 single-nucleotide polymorphism genotypes in patients with systemic lupus erythematosus (SLE) and healthy controls, by ethnicity*
 CaucasianAfrican American
SLE patientsHealthy controlsPSLE patientsHealthy controlsP
  • *

    Values are the number (%) of subjects.

  • Comprising −173 G/C and −173 C/C.

−173 MIF genotype      
 G/G701 (67.3)913 (65.4) 90 (50.3)62 (35.8) 
 G/C300 (28.8)440 (31.5) 64 (35.8)89 (51.4) 
 C/C41 (3.9)42 (3.0) 25 (14.0)22 (12.7) 
 Total1,0421,395 179173 
−173*C-containing genotypes341 (32.7)482 (34.6)0.3689 (49.7)111 (64.2)0.007

The frequencies of the −794 CATT repeat genotype are displayed in Table 3. The high-expression −794 CATT7–containing MIF genotypes were significantly underrepresented in the Caucasian SLE group when compared with controls (21% versus 24%; P = 0.049), but this difference was not noted in the African American SLE patients compared with controls (14.5% versus 19.5%; P = 0.256). After permutation testing, however, the P values were 0.147 in Caucasian patients and 0.383 in African American patients compared with controls. These results, although suggestive, did not support the hypothesis that the high-expression −794 CATT7–containing genotypes protect against the development of SLE.

Table 3. Frequencies of MIF −794 CATT5–8genotypes in patients with systemic lupus erythematosus (SLE) and healthy controls, by ethnicity*
 CaucasianAfrican American
SLE patientsHealthy controlsPSLE patientsHealthy controlsP
  • *

    Values are the number (%) of subjects.

  • Comprising −794 CATT5/7, −794 CATT6/7, and −794 CATT7/7.

−794 CATT MIF genotype      
 CATT5/577 (7.7)76 (5.7) 39 (22.3)30 (16.8) 
 CATT5/6298 (29.8)405 (30.5) 48 (27.4)65 (36.3) 
 CATT5/750 (5.0)108 (8.1) 12 (6.9)13 (7.3) 
 CATT5/81 (0.1)2 (0.2) 00 
 CATT6/6414 (41.4)519 (39.1) 60 (34.3)48 (26.8) 
 CATT6/7148 (14.8)188 (14.2) 9 (5.1)18 (10.1) 
 CATT6/81 (0.15)5 (0.4) 2 (1.1)1 (0.6) 
 CATT7/712 (1.2)24 (1.8) 4 (2.3)4 (2.2) 
 CATT8/800 1 (0.6)0 
 Total1,0011,327 175179 
−794 CATT7–containing genotypes210 (21.0)320 (24.2)0.04925 (14.5)35 (19.5)0.25

MIF haplotypes in both ethnic groups were reconstructed computationally. The estimated frequencies of all possible MIF haplotypes and their relative odds ratios (ORs) are shown in Table 4. The −173 G/C and −794 CATT polymorphisms were in linkage disequilibrium, with the −173*C allele being more frequently associated with CATT7 than with the CATT5 or CATT6 alleles (D′ = 0.74 for Caucasians and D′ = 0.80 for African Americans). After adjusting for sex, age, geographic location, MIF polymorphisms, and ANA status, the frequency of the high-expression 7C haplotype was found to be lower in SLE patients than in healthy controls, with differences being statistically significant in both ethnic groups (in Caucasians, OR 0.63, 95% CI 0.53–0.89, P = 0.001 versus controls; in African Americans, OR 0.46, 95% CI 0.23–0.95, P = 0.012 versus controls). The P values on permutation testing were 0.0016 in Caucasian patients and 0.06 in African American patients compared with controls.

Table 4. Frequencies of MIF haplotypes in Caucasian and African American patients with systemic lupus erythematosus (SLE) and healthy controls*
 CaucasianAfrican American
MIF haplotypePatients (n = 1,988)Controls (n = 2,582)OR (95% CI)PPatients (n = 348)Controls (n = 348)OR (95% CI)P
  • *

    Values are the number (%) of haplotypes. Odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated by logistic regression. The reported P values were adjusted for sex, age, geographic location, MIF polymorphisms, and antinuclear antibody status. n = number of haplotypes examined.

−794 CATT5/−173*G (5G)452 (22.8)618 (23.9)1 85 (24.0)71 (20.4)1 
−794 CATT6/−173*G (6G)1,123 (56.5)1,449 (56.1)1.08 (0.91–1.28)0.395140 (40.1)141 (40.2)0.85 (0.57–1.27)0.43
−794 CATT7/−173*G (7G)48 (2.4)26 (1.0)1.67 (0.82–3.37)0.15613 (3.5)3 (0.7)4.42 (0.99–19.74)0.052
−794 CATT5/−173*C (5C)50 (2.5)30 (1.2)1.85 (0.81–4.25)0.14555 (15.3)65 (18.8)0.69 (0.40–1.20)0.188
−794 CATT6/−173*C (6C)146 (7.3)142 (5.5)1.24 (0.90–1.70)0.19437 (10.3)33 (9.5)0.88 (0.52–1.51)0.648
−794 CATT7/−173*C (7C)169 (8.5)317 (12.3)0.63 (0.53–0.89)0.00118 (4.8)35 (10.0)0.46 (0.23–0.95)0.012

To account for population stratification bias, we used the formulas established by Lee and Wang (22). The prevalence of SLE is reported to be 40–90 per 100,000 population in Caucasians and 100–300 per 100,000 population in blacks (26, 27). The frequency of the 7C haplotype in Caucasians is reported to be 5–12% (28, 29), which is within the range of frequencies reported herein. The frequency of the 7C haplotype in African Americans has not been previously reported, and therefore we adopted the frequencies of 4–11% reported in our study. Based on the prevalence of SLE and the frequency of the 7C haplotype, we calculated the highest OR (“U”) and the lowest OR (“1/U”) that can be attributed to population stratification. In Caucasians, “U” is 1.2 and “1/U” is 0.83, while in African Americans, “U” is 1.33 and “1/U” is 0.75. The ORs observed in the present study, 0.63 in Caucasians and 0.46 in African Americans, are below the lowest calculated ORs for these respective populations (the ORs of 0.83 and 0.75 reported above), indicating that the ORs reported herein cannot be explained simply by population stratification. These results, taken together, further suggest that the high-expression MIF haplotype 7C confers protection against the development of SLE in both Caucasian and African American subjects.

MIF polymorphisms and SLE clinical manifestations.

To determine the effect of MIF gene polymorphisms on disease manifestations, SLE patients were stratified according to the presence of documented clinical manifestations. The frequencies of the low-expression −794 CATT5–containing and −173*G-containing MIF genotypes were measured and compared between patients with significant organ involvement (nephritis, serositis, or CNS involvement) and those with mild disease (consisting of arthralgias/arthritis and mucocutaneous manifestations). Since the majority of the patients in the SLE cohort were Caucasian, our investigation of the potential association between MIF polymorphisms and clinical manifestations focused primarily on this ethnic group, in order to allow for higher sample numbers for statistical analysis.

Patients with nephritis, patients with serositis, and male patients with CNS involvement had a lower frequency of the low-expression −794 CATT5–containing MIF genotypes when compared with patients without these disease complications (for nephritis, 36% versus 64% [P = 0.023]; for serositis, 42% versus 58% [P = 0.005]; and for CNS involvement, 11% versus 89% [P = 0.040]). No differences in the distribution of the low-expression −173*G-containing MIF genotype were observed in patients with and those without significant end-organ involvement. These groups were also analyzed for differences in the high-expression −794 CATT7–containing and −173*C-containing genotypes and the 7C haplotype, but no statistically differences were detected.

MIF polymorphisms and autoantibody status.

To determine the relationship between MIF polymorphisms and ANA status, the frequencies of ANA positivity (defined as a titer ≥1:40) were determined in SLE patients and control subjects, and ANA status was then assessed with respect to the types of MIF genotypes and haplotypes present. Both patients and healthy controls with the high-expression −794 CATT7–containing or −173*C-containing MIF genotypes and the high-expression 7C haplotype were observed to be less likely to be ANA positive when compared with subjects without these combinations (for those with the −794 CATT7–containing genotypes, 17.7% versus 25.8% [P = 0.03]; for those with the −173*C-containing genotypes, 31.8% versus 43.8% [P = 0.008]; and for those with the 7C haplotype, 63% versus 78% [P = 0.0001]). The observation that high-expression MIF alleles are also associated with a lower incidence of ANA positivity further supports the conclusion that these alleles may confer protection from SLE. Additional analyses for the potential association between anti–double-stranded DNA or extractable ANAs and MIF polymorphisms were not performed because of incomplete data for many patients.

Plasma levels of MIF and TNFα.

MIF levels were measured in plasma samples from each group, obtained at the time of study entry, to determine whether there was a relationship between circulating MIF concentrations and MIF haplotypes. The mean levels of MIF in the plasma of Caucasian SLE patients as a group were lower than those in matched disease-free controls (mean ± SD 4.6 ± 4.6 ng/ml in patients [n = 116] versus 8.9 ± 7.0 ng/ml in controls [n = 55]; P < 0.001) (Figure 2A). Circulating MIF levels were also lower in the African American SLE patients compared with matched controls, but this difference did not reach statistical significance (mean ± SD 5.6 ± 6.3 ng/ml in SLE patients versus 6.4 ± 6.8 ng/ml in controls [n = 44 in each group]; P = 0.580).

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Figure 2. A and B, Macrophage migration inhibitory factor (MIF) plasma levels in patients with systemic lupus erythematosus (SLE) and healthy controls, grouped by ethnicity (Caucasian and African American) (A) and by MIF haplotype (high-expression −794 CATT7/−173*C [7C] or low-expression −794 CATT5/−173*G [5G]) (B). Bars in A show the mean ± SD level of MIF in 116 Caucasian patients and 55 healthy controls, and 44 African American patients and 44 healthy controls. Bars in B show the mean ± SD level of MIF in 17 Caucasian patients and 41 African American patients with either the high-expression 7C or low-expression 5G MIF haplotype. C, Positive correlation between MIF plasma levels and tumor necrosis factor α (TNFα) plasma levels in 160 Caucasian and African American patients with SLE. Correlations were assessed by Spearman's correlation test. D, Regulation of Toll-like receptor 7–induced MIF release from peripheral blood mononuclear cells (PBMCs) according to MIF haplotype (7C versus 5G). Bars show the mean ± SD level of MIF released by PBMCs from Caucasian individuals with the low-expression 5G haplotype (n = 20) or high-expression 7C haplotype (n = 10). = P < 0.001; ∗∗ = P = 0.04; ∗∗∗ = P = 0.046 versus control or 7C, by unpaired t-test.

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We next measured plasma MIF levels in SLE patients identified as having the high-expression MIF haplotype 7C and compared the values with patients known to have the low-expression MIF haplotype 5G. Notably, both Caucasian and African American patients with the high-expression 7C haplotype had higher plasma levels of MIF when compared to those in patients with the low-expression 5G haplotype (in Caucasians, mean ± SD 2.4 ± 1.7 ng/ml versus 1.6 ± 1.6 ng/ml [P = 0.041]; in African Americans, 8.9 ± 7.6 ng/ml versus 4.8 ± 4.3 ng/ml [P = 0.040]) (Figure 2B).

To minimize the effect of medications and other disease-related factors on MIF plasma levels, we also measured the concentrations of circulating MIF in Caucasian and African American healthy controls, all of whom had been identified as carriers of either the high-expression 7C MIF haplotype or the low-expression 5G MIF haplotype. Of the 100 control subjects studied, 17 were identified as having the 7C haplotype and 36 as having the 5G haplotype. The circulating MIF plasma levels were higher in the group with the high-expression 7C haplotype (mean ± SD 10.3 ± 8.1 ng/ml) than in the group with the low-expression 5G haplotype (6.4 ± 5.5 ng/ml) (P = 0.048). These data suggest that the MIF haplotype influences the levels of circulating MIF at baseline, irrespective of underlying disease or autoimmunity.

MIF may be distinguished functionally from other cytokines because of its action as a regulatory mediator that sustains the activation of immune cells and the production of downstream effectors such as TNFα (30). TNFα is considered to be a major initiator of vascular endothelial cell and end-organ damage. Among both the Caucasian group and the African American group, we observed higher TNFα levels in SLE patients than in controls (mean ± SD 64.9 ± 192.4 pg/ml versus 14.3 ± 42.3 pg/ml; P = 0.05), which is consistent with the findings previously reported (31). Moreover, a comparative analysis of the levels of TNFα and MIF in SLE patients showed a weak positive correlation between the 2 cytokines (r2 = 0.19, P = 0.04) (Figure 2C).

In separate analyses, we also examined circulating MIF concentrations in relation to SLE clinical manifestations. No significant associations were observed. However, this comparison may have been confounded by the extent of disease activity, the effect of immunosuppressive treatments, and the influence of endogenous or administered glucocorticoids on circulating MIF levels (32).

Influence of MIF polymorphisms on immune cell activation.

Activated monocyte/macrophages are an abundant source of MIF in vivo and have been shown to release MIF in response to different microbial products (6). There is evidence that viral infections and nucleic acid–containing immune complexes contribute to the immunopathology of SLE by providing recurrent or self-sustaining stimulatory signals through specific TLRs (33, 34).

We tested the influence of the MIF genotype on the ability of human peripheral blood mononuclear cells to produce MIF in response to TLR-7 agonism. Monocytes were isolated from 30 healthy Caucasian control subjects with the low-expression 5G MIF haplotype or the high-expression 7C MIF haplotype, and the cells were then stimulated with the TLR-7 agonist polyU. As shown in Figure 2D, human peripheral blood monocytes with the high-expression 7C haplotype produced almost 80% more MIF than did cells with the low-expression 5G haplotype (mean ± SD 10.5 ± 3.8 ng/ml versus 5.8 ± 4.4 ng/ml; P = 0.046). These data suggest that circulating RNA, whether of viral or endogenous origin, may provide a relevant source of MIF haplotype–dependent immunologic stimulation through TLR-7.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

In the present study, we have demonstrated a significant relationship between two known promoter polymorphisms in MIF, neither of which has been studied previously in genome-wide association studies, and the incidence and clinical severity of SLE (19). The presence of the high-expression 7C MIF haplotype was associated with a lower incidence of SLE in Caucasians and, to a lesser extent, in African Americans. When compared with ethnically matched controls, African Americans with SLE had lower frequencies of the high-expression −173*C-containing MIF genotype. We further observed that high-expression MIF genotypes are associated with a reduced frequency of ANAs, the production of which is a hallmark serologic abnormality of SLE (35). When the SLE group and control group were stratified by MIF haplotype, a significant association between plasma MIF levels and MIF haplotype was evident. These results, collectively, suggest that a genetic predisposition to increased MIF production may confer protection against the development of an autoimmune response leading to SLE.

MIF is a proinflammatory mediator and an upstream regulator of TNFα expression. The possibility that an increase in TNFα production may confer protection against SLE was suggested more than 20 years ago in a study by Jacob and colleagues, who reported that monocytes with the HLA–DR3 or HLA–DR4 SLE susceptibility loci showed low levels of inducible TNFα production (36). Those authors also found that TNFα administration to lupus-prone mice delayed the onset of autoimmunity (37). More recently, it has been noted that the administration of anti-TNFα therapies can lead to the development of ANAs (38), further pointing to the potentially protective role of TNFα in SLE.

Our data also indicate that patients with established SLE who have low-expression −794 CATT5–containing MIF genotypes may be protected from the development of the inflammatory clinical manifestations of serositis, nephritis, and CNS involvement. These conclusions mirror the associations that have been reported previously between high-expression MIF alleles and disease severity in such inflammatory disorders as rheumatoid arthritis (10), systemic sclerosis (14), and asthma (15). Thus, once autoimmunity develops, a genetic propensity for increased MIF expression likely contributes to specific disease manifestations and end-organ damage.

What is less evident, however, is how a genetic predisposition to increased MIF expression and a downstream response that includes TNFα production (6) may protect against the development of autoimmunity or clinical progression to SLE. High-expression MIF alleles have been shown to be associated with improved survival from certain infections, in part by augmenting innate immune responses (28). It has been hypothesized that antecedent infections play a role in SLE, through mechanisms that may involve antigenic mimicry, oligoclonal B or T cell activation, and loss of tolerance (39). Thus, a more robust, MIF-dependent antimicrobial response may promote the clearance and the timely resolution of infection, thereby protecting against the development of autoimmunity. Our in vitro data provide some support for this notion, since monocytes with the high-expression 7C MIF haplotype produced more MIF upon stimulation of TLR-7.

Finally, recent data support a role for excessive apoptosis or defective clearance of apoptotic cells in the immunopathogenesis of SLE. Apoptotic nuclei may overwhelm the reticuloendothelial system, break immune tolerance, and induce autoantibody production against nuclear components (40). A higher level of MIF expression may reduce the apoptotic response during inflammation and decrease the likelihood of an autoimmune response progressing to SLE (30).

Our ELISA analyses did not reveal a significant difference in circulating MIF levels among SLE patients with different disease manifestations. Although higher MIF levels in plasma may be anticipated in patients with active end-organ disease, the measurement of MIF in the blood may be confounded by several factors. Measurements in plasma may not accurately reflect elevated levels of MIF expression in sites of tissue inflammation, and baseline MIF levels are known to vary in a circadian rhythm (41). While high-dose, exogenous glucocorticoids suppress MIF secretion, low doses may actually induce MIF release in vivo. Thus, any conclusions based on the measurement of plasma MIF must be tempered by the heterogeneity of disease activity and the different treatment protocols, including glucocorticoids, in patients. Finally, lymphopenia is a feature of SLE and may reduce circulating immune cell sources of MIF production.

Recent genome-wide association scans have identified several gene polymorphisms that are associated with susceptibility to SLE (4). These analyses have been limited to the study of selected SNPs and have not examined microsatellite repeats such as the MIF −794 CATT site. Moreover, these studies have focused on disease susceptibility and not clinical manifestations, and they have so far been limited to Caucasian populations.

The main limitation of the present study is its cross-sectional design. Thus, the clinical phenotype was determined up to the point in time that the study was performed. Internationally established SLE activity and damage indices also were not available for many patients; therefore, the inclusion of end-organ involvement served as our assessment of disease severity. Furthermore, our findings do not replicate those from the study by Sanchez et al, who found that, in a smaller Spanish population, there was an increased incidence of SLE in those individuals with the homozygous −173 C/C MIF genotype (29). It is possible that our conclusions differ because of sample size or unknown genetic variations between populations.

In summary, our genetic association analyses of prevalent and functional MIF polymorphisms suggest that MIF plays a dual role in SLE. In Caucasians and African Americans, high-expression MIF polymorphisms are associated with a lower incidence of SLE. In patients with established SLE, however, low-expression MIF polymorphisms are associated with a lower incidence of end-organ injury. The possibility that some SLE patients demonstrate a propensity for end-organ disease based on the type of MIF allele present may support a MIF-directed pharmacogenomic approach in patients with SLE entering clinical evaluation.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Bucala had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Sreih, Leng, Yu, Zhang, Alarcón-Riquelme, Harley, Bucala.

Acquisition of data. Sreih, Leng, LaChance, Yu, Mizue, Pons-Estel, Abelson, Gunnarsson, Svenungsson, Cavett, Glenn, Zhang, Montgomery, Perl, Salmon, Harley, Bucala.

Analysis and interpretation of data. Sreih, Ezzeddine, Leng, Subrahmanyan, Abelson, Perl, Harley, Bucala.

ROLE OF THE STUDY SPONSOR

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

Bristol-Myers Squibb had no role in the study design or in the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication. Publication of this article was not contingent upon approval by Bristol-Myers Squibb.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES
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