Published ahead of print on September 22, 2005, doi:10.1164/rccm.200412-1714OC
© 2005 American Thoracic Society doi: 10.1164/rccm.200412-1714OC
Cystic Fibrosis, Disease Severity, and a Macrophage Migration Inhibitory Factor PolymorphismThe Conway Institute of Biomolecular and Biomedical Research and the Dublin Molecular Medicine Centre, Department of Medicine, University College Dublin, Dublin, Ireland; Institute of Genetics, School of Molecular Medical Sciences, Queen's Medical Centre,University of Nottingham, Nottingham, England; and Department of Medicine and Pathology, Yale University School of Medicine, New Haven, Connecticut Correspondence and requests for reprints should be addressed to Dr. Seamas C. Donnelly, M.D., F.R.C.P.I., Department of Medicine and Therapeutics, The Education Research Centre, St. Vincent's University Hospital, Elm Park, Dublin 4, Ireland. E-mail: seamas.donnelly{at}ucd.ie
Rationale: Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator. It contributes toward an exaggerated gram-negative inflammatory response via its ability to induce Toll-like receptor–4 expression. Studies have shown that MIF knockout mice have less aggressive Pseudomonas infection (compared with wild-type). Objectives: To assess whether a novel functional MIF polymorphism was associated with clinical prognosis in a patient cohort with chronic gram-negative infection, namely cystic fibrosis (CF). Methods: Collected genomic DNA was analyzed via polymerase chain reaction amplification for the polymorphic region for the CATT repeat polymorphism. Individuals may have a 5-, 6-, 7-, or 8-CATT tetranucleotide repeat unit on each allele. The 5-CATT repeat allele exhibits the lowest MIF promoter activity. Measurements and Main Results: Patients with stable CF (n = 167) and a matched control group (n = 166) were enrolled. In patients with CF, the MIF5+ group had a decreased incidence of Pseudomonas aeruginosa colonization (odds ratio, 0.25; 95% confidence interval, 0.09–0.65; p = 0.004) and a significant reduction in the risk of pancreatic insufficiency (odds ratio, 0.27; 95% confidence interval, 0.07–1.0; p = 0.05). A trend toward milder disease activity in the MIF5+ group was seen with all other parameters. Conclusions: The results support the concept of a regulatory role for MIF in CF.
Key Words: cystic fibrosis • macrophage migration inhibitory factor • polymorphism Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator (1). It contributes toward an excessive inflammatory response both directly via an induction of proinflammatory cytokine secretion (2) and indirectly through its ability to override the antiinflammatory activity of glucocorticoids (3). MIF is implicated in a wide number of immune and inflammatory diseases, including acute respiratory distress syndrome (3), asthma (4), septic shock (5), and rheumatoid arthritis (6). MIF is required to combat serious infections; however, high-level production of MIF may be harmful during acute infections. MIF has been shown to contribute toward an exaggerated gram-negative response through its ability to induce Toll-like receptor 4 (TLR4), a key receptor responsible for LPS-induced inflammatory cytokine production (7). Recombinant MIF has been shown to exacerbate lethal sepsis when injected with LPS or Escherichia coli into mice (8). Anti–MIF neutralizing antibodies protect mice from the lethal endotoxic sepsis induced by intraperitoneal E. coli or cecal ligation and puncture for up to 8 h after the onset of the bacterial peritonitis (2), highlighting the potential role of an anti-MIF strategy as a therapeutic target clinically. MIF knockout mice in vivo cleared Pseudomonas aeruginosa (PA) instilled into the trachea better than wild-type mice with diminished neutrophil accumulation in their bronchoalveolar fluid, suggesting neutralization of MIF may enhance resistance to PA (5). We recently described a functional CATT repeat promoter polymorphism in the MIF gene (9). Individuals were identified who were homozygous or heterozygous for 5, 6, 7, or 8 CATT repeats at position –794, designated 5-CATT, 6-CATT, and so forth. The 5-CATT repeat allele exhibited lowest MIF promoter activity in vitro. Patients with rheumatoid arthritis with the 5-CATT allele had less-aggressive disease (p < 0.02) (9). Cystic fibrosis (CF), caused by mutations in the CF transmembrane regulator (CFTR) gene (10), is the most common fatal inherited disease in whites, with a frequency of 1 in 1,461 live births in Ireland (11). Most patients succumb in young adulthood from progressive lung disease (12). Attempts to link CFTR mutation to disease severity have not been very successful (13). Patients with identical CF genotypes can have marked differences clinically (14). It is likely that variations in genes other than the gene coding for CFTR modify clinical course (i.e., modifier genes) (15). Putative modifier genes in CF have been reported (16–18). We propose that the regulation of MIF gene activity may modify the clinical course in adult CF, as this cohort has significant chronic gram-negative infection, namely PA colonization. Our hypothesis is that patients with CF exhibiting the 5-CATT repeat polymorphism in the MIF gene and consequently less MIF promoter activity would have milder clinical disease. Preliminary data relating to this work were previously presented (19, 20).
Study Population In a case-control study, adult patients with stable CF (age  18 yr) from the Irish National Adult Referral Center for Cystic Fibrosis, St. Vincent's University Hospital, provided genomic DNA for analysis. A proven diagnosis of CF was based on CFTR genotyping (available on all patients), sweat testing, and clinical phenotype. Clinical stability was defined by the absence of active acute infection requiring back-up antibiotic treatment for a minimum of 6 wk and was determined by the principal investigator by a clinical review. Treatment of all patients was standardized, in keeping with best international practice (21, 22), and was overseen by a single attending pulmonologist. At recruitment, markers of disease activity and severity were assessed. These included the following: sputa colonization, including the presence or absence of PA and Staphylococcus aureus; pancreatic sufficiency (case not requiring pancreatic supplemental enzymes); liver disease (determined by ultrasound); and diabetes mellitus (post–oral glucose tolerance test). Measuring baseline spirometry assessed lung function, and nutritional status was evaluated by calculating the percentage of ideal bodyweight of the patients. Genomic DNA from a healthy age- and sex-matched adult (age 18 yr) white control group recruited from the same population was also evaluated. Individuals in the control group had no active medical problems and no known history of lung disease, including asthma, and were not receiving regular medications, including inhaled medication. Informed consent was obtained from all participants. The ethics committee of St. Vincent's University Hospital approved the study.
Identification of the Polymorphism
Statistical Analysis
MIF Genotype and CF Phenotype Correlations In this study, 167 patients with CF and 166 age- and sex-matched control subjects were enrolled (Table 1). MIF genotype frequencies did not differ significantly between males and females in case or control groups (  2 test, p > 0.6). There was no association between MIF genotype and age in cases or control subjects (Mann-Whitney test, p > 0.2). MIF genotype frequencies did not differ significantly between cases and control subjects (2 test, p = 0.48; Table 2). In the CF group, 39% (65/167) were MIF5+ and 61% (102/167) MIF5–. Comparable figures for the control group were 43% (71/166) and 57% (95/166), respectively. Table 3 summarizes pulmonary function and nutritional status in the CF group.
In the initial logistic regression analysis of interaction between CFTR and MIF, genotype was included as a further explanatory variable. There was no evidence of interaction between DF508 and MIF5+ in any of the outcome variables studied, so this variable was dropped from the model. Looking at the presence or absence of the respiratory pathogens in patients with CF, the MIF5+ group was associated with a significant decreased incidence of Pseudomonas colonization, with 75% of this group colonized with PA versus 91% of the MIF5– group (odds ratio [OR], 0.25; 95% confidence interval [CI], 0.09–0.65; p = 0.004). No significant association was found between the MIF5+ group and a decreased incidence of other respiratory pathogen colonizations, with 34% of the MIF5+ group colonized with S. aureus versus 38% of the MIF5– group (OR, 0.79; 95% CI, 0.41–1.47; p = 1.55) and 32% of the MIF5+ group colonized with Candida versus 40% of the MIF5– group (OR, 0.73; 95% CI, 0.38–1.43; p = 0.36). The MIF5+ group had a significant reduction in the risk of pancreatic insufficiency, with 86%, compared with 96% of the MIF5– group (OR, 0.27; 95% CI, 0.07–1.0; p = 0.05). There was no significant association between pancreatic insufficiency and age (p = 0.09) or sex (p = 0.84). Inclusion of heterozygosity for the CFTR mutation R117H in the regression model confirmed previous reports of pancreas-sparing with this mutation (OR, 0.04; 95% CI, 0.01–0.24), but the protective effects of the MIF5-CATT genotypes remained after adjusting for R117H heterozygosity. In patients carrying the MIF5-CATT allele, a nonsignificant reduced incidence was observed in liver disease (9 vs. 18%; OR, 0.48; 95% CI, 0.18–1.29; p = 0.15) and diabetes (14 vs. 18%; OR, 0.79; 95% CI, 0.32–1.95; p = 0.61; Table 4).
Milder disease activity in the MIF5+ group was also reflected in the baseline spirometry with a nonsignificant trend toward better lung function (FEV1 80%: 33 vs. 23%; 2 = 2.2; p = 0.14) and better nutritional status (% ideal bodyweight 90%: 86 vs. 76%; 2 = 2.35; p = 0.13) in the MIF5+ group (Table 4) (22).
CFTR Genotype
Patients with CF with PA colonization and pancreatic insufficiency are well recognized to be associated with an adverse prognosis (21–25). In this case-control study of an Irish population with CF, we found a significant association between the presence of the 5-CATT MIF-promoter polymorphism and a decreased incidence of Pseudomonas colonization and pancreatic insufficiency. In a previous in vitro study, we showed that the 5-CATT repeat allele exhibited the lowest MIF promoter activity in vitro (9). This supports the concept of a regulatory role for MIF driving an aggressive inflammatory reaction in CF. In a recent study, mean MIF levels were 23-fold higher in the plasma of patients with CF compared with control subjects (25). The exact mechanism whereby possession of the 5-CATT repeat allele, and hence lowest promoter activity, leads to a milder clinical phenotype is unknown; however, MIF contributes toward a sustained inflammatory response by both directly inducing enhanced proinflammatory cytokine secretion (1–3), and indirectly by overriding the antiinflammatory activity of glucocorticoids (1, 3). In the context of gram-negative infection, MIF has the additional capacity to drive an exaggerated inflammatory response by augmenting TLR4 expression on inflammatory cells, thus contributing toward a maximal LPS response (7). Specifically, in PA infection, MIF knockout mice in vivo cleared PA instilled into the trachea better than wild-type mice and had diminished neutrophil accumulation in their bronchoalveolar fluid, suggesting that neutralization of MIF may enhance resistance to PA (5). In CF lung disease, an exaggerated, sustained, and extended inflammatory response to gram-negative bacteria characterized by neutrophil-dominated airway inflammation is well recognized (21–23). Reducing neutrophilic inflammation per se may not itself reduce Pseudomonas colonization, but it may attenuate the preservation of lung function (21–25) and limit systemic end organ injury. Although antiinflammatory therapies to date have shown a reduction in the decline of lung function, the risk/benefit ratio does not favor long-term prednisolone treatment (21, 23, 26), and results from high-dose nonsteroidal antiinflammatory therapies have been associated with significantly enhanced side-effect profiles, including gastrointestinal bleeding (24, 27). Therefore, the identification of a specific targeted antiinflammatory therapy based on individual patient MIF genotype may prove clinically useful. In the context of CF, an anti–MIF antibody strategy would potentially attenuate the inflammatory reaction by both directly attenuating proinflammatory cytokine production by inflammatory cells and indirectly via downregulation of TLR4 expression and blunting of the LPS response, and removal of MIF inhibition on glucocorticoid function, thus contributing to maximal in vivo glucocorticoid antiinflammatory activity and potentially enhance resistance to PA. Although pancreatic insufficiency and diabetes mellitus in CF are believed to result from a reduced volume of pancreatic secretion with low concentrations of bicarbonate, resulting in the retention and premature activation of digestive proenzymes leading to tissue destruction and fibrosis (23, 28); it is worth noting that MIF5+ may attenuate this process. A recent article highlighted the role of MIF in a rat model of acute pancreatitis and also demonstrated a survival benefit with prophylactic administration of anti–MIF antibody in this group (29). Also in this article, serum MIF levels in healthy volunteers and patients suffering from mild or severe pancreatitis directly correlated with disease severity (29). The 5-CATT repeat group also showed a nonsignificant trend toward milder disease activity with less S. aureus and Candida colonization, liver disease, and diabetes, and better nutritional status and baseline FEV1, all of which have important prognostic implications (23, 25, 30, 31). Larger studies are needed to definitively address these specific issues.
Although attempts to link CFTR mutation to disease severity have not been very successful (13, 14), in analyzing our results it is important to consider the effect CFTR genotype may have on disease severity. Our study was that of an ethnically distinct, white group from a single center in an island nation. With 57% of individuals homozygous for It should be noted that there were eight patients with CF homozygous for the 5-CATT repeat allele and seven control subjects. Because of the small number of MIF5+ homozygotes, it was not possible to obtain reliable estimates of gene dose effects. A further larger cohort study would be useful to address this question. In conclusion, MIF has a biologically plausible role as a modifying gene in CF. We describe a 5-CATT repeat polymorphism in the promoter region of the MIF gene, which is associated with a significant decrease in PA colonization and pancreatic insufficiency in adult CF. This work supports the potential role of an anti–MIF antibody therapy as part of a targeted therapeutic strategy in CF.
Supported by the Science Foundation Ireland, Irish Lung Foundation, Health Research Board Ireland, and Cystic Fibrosis Association of Ireland. Originally Published in Press as DOI: 10.1164/rccm.200412-1714OC on September 22, 2005 Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form December 21, 2004; accepted in final form September 12, 2005
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ABSTRACT
METHODS
F508+ (carrying one or two copies of the 
T (