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. 2012 Jul 15;186(2):162-9.
doi: 10.1164/rccm.201110-1864OC. Epub 2012 May 16.

Macrophage migration inhibitory factor enzymatic activity, lung inflammation, and cystic fibrosis

Huzaifa Adamali  1 , Michelle E Armstrong, Anne Marie McLaughlin, Gordon Cooke, Edward McKone, Christine M Costello, Charles G Gallagher, Lin Leng, John A Baugh, Günter Fingerle-Rowson, Richard J Bucala, Paul McLoughlin, Seamas C Donnelly
Affiliations

Macrophage migration inhibitory factor enzymatic activity, lung inflammation, and cystic fibrosis

Huzaifa Adamali et al. Am J Respir Crit Care Med. .

"V体育安卓版" Abstract

Rationale: Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator with unique tautomerase enzymatic activity; the precise function has not been clearly defined. We previously demonstrated that individual patients with cystic fibrosis (CF) who are genetically predisposed to be high MIF producers develop accelerated end-organ injury VSports手机版. .

Objectives: To characterize the effects of the MIF-CATT polymorphism in patients with CF ex vivo. To investigate the role of MIF's tautomerase activity in a murine model of Pseudomonas aeruginosa infection V体育安卓版. .

Methods: MIF and tumor necrosis factor (TNF)-α protein levels were assessed in plasma or peripheral blood mononuclear cell (PBMC) supernatants by ELISA V体育ios版. A murine pulmonary model of chronic Pseudomonas infection was used in MIF wild-type mice (mif(+/+)) and in tautomerase-null, MIF gene knockin mice (mif (P1G/P1G)). .

Measurements and main results: MIF protein was measured in plasma and PBMCs from 5- and 6-CATT patients with CF; LPS-induced TNF-α production from PBMCs was also assessed. The effect of a specific inhibitor of MIF-tautomerase activity, ISO-1, was investigated in PBMCs. In the murine infection model, total weight loss, differential cell counts, bacterial load, and intraacinar airspace/tissue volume were measured. MIF and TNF-α levels were increased in 6-CATT compared with 5-CATT patients with CF VSports最新版本. LPS-induced TNF-α production from PBMCs was attenuated in the presence of ISO-1. In a murine model of Pseudomonas infection, significantly less pulmonary inflammation and bacterial load was observed in mif(P1G/P1G) compared with mif(+/+) mice. .

Conclusions: MIF-tautomerase activity may provide a novel therapeutic target in patients with chronic inflammatory diseases such as CF, particularly those patients who are genetically predisposed to produce increased levels of this cytokine. V体育平台登录.

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Figures

Figure 1.
Figure 1.
Patients with cystic fibrosis (CF) with 5, X-CATT low–macrophage migration inhibitory factor (MIF) expression alleles have a significantly lower rate of decline in FEV1 (% predicted) compared with patients with CF with X, X-CATT high-MIF expression alleles over time. (A) Average rate of decline in FEV1 (% predicted) is significantly less (P = 0.013; nonlinear association) in 5, X-CATT (n = 50) compared with X, X-CATT patients with CF (n = 93), who had an average follow-up period of 3.13 years and were aged 25 to 30 years. A median of seven measures of lung function (interquartile range, 3–11) were recorded per patient. Results were adjusted for age and sex. (B) There was no significant association between rate of change in FVC and MIF genotype over time. (A, B) There was no significant difference in FEV1 and FVC across MIF genotype groups at time of study entry.
Figure 2.
Figure 2.
Macrophage migration inhibitory factor (MIF) is significantly increased in vivo and in ex vivo peripheral blood mononuclear cell (PBMC) cultures from 6, 6-CATT patients with cystic fibrosis (CF) with high-MIF expression alleles compared with 5, 5-CATT low-MIF expressers. (A) MIF is significantly increased in the plasma of patients with CF (n = 70) compared with control subjects (n = 72). ***P < 0.001, plasma from control versus CF. (B) MIF is significantly increased in the plasma of patients with CF with 6, 6-CATT alleles (high-MIF expressers; n = 18) compared with patients with 5, 5-CATT alleles (low-MIF expressers; n = 6). *P < 0.05, plasma from 6, 6-CATT- versus 5, 5-CATT-patients with CF. (C) PBMCs derived from patients with CF with 6, 6-CATT alleles (n = 6) secrete increased MIF compared with patients with 5, 5-CATT alleles (n = 6). *P < 0.05, PBMCs from 6, 6-CATT- versus 5, 5-CATT-patients with CF. MIF protein concentrations were quantitated by ELISA. Values represent mean ± SEM of MIF protein (pg/ml) and are expressed as fold increase over control subjects.
Figure 3.
Figure 3.
Tumor necrosis factor (TNF)-α mRNA expression and protein production are increased in peripheral blood mononuclear cells (PBMCs) from 6, 6-CATT compared with 5, 5-CATT patients with cystic fibrosis (CF) after LPS treatment. (A) TNF-α mRNA expression is significantly increased in PBMCs from patients with CF with 6,6-CATT alleles (high–macrophage migration inhibitory factor [MIF] expressers; n = 6) compared with patients with 5,5-CATT alleles (low-MIF expressers; n = 6) after LPS treatment (100 ng/ml) for 6 hours. (B) TNF-α protein production is increased from PBMCs from patients with CF with 6, 6-CATT alleles (high-MIF expressers; n = 6) compared with patients with 5, 5-CATT alleles (low-MIF expressers; n = 6) after LPS treatment (100 ng/ml) for 24 hours. TNF-α mRNA expression was quantitated by quantitative real-time polymerase chain reaction. Values represent mean ± SEM for TNF-α mRNA and are expressed per unit of 18S ribosomal mRNA and are expressed as fold increase over medium only control. TNF-α protein concentrations were quantitated by ELISA. Values represent mean ± SEM of TNF-α protein (pg/ml). **P < 0.01, *P < 0.05: LPS versus medium only in PBMCs from 6, 6-CATT patients with CF; ***P < 0.001: LPS versus medium only in PBMCs from 5, 5-CATT-patients with CF.
Figure 4.
Figure 4.
LPS-induced tumor necrosis factor (TNF)-α production from peripheral blood mononuclear cells (PBMCs) from healthy donors, and 5, 5-CATT and 6, 6-CATT patients with cystic fibrosis (CF), is attenuated after pretreatment with ISO-1, a small molecular weight inhibitor of macrophage migration inhibitory factor (MIF) enzymatic activity. A significant increase in TNF-α production was observed in PBMCs from (A) healthy donors, (B) 5, 5-CATT, and (C) 6, 6-CATT patients with CF (A–C, P < 0.001) after 24 hours of treatment with LPS (100 ng/ml) compared with medium-only cells. After a 30-minute pretreatment with 10 to 100 μM ISO-1, a specific inhibitor of MIF-tautomerase activity, a significant decrease in LPS-induced TNF-α was observed in PBMCs from (A) healthy donors (100 μM ISO-1; P < 0.01), (B) 5, 5-CATT patients with CF (100 μM; P < 0.01), and (C) 6, 6-CATT patients with CF (10 μM, P < 0.01; 100 μM, P < 0.05). The fold decrease in LPS-induced TNF-α production using 100 μM pretreatment with ISO-1 was (A) 16.3-fold in healthy donors, (B) 8.6-fold in 5, 5-CATT-patients with CF, and (C) 4.2-fold in 6, 6-CATT patients with CF. TNF-α protein concentrations were quantitated by ELISA. Values represent mean ± SEM of TNF-α protein (pg/ml). ***P < 0.001, LPS versus medium only; **P < 0.01, LPS versus pretreatment with ISO-1 before LPS. Results are representative of a minimum of four replicate samples from n = 3 healthy donors and n = 4 patients with CF (both genotypes). DMSO = dimethyl sulfoxide.
Figure 5.
Figure 5.
Mice devoid of macrophage migration inhibitory factor (MIF) enzymatic activity reveal less weight loss compared with wild-type mice after Pseudomonas aeruginosa infection. Enzymatically devoid mice (mifP1G/P1G) experience less weight loss after infection compared with wild-type mice at Day 3 and Day 14 postinfection (Day 3: n = 8/group; Day 14: n = 10/group). Change in mean body weight is expressed as a percentage ± SEM. **P < 0.01, mif P1G/P1G enzymatic-knockout (n = 8) versus mif+/+ wild-type mice at Day 3 postinfection.
Figure 6.
Figure 6.
Tautomerase-null mice experience reduced pulmonary neutrophil infiltration, tumor necrosis factor (TNF)-α mRNA expression, TNF-α protein levels, and bacterial load compared with macrophage migration inhibitory factor (MIF) wild-type mice after Pseudomonas aeruginosa infection. (A) Differential cell counts revealed a significant decrease in the percentage of neutrophils at Day 3 postinfection in Pseudomonas-infected lungs from mifP1G/P1G mice versus wild-type mice (Day 3: n = 8/group; Day 14: n = 10/group). Neutrophil numbers were assessed by differential cell count using Diff-Quick and are expressed as a percentage ± SEM of total cell count. (B) A significant decrease in pulmonary TNF-α mRNA expression was observed at Days 3 and 14 in lungs from tautomerase-null mice compared with wild-type mice after P. aeruginosa infection. TNF-α mRNA expression was quantitated by quantitative real-time polymerase chain reaction. Values represent mean ± SEM for TNF-α mRNA and are expressed per unit of 18S ribosomal mRNA and are expressed as fold increase over medium only control. **P < 0.01, ***P < 0.001; mifP1G/P1G enzymatic-knockout versus wild-type mice. (C) A significant decrease in P. aeruginosa load was observed in mifP1G/P1G mice compared with wild-type mice at 3 days postinfection (n = 8 per group; P < 0.05). Bacterial load was quantitated by TaqMan assay for P. aeruginosa detection (Applied Biosystems, Foster City, CA) (24, 25), using genomic DNA, which was extracted from formalin-fixed and paraffin-embedded, murine lung sections at 3 days post P. aeruginosa infection (23) (see online supplement). A 103 to 108 serial dilution of an overnight culture of P. aeruginosa (1.27 × 109 cfu/ml) was used to generate a standard curve for analyses (24, 25). Results are expressed in arbitrary units and show the relative fold-change in P. aeruginosa load in mif+/+ mice compared with mifP1G/P1G mice at 3 days postinfection. (D–F) Using immunohistochemistry, a trend toward a decrease in TNF-α protein levels was observed in lungs using from tautomerase-null mice (E) compared with wild-type mice (D) at 3 days post P. aeruginosa infection (see online supplement). No staining was observed with irrelevant rabbit polyclonal IgG (F). ×10 Objective, scale bar = 100 μm.
Figure 7.
Figure 7.
Tautomerase-null mice reveal less inflammation, alveolar wall thickening, and inflammatory cell infiltrate compared with mif+/+ mice after chronic Pseudomonas aeruginosa infection. Histological analysis was performed on lung sections from (A–C) mif+/+ and (D–F) tautomerase-null mice, respectively (hematoxylin and eosin stain; ×1.25 or ×10 objective). (A, D) Day 3 postinfection, ×1.25 objective; (B, E) Day 3 postinfection, ×10 objective; (C, F) Day 14 postinfection, ×10 objective. (B, E) Scale bar = 200 μm and (D) 1,500 μm.
Figure 8.
Figure 8.
Tautomerase-null mice reveal reduced pulmonary intraacinar tissue volume and reduced pathology compared with mif +/+ mice after chronic Pseudomonas aeruginosa infection. (A, B) Stereological examination reveals that mif P1G/P1G mice have decreased intraacinar tissue volume, or an increased air/tissue ratio, compared with wild-type mice at (A) Day 3 and (B) Day 14 postinfection (Day 3: n = 8/group; Day 14: n = 10/group). Volumes (ml) of intraacinar airspace (air volume) and intraacinar tissue (tissue volume) of left lungs are expressed as mean ± SEM. **P < 0.01, ***P < 0.001: intraacinar tissue volumes of left lungs from mif P1G/P1G versus mif+/+ wild-type mice at Day 3 postinfection.
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