Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis

Abstract

To study the biologic role of migration inhibitory factor (MIF), a pleiotropic cytokine, we generated a mouse strain lacking MIF by gene targeting in embryonic stem cells. Analysis of the role of MIF during sepsis showed that MIF-/- mice were resistant to the lethal effects of high dose bacterial lipopolysaccharide (LPS), or Staphylococcus aureus enterotoxin B (SEB) with D-galactosamine and had lower plasma levels of tumor necrosis factor alpha (TNF-alpha) than did wild-type mice, but normal levels of interleukin (IL)-6 and IL-10. When stimulated with LPS and interferon gamma, macrophages from MIF-/- mice showed diminished production of TNF-alpha, normal IL-6 and IL-12, and increased production of nitric oxide. MIF-/- animals cleared gram-negative bacteria Pseudomonas aeruginosa instilled into the trachea better than did wild-type mice and had diminished neutrophil accumulation in their bronchoalveolar fluid compared to the wild-type mice. Thioglycollate elicited peritoneal exudates in uninfected MIF-/- mice, but showed normal neutrophil accumulation. Finally, the findings of enhanced resistance to P. aeruginosa and resistance to endotoxin-induced lethal shock suggest that the counteraction or neutralization of MIF may serve as an adjunct therapy in sepsis.

Figure 1

Disruption of the Mif gene in mice. (A) Gene-targeting strategy: structure and partial restriction maps of Mif gene, targeting vector, mutated allele, and probe used for Southern blot hybridization. (B) Southern blot analysis of tail DNA from MIF^+/+, MIF^+/−, and MIF^−/− mice. The 6.1-kb wild-type and the 7-kb target XbaI fragments were identified using the 0.7-kb EcoRV/XbaI probe shown in A. (C) Northern blot hybridization of liver RNA obtained from wild-type and MIF^−/− mice 1 h after intraperitoneal treatment with 500 μg of LPS was performed using probes for MIF and β-actin. (D) ELISA shows absence of MIF protein in the supernatants obtained from MIF^−/− spleen cells 72 h after stimulation with Con A (1 μg/ml).

Figure 1

Disruption of the Mif gene in mice. (A) Gene-targeting strategy: structure and partial restriction maps of Mif gene, targeting vector, mutated allele, and probe used for Southern blot hybridization. (B) Southern blot analysis of tail DNA from MIF^+/+, MIF^+/−, and MIF^−/− mice. The 6.1-kb wild-type and the 7-kb target XbaI fragments were identified using the 0.7-kb EcoRV/XbaI probe shown in A. (C) Northern blot hybridization of liver RNA obtained from wild-type and MIF^−/− mice 1 h after intraperitoneal treatment with 500 μg of LPS was performed using probes for MIF and β-actin. (D) ELISA shows absence of MIF protein in the supernatants obtained from MIF^−/− spleen cells 72 h after stimulation with Con A (1 μg/ml).

Figure 1

Disruption of the Mif gene in mice. (A) Gene-targeting strategy: structure and partial restriction maps of Mif gene, targeting vector, mutated allele, and probe used for Southern blot hybridization. (B) Southern blot analysis of tail DNA from MIF^+/+, MIF^+/−, and MIF^−/− mice. The 6.1-kb wild-type and the 7-kb target XbaI fragments were identified using the 0.7-kb EcoRV/XbaI probe shown in A. (C) Northern blot hybridization of liver RNA obtained from wild-type and MIF^−/− mice 1 h after intraperitoneal treatment with 500 μg of LPS was performed using probes for MIF and β-actin. (D) ELISA shows absence of MIF protein in the supernatants obtained from MIF^−/− spleen cells 72 h after stimulation with Con A (1 μg/ml).

Figure 1

Disruption of the Mif gene in mice. (A) Gene-targeting strategy: structure and partial restriction maps of Mif gene, targeting vector, mutated allele, and probe used for Southern blot hybridization. (B) Southern blot analysis of tail DNA from MIF^+/+, MIF^+/−, and MIF^−/− mice. The 6.1-kb wild-type and the 7-kb target XbaI fragments were identified using the 0.7-kb EcoRV/XbaI probe shown in A. (C) Northern blot hybridization of liver RNA obtained from wild-type and MIF^−/− mice 1 h after intraperitoneal treatment with 500 μg of LPS was performed using probes for MIF and β-actin. (D) ELISA shows absence of MIF protein in the supernatants obtained from MIF^−/− spleen cells 72 h after stimulation with Con A (1 μg/ml).

Figure 2

MIF-deficient mice are more resistant to LPS-induced lethality and have less plasma TNF-α. (A) Survival curves of mice that received 25 mg/kg LPS intraperitoneally. (B) Plasma levels of cytokines at 90 min after LPS. *P < 0.001.

Figure 2

MIF-deficient mice are more resistant to LPS-induced lethality and have less plasma TNF-α. (A) Survival curves of mice that received 25 mg/kg LPS intraperitoneally. (B) Plasma levels of cytokines at 90 min after LPS. *P < 0.001.

Figure 3

Stimulated macrophages from MIF-deficient mice produce less TNF-α but more NO. Thioglycollate-elicited macrophages were stimulated with 100 μg/ml of LPS and 100 U/ml of IFN-γ. Supernatants were removed at 6, 12, 18, and 24 h and tested for (A) TNF-α, (B) IL-12, (C) IL-6, and (D) NO. Similar results were obtained in three other experiments at 24 h.

Figure 4

MIF^−/− mice clear P. aeruginosa instilled into the lungs better than do wild-type mice, and show less neutrophil accumulation. (A) Clearance of P. aeruginosa at 6 and 24 h. Data from wild-type (WT) and heterozygous mice were combined as they showed no difference. For 24 h: WT, n = 15; MIF^−/−, n = 14. For 6 h: WT, n = 8; MIF^−/−, n = 6. □, +/+; ▪, −/−. (B) Neutrophils accumulating in the BAL fluid at 6 h after instillation of bacteria.

Figure 4

MIF^−/− mice clear P. aeruginosa instilled into the lungs better than do wild-type mice, and show less neutrophil accumulation. (A) Clearance of P. aeruginosa at 6 and 24 h. Data from wild-type (WT) and heterozygous mice were combined as they showed no difference. For 24 h: WT, n = 15; MIF^−/−, n = 14. For 6 h: WT, n = 8; MIF^−/−, n = 6. □, +/+; ▪, −/−. (B) Neutrophils accumulating in the BAL fluid at 6 h after instillation of bacteria.