The role of macrophage migration inhibitory factor in the cascade of events leading to reperfusion-induced inflammatory injury and lethality

Abstract

Ischemia and reperfusion (I/R) injury is associated with a systemic inflammatory response, characterized by intense tumor necrosis factor (TNF)-alpha production and TNF-alpha-dependent tissue injury. Macrophage migration inhibitory factor (MIF) is a potent proinflammatory cytokine that may induce TNF-alpha release and play an important role in innate immune and inflammatory responses. The aim of this work was to assess whether MIF was involved the inflammatory cascade and injury that follows intestinal I/R. To this end, wild-type (WT) and MIF-deficient (MIF(-/-)) mice underwent 60 minutes of ischemia followed by 60 minutes of reperfusion, after which they were culled for the assessment of inflammatory parameters. I/R was accompanied by an increase in circulating levels of MIF and an increase of vascular permeability, hemorrhage, and production of TNF-alpha in the intestine and lungs. The latter parameters were markedly suppressed in reperfused MIF(-/-) mice, and this was associated with decreased lethality (80% in WT versus 20% in MIF(-/-) mice). Interestingly, the reperfusion-associated neutrophil accumulation in the intestine and lungs was similar in WT and MIF(-/-) mice. Leukocytes isolated from lungs of MIF(-/-) mice were less activated, as assessed by their response to zymosan in a luminol-enhanced chemiluminescence assay. In conclusion, our results suggest that MIF plays an important role in the cascade of events leading to TNF-alpha production and reperfusion-induced tissue injury and lethality in mice.

Figure 1

Concentration of MIF in serum of mice undergoing intestinal I/R injury. The concentration of MIF was determined by ELISA in serum of sham-operated mice (Sham) and mice undergoing 60 minutes of ischemia and 60 minutes of reperfusion (I/R) of the superior mesenteric artery. Results are shown as pg of MIF per ml of serum and are mean ± SEM of five animals. *P < 0.01 when compared to sham-operated animals.

Figure 2

Concentrations of TNF-α in the intestine and serum of WT and MIF^−/− mice undergoing intestinal I/R injury. The concentrations of TNF-α in the intestine (A) and serum (B) of sham-operated mice (Sham) and mice undergoing 60 minutes of ischemia and 60 minutes of reperfusion (I/R) of the superior mesenteric artery were determined by ELISA. Results are shown as pg of TNF-α per ml of serum or per 100 mg of intestine and are mean ± SEM of five animals. ND = not detectable.

Figure 3

Changes in vascular permeability (A), hemorrhage (B), and neutrophil infiltration (C) in the intestine of WT and MIF^−/− mice undergoing intestinal ischemia and reperfusion injury. All parameters were measured in sham-operated mice (Sham) and after 60 minutes of ischemia followed by 60 minutes of reperfusion (I/R) of the superior mesenteric artery. A: Changes in vascular permeability were assessed as the amount of Evans blue that extravasated into the tissue and is expressed as μg of Evans blue per 100 mg of tissue. Hemorrhage was evaluated as the amount of hemoglobin in 100 mg of tissue (B) and neutrophil influx as the tissue myeloperoxidase content (C). Neutrophil influx is given as relative units as shown in the Materials and Methods section. Results are mean ± SEM of five animals in each group. *P < 0.01 when compared to Sham-operated mice and ^#P < 0.01 when compared to WT undergoing I/R. All experiments were repeated twice.

Figure 4

Histopathological analysis of the intestines (a–d) and lungs (e–h) of WT and MIF^−/− mice undergoing intestinal I/R injury. Tissues were obtained from sham-operated mice (Sham) and after 60 minutes of ischemia followed by 60 minutes of reperfusion (I/R) of the superior mesenteric artery. Sections were stained with H&E. Original magnifications, ×100.

Figure 5

Time course of luminol-enhanced chemiluminescence in leukocytes obtained from lungs of WT and MIF^−/− mice undergoing intestinal I/R injury. Leukocytes pooled from lungs of five mice after 60 minutes of ischemia followed by 60 minutes of reperfusion (I/R) of the superior mesenteric artery. Leukocytes (3 × 10⁵ cells/well) were left unstimulated (basal) or were stimulated with zymosan (20 particles/cell). The relative luminescence units (RLUs) were assayed in triplicate and results are representative of two different experiments.

Figure 6

Survival curves of WT and MIF^−/− mice undergoing intestinal I/R injury. Mice were anesthetized and submitted to 60 minutes of ischemia of the superior mesenteric artery. The vessel was reperfused, and survival was monitored as indicated. This graph represents one of two similar experiments, and there was a significant difference (P < 0.01) between WT and MIF^−/− mice.