Discovery of a natural antagonist of macrophage migration inhibitory factor

Introduction

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays a critical role in the pathogenesis of sepsis. Plasma MIF concentrations are significantly higher in nonsurvivors than survivors of severe sepsis, and administration of antibodies that neutralize MIF activity improves survival in an experimental model of sepsis. Three-dimensional X-ray crystallography shows MIF has a homotrimeric conformation and we have determined that the hydrophobic cavity formed between two adjacent subunits of the homotrimer is required for the pro-inflammatory activity of the molecule. We have designed several small molecules that fit into the site critical for the proinflammatory action of MIF, and confirmed the interaction by the crystal structure of the MIF complex. Binding of MIF in this way inhibits its proinflammatory activity, improves the clinical outcome in sepsis, and recapitulates immunotherapy and gene deletion. However, no natural soluble ligand of MIF has been reported previously.

Hypothesis

MIFnl1 binds to the hydrophobic cavity of MIF. Increased concentrations of MIF in sepsis deplete plasma MIFnl1 and lead to a critical MIF:MIFnl1 imbalance.

Results

We have discovered a natural ligand, designated MIFnl1, that binds the proinflammatory site of MIF with high affinity, and effectively modulates its activity. We examined several classes of endogenous small molecules and their metabolites and observed that MIFnl1 binds to, and inhibits, the hydrophobic cavity of MIF in a dose-dependent manner with an IC₅₀ of 15.8 μM. Importantly, MIFnl1 was a more potent inhibitor of MIF than ISO-1 (IC₅₀ = 25 μM), the gold standard synthetic inhibitor of MIF. In addition, in plasma from patients with sepsis, we found an inverse correlation between the increased level of MIF and the decreased concentration of MIFnl1. Therefore, we hypothesized that supplementation of this ligand during sepsis should compensate for its dramatic reduction and improve survival in our peritonitis model of sepsis in C57/Bl6 mice. Administration of MIFnl1 improved the 7-day survival rate to 60% compared with 20% observed for the vehicle-treated mice.

Conclusion

Our data identify for the first time the presence of a natural, ligand antagonist of MIF in plasma. This suggests that, during severe sepsis, increased production and release of MIF leads to an imbalance of the MIF:MIFnl1 regulatory mechanism resulting in the development of an overwhelming systemic inflammatory response leading to cardiovascular collapse and death. A better understanding of the kinetics of MIF/ligand regulation in patients with sepsis may lead to improved outcome in this devastating disease.