Direct and Specific Interaction of MIF and Bacterial Endotoxin May Play an Important Role in the Pathogenesis of Sepsis

Inflammatory response is triggered in the body as a result of infections by bacteria and other pathogens. Especially, a bacterial outer membrane component, lipopolysaccharide (LPS), is a major virulence factor. LPS activates the secretion of various inflammatory cytokines. Among the secreted cytokines, macrophage migration inhibitory factor (MIF) is a cytokine‐like protein that amplifies inflammation by activating the secretion of other inflammatory cytokines. MIF levels are elevated in severe sepsis and septic shock. MIF−/− mice demonstrated improved survival from sepsis. This implies that MIF plays an important role in sepsis, but the underlying molecular mechanism is unknown. Our recent data demonstrated evidence that MIF dramatically improved CXCR4‐mediated cell responses only when it makes a complex with LPS. Based on this observation, we hypothesized that LPS released during bacterial infection forms a complex with MIF and the complex plays a deteriorating role in the pathogenesis of sepsis. Removal of bacterial endotoxin from recombinant MIF is challenging and it is controversial whether MIF alone or MIF complexed with a binding partner mediates cell responses leading to various inflammation diseases. We noticed that a conventional endotoxin‐removal column failed to separate endotoxin from MIF. This led us to investigate whether MIF and LPS directly bind each other. MIF has one tryptophan (W109) exposed to the solvent. We demonstrated that the intrinsic fluorescence of W109 was quenched when titrated with increasing concentrations of LPS. We confirmed the MIF‐LPS interaction by using isothermal titration calorimetry and determined the affinity (K D ) of 2.9 micromolar. We also proved that MIF and LPS formed a complex during protein purification and in an animal model of sepsis. Finally, we identified a LPS binding site located at the trimeric interface of MIF by using NMR. To elucidate potential biological consequences of the MIF‐LPS interaction, we tested whether this interaction alters the effect of each molecule on endothelial barrier function, which is damaged during conditions such as sepsis. Intriguingly, LPS together with MIF revealed less endothelial permeability than LPS alone. MIF alone did not significantly change endothelial barrier permeability. Our findings on the MIF‐LPS interaction could serve as a novel paradigm to better understand physiological functions of this important and enigmatic immune regulator. Support or Funding Information NIH NIAMS