Macrophage migration inhibitory factor (MIF) is a pivotal regulator of the immune response. Neutralization or genetic deletion of MIF does not completely abrogate activation responses, however, and deletion of the MIF receptor, CD74, produces a more pronounced phenotype than MIF deficiency. We hypothesized that these observations may be explained by a second MIF-like ligand, and we considered a probable candidate to be the protein encoded by the homologous, D-dopachrome tautomerase (D-DT) gene. We show that recombinant D-DT protein binds CD74 with high affinity, leading to activation of ERK1/2 MAP kinase and downstream proinflammatory pathways. Circulating D-DT levels correlate with disease severity in sepsis or malignancy, and the specific immunoneutralization of D-DT protects mice from lethal endotoxemia by reducing the expression of downstream effector cytokines. These data indicate that D-DT is a MIF-like cytokine with an overlapping spectrum of activities that are important for our understanding of MIF-dependent physiology and pathology.inflammation | lipopolysaccharide | septic shock M acrophage migration inhibitory factor (MIF) is the first cytokine activity described and a key regulatory mediator that is released upon activation of different cell types (1-3). MIF increases macrophage antimicrobial responses and it is expressed upstream of cytokines such as tumor necrosis factor (TNF)-α, IFN-γ, and IL-1β (4). MIF activates immune cells by binding to CD74, leading to the recruitment of CD44 into a signaling complex, the stimulation of nonreceptor tyrosine kinases, and initiation of the ERK1/2 MAP kinase pathway (5, 6). The chemokine receptors CXCR2 and CXCR4 also become activated by MIF via noncognate interactions that are reinforced in the presence of CD74 (7). Among mesenchymal cell types, MIF binding to cardiomyocyte CD74 stimulates the AMP-activated kinase (AMPK) cascade to mediate protection from ischemic injury (8, 9).Although MIF receptor knockout mice (CD74) phenocopy features of MIF deficiency (10-12), recent observations have led to the hypothesis that there may be a second ligand for CD74. MIF-deficient B cells, for example, are more sensitive to apoptosis than wild-type B cells, but the magnitude of this defect is twofold more pronounced in CD74-deficient cells (13). Intravital microscopy studies also have shown a more pronounced effect of antagonism of CD74 than MIF in monocyte arrest (7). Finally, anti-MIF antibodies, although highly effective in experimental studies, do not completely inhibit CD74-dependent cellular activation responses (14).We hypothesized that these observations may be explained by a second MIF-like ligand, and we considered a likely candidate to be the protein encoded by the DDT gene, D-dopachrome tautomerase (D-DT). DDT and MIF show a conserved intron-exon structure and their coding regions are highly homologous. The genes for MIF and D-DT are in close apposition to each other and to two theta-class glutathione S-transferases, suggesting that these gene clusters arose by...

Although chemokine and growth factor receptors are attractive and popular targets for cancer therapeutic intervention, structure-based targeting of the ligands themselves is generally not considered practical. New evidence indicates that a notable exception to this is macrophage migration inhibitory factor (MIF). MIF, an autocrine-and paracrine-acting cytokine/growth factor, plays a pivotal role in both the initiation and maintenance of neoplastic diseases. MIF possesses a nonphysiologic enzymatic activity that is evolutionarily wellconserved. Although small molecule antagonists of MIFs enzymatic active site have been reported to inhibit biological activities of MIF, universally high IC 50 s have limited their clinical appeal. Using a computational virtual screening strategy, we have identified a unique small molecule inhibitor that serves as a suicide substrate for MIF, resulting in the covalent modification of the catalytically active NH 2 -terminal proline. Our studies further reveal that this compound, 4-iodo-6-phenylpyrimidine (4-IPP), is f5Â to 10Â times more potent in blocking MIF-dependent catalysis and lung adenocarcinoma cell migration and anchorage-independent growth than the prototypical MIF inhibitor, ISO-1. Finally, using an in silico combinatorial optimization strategy, we have identified four unique congeners of 4-IPP that exhibit MIF inhibitory activity at concentrations 10Â to 20Â lower than that of parental 4-IPP. [Cancer Res 2008;68(18):7253-7]

Background-Elderly patients are more sensitive than younger patients to myocardial ischemia, which results in higher mortality. We investigated how aging affects the cardioprotective AMP-activated protein kinase (AMPK) signaling pathway. Methods and Results-Ischemic AMPK activation was impaired in aged compared with young murine hearts. The expression and secretion of the AMPK upstream regulator, macrophage migration inhibitory factor (MIF), were lower in aged compared with young adult hearts. Additionally, the levels of hypoxia-inducible factor 1␣, a known transcriptional activator of MIF, were reduced in aged compared with young hearts. Ischemia-induced AMPK activation in MIF knockout mice was blunted, leading to greater contractile dysfunction in MIF-deficient than in wild-type hearts. Furthermore, intramyocardial injection of adenovirus encoding MIF in aged mice increased MIF expression and ischemic AMPK activation and reduced infarct size. Conclusions-An impaired MIF-AMPK activation response in senescence thus may be attributed to an agingassociated defect in hypoxia-inducible factor 1␣, the transcription factor for MIF. In the clinical setting, impaired cardiac hypoxia-inducible factor 1␣ activation and consequent reduced MIF expression may play an important role in the increased susceptibility to myocardial ischemia observed in older cardiac patients. (Circulation. 2010;122: 282-292.)

Parasitic organisms have evolved specialized strategies to evade immune defense mechanisms. We describe herein an ortholog of the cytokine, macrophage migration inhibitory factor (MIF), which is produced by the obligate intracellular parasite, Leishmania major. The Leishmania MIF protein, Lm1740MIF, shows significant structural homology with human MIF as revealed by a high-resolution x-ray crystal structure (1.03 Å). Differences between the two proteins in the N-terminal tautomerization site are evident, and we provide evidence for the selective, species-specific inhibition of MIF by small-molecule antagonists that target this site. Lm1740MIF shows significant binding interaction with the MIF receptor, CD74 (Kd = 2.9 × 10−8 M). Like its mammalian counterpart, Lm1740MIF induces ERK1/2 MAP kinase activation in a CD74-dependent manner and inhibits the activation-induced apoptosis of macrophages. The ability of Lm1740MIF to inhibit apoptosis may facilitate the persistence of Leishmania within the macrophage and contribute to its evasion from immune destruction.