A Novel Allosteric Inhibitor of Macrophage Migration Inhibitory Factor (MIF)

Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.     

Expression and Function of Macrophage Migration Inhibitory Factor (MIF) in Melioidosis

Background

Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast-Asia. We aimed to characterize the expression and function of MIF in melioidosis.

Methodology and Principal Findings

MIF expression was determined in leukocytes and plasma from 34 melioidosis patients and 32 controls, and in mice infected with B. pseudomallei. MIF function was investigated in experimental murine melioidosis using anti-MIF antibodies and recombinant MIF. Patients demonstrated markedly increased MIF mRNA leukocyte and MIF plasma concentrations. Elevated MIF concentrations were associated with mortality. Mice inoculated intranasally with B. pseudomallei displayed a robust increase in pulmonary and systemic MIF expression. Anti-MIF treated mice showed lower bacterial loads in their lungs upon infection with a low inoculum. Conversely, mice treated with recombinant MIF displayed a modestly impaired clearance of B. pseudomallei. MIF exerted no direct effects on bacterial outgrowth or phagocytosis of B. pseudomallei.

Conclusions

MIF concentrations are markedly elevated during clinical melioidosis and correlate with patients'' outcomes. In experimental melioidosis MIF impaired antibacterial defense.     

Negative Regulation of AMP-activated Protein Kinase (AMPK) Activity by Macrophage Migration Inhibitory Factor (MIF) Family Members in Non-small Cell Lung Carcinomas

AMP-activated protein kinase (AMPK) is a nutrient- and metabolic stress-sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, d-dopachrome tautomerase (d-DT), have protumorigenic functions in non-small cell lung carcinomas (NSCLCs) but have AMPK-activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and d-DTs AMPK-activating properties in nontransformed cells, MIF and d-DT act cooperatively to inhibit steady-state phosphorylation and activation of AMPK in LKB1 wild type and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and d-DT, acting through their shared cell surface receptor, CD74, antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production, and redox balance, resulting in reduced Ca²⁺/calmodulin-dependent kinase kinase β-dependent AMPK activation. Combined, these studies indicate that MIF and d-DT cooperate to inhibit AMPK activation in an LKB1-independent manner.     

Role of Macrophage Migration Inhibitory Factor (MIF) in Pollen-Induced Allergic Conjunctivitis and Pollen Dermatitis in Mice

Pollen is a clinically important airborne allergen and one of the major causes of allergic conjunctivitis. A subpopulation of patients with atopic dermatitis (AD) are also known to have exacerbated skin eruptions on the face, especially around the eyelids, after contact with pollen. This pollen-induced skin reaction is now known as pollen dermatitis. Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that plays an essential role in allergic inflammation. Recent findings suggest that MIF is involved in several allergic disorders, including AD. In this study, MIF knockout (KO), MIF transgenic (Tg) and WT littermate mice were immunized with ragweed (RW) pollen or Japanese cedar (JC) pollen and challenged via eye drops. We observed that the numbers of conjunctiva- and eyelid-infiltrating eosinophils were significantly increased in RW and JC pollen-sensitized MIF Tg compared with WT mice or MIF KO mice. The mRNA expression levels of eotaxin, interleukin (IL)-5 and IL-13 were increased in pollen-sensitized eyelid skin sites of MIF Tg mice. An in vitro analysis revealed that high eotaxin expression was induced in dermal fibroblasts by MIF combined with stimulation of IL-4 or IL-13. This eotaxin expression was inhibited by the treatment with CD74 siRNA in fibroblasts. These findings indicate that MIF can induce eosinophil accumulation in the conjunctiva and eyelid dermis exposed to pollen. Therefore, targeted inhibition of MIF might result as a new option to control pollen-induced allergic conjunctivitis and pollen dermatitis.     

Filtration of Macrophage Migration Inhibitory Factor (MIF) in Patients with End Stage Renal Disease Undergoing Hemodialysis

BackgroundEnd stage renal disease (ESRD) patients are characterized by increased morbidity and mortality due to highest prevalence of cardiovascular disease. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that controls cellular signaling in human physiology, pathophysiology, and diseases. Increased MIF plasma levels promote vascular inflammation and development of atherosclerosis. We have shown that MIF is associated with vascular dysfunction in ESRD patients. Whether hemodialysis (HD) affects circulating MIF plasma levels is unknown. We here aimed to investigate whether HD influences the circulating MIF pool in ESRD patients.ConclusionMIF is a dialyzable plasma component that is effectively filtrated during HD from the patient blood pool in large amounts. After removal of remarkable amounts of MIF during a single HD session, MIF plasma pool is early reconstituted after termination of HD from unknown sources.     

Macrophage Migration Inhibitory Factor (MIF) and Thyroid Hormone Alterations in Antineutrophil Cytoplasmic Antibody (ANCA)-Associated Vasculitis (AAV)

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine known to be released from lymphocytes, macrophages and endothelial cells and also in animal models shown to be inducible with glucocorticoids (GC). In contrast, thyroxine seems to antagonize MIF activity. To investigate whether MIF is increased in active antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and possible correlations with GC dosing and thyroid hormone levels, 27 consecutive patients with active AAV were studied and followed prospectively. Disease activity was assessed using Birmingham Vasculitis Activity Score 2003 (BVAS) at baseline and at follow-up at 3 and 6 months, along with MIF, thyroid hormones free triiodothyronine (fT3) and free thyroxine (fT4), C-reactive protein (CRP) and creatinine. MIF was elevated significantly at baseline compared with follow-up at 3 and 6 months (8,618 pg/mL versus 5,696 and 6,212 respectively; P < 0.002) but did not correlate to CRP, GC dose, creatinine or organ involvement. fT3 was depressed significantly at baseline compared with follow-up (1.99 pg/mL versus 2.31 and 2.67 respectively; P = 0.01) and correlated inversely to the BVAS score at baseline. We found a significant correlation between the MIF/fT4 ratio at baseline versus MIF/fT4 ratio at 6 months (ρ = 0.52, P < 0.005) and a trend between the baseline MIF/fT3 ratio versus MIF/fT3 ratio at 6 months (ρ = 0.39, P = 0.05). These results suggest a possible role for MIF and thyroid status in AAV. Further studies could reveal whether the association between AAV and thyroid hormone levels in the context of elevated MIF may present a link as well as a target of treatment.     

Structural and functional characterization of a secreted hookworm Macrophage Migration Inhibitory Factor (MIF) that interacts with the human MIF receptor CD74

Hookworms, parasitic nematodes that infect nearly one billion people worldwide, are a major cause of anemia and malnutrition. We hypothesize that hookworms actively manipulate the host immune response through the production of specific molecules designed to facilitate infection by larval stages and adult worm survival within the intestine. A full-length cDNA encoding a secreted orthologue of the human cytokine, Macrophage Migration Inhibitory Factor (MIF) has been cloned from the hookworm Ancylostoma ceylanicum. Elucidation of the three-dimensional crystal structure of recombinant AceMIF (rAceMIF) revealed an overall structural homology with significant differences in the tautomerase sites of the human and hookworm proteins. The relative bioactivities of human and hookworm MIF proteins were compared using in vitro assays of tautomerase activity, macrophage migration, and binding to MIF receptor CD74. The activity of rAceMIF was not inhibited by the ligand ISO-1, which was previously determined to be an inhibitor of the catalytic site of human MIF. These data define unique immunological, structural, and functional characteristics of AceMIF, thereby establishing the potential for selectively inhibiting the hookworm cytokine as a means of reducing parasite survival and disease pathogenesis.     

Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC₅₀ values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro¹; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.     

Genotyping of Macrophage Migration Inhibitory Factor (MIF) CATT5–8 Repeat Polymorphism by Denaturing High-Performance Liquid Chromatography (DHPLC)

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed in many different cell types and implicated in the pathogenesis of numerous acute and chronic inflammatory diseases. Variable Number of Tandem Repeat (VNTR) CATT_5–8 at position ?794 in the promoter of the MIF gene has been associated with several human pathological conditions. Different methods for genotyping the CATT tetranucleotide repeats have been described. Here, we report, for the first time, the complete characterization of the CATT_5–8 repeat polymorphism using exclusively the denaturing high-performance liquid chromatography (DHPLC) technique under partially denaturing conditions. This approach, based on a step-by-step DHPLC protocol, allowed the accurate determination of all the homozygous and heterozygous genotypes in 350 DNA samples from control subjects. The results were validated by comparison to DNA sequencing, and the DHPLC approach was accurate, sensitive, and highly reproducible. Data from the current study demonstrate that this method of analysis by DHPLC may represent a powerful and sensitive alternative tool for a rapid and efficient genotyping of short tandem repeats presenting a limited number of alleles.     

Activity and Properties of Macrophage Migration Inhibitory Factor produced by Mixed Lymphocyte Cultures

THE macrophage migration test is an in vitro demonstration of delayed hypersensitivity. Supernatant fluids of sensitive lymphocytes cultured for 24 h in the presence of specific antigen contain migration inhibitory factor (MIF) that arrests the migration of macrophages of unsensitized animals in vitro¹,². In vivo, it induces delayed skin reactions³. The use of the macrophage migration test, based on differences of transplantation antigens in donor and recipient, to show histocompatibility has been suggested⁴. The test was also recommended as an indicator of immunological reactivity after organ transplantation, to demonstrate impending rejection⁵. It can demonstrate homograft sensitivity, for migration of peritoneal exudate cells (containing lymphocytes and macrophages) of CBA mice previously sensitized by grafts from A/Jax donors was inhibited when they were mixed with peritoneal exudate cells of the donor strain. However, histocompatibility was not demonstrated, for mixtures of peritoneal exudate cells of ungrafted CBA mice and A/Jax mice migrated regularly during the 24 h test⁶.     

调控巨噬细胞迁移抑制因子基因表达的影响因素

巨噬细胞迁移抑制因子(macrophage migration inhibitory factor, MIF)是一种广泛表达的多效性细胞因子,参与多种炎症和免疫疾病的过程并在其中发挥重要作用,是许多疾病的生物标志物或治疗靶点。MIF基因在系统发育中高度保守,在其启动子区有多种不同转录因子的特定结合位点,借此调节MIF的表达。MIF在细胞内外均发挥作用,且MIF是组成型表达。因此,研究调控MIF基因表达和刺激MIF分泌的相关因素具有重要意义。本文通过对MIF基因和MIF启动子上的结合位点的简述,对影响MIF基因表达的相关因素进行总结和归类。根据与MIF基因结合的方式,可分为：(1)与MIF基因启动子特定位点结合,改变转录活性;(2)与MIF CATT5-8微卫星重复序列结合,改变高表达MIF等位基因;(3)非编码RNA调控MIF表达;(4)影响MIF分泌的相关因素。通过对这4类调控MIF基因表达的相关因素的综述,进而认识MIF基因表达的调控机制和影响因素,以期对其治疗相关疾病提供理论基础。     

The Dexamethasone-induced Inhibition of Proliferation, Migration, and Invasion in Glioma Cell Lines Is Antagonized by Macrophage Migration Inhibitory Factor (MIF) and Can Be Enhanced by Specific MIF Inhibitors

Glioblastomas (GBMs) are the most frequent and malignant brain tumors in adults. Glucocorticoids (GCs) are routinely used in the treatment of GBMs for their capacity to reduce the tumor-associated edema. Few in vitro studies have suggested that GCs inhibit the migration and invasion of GBM cells through the induction of MAPK phosphatase 1 (MKP-1). Macrophage migration inhibitory factor (MIF), an endogenous GC antagonist is up-regulated in GBMs. Recently, MIF has been involved in tumor growth and migration/invasion and specific MIF inhibitors have been developed on their capacity to block its enzymatic tautomerase activity site. In this study, we characterized several glioma cell lines for their MIF production. U373 MG cells were selected for their very low endogenous levels of MIF. We showed that dexamethasone inhibits the migration and invasion of U373 MG cells, through a glucocorticoid receptor (GR)- dependent inhibition of the ERK1/2 MAPK pathway. Oppositely, we found that exogenous MIF increases U373 MG migration and invasion through the stimulation of the ERK1/2 MAP kinase pathway and that this activation is CD74 independent. Finally, we used the Hs 683 glioma cells that are resistant to GCs and produce high levels of endogenous MIF, and showed that the specific MIF inhibitor ISO-1 could restore dexamethasone sensitivity in these cells. Collectively, our results indicate an intricate pathway between MIF expression and GC resistance. They suggest that MIF inhibitors could increase the response of GBMs to corticotherapy.     

Macrophage Migration Inhibitory Factor Mediates PAR-Induced Bladder Pain

IntroductionMacrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is constitutively expressed in urothelial cells that also express protease-activated receptors (PAR). Urothelial PAR1 receptors were shown to mediate bladder inflammation. We showed that PAR1 and PAR4 activator, thrombin, also mediates urothelial MIF release. We hypothesized that stimulation of urothelial PAR1 or PAR4 receptors elicits release of urothelial MIF that acts on MIF receptors in the urothelium to mediate bladder inflammation and pain. Thus, we examined the effect of activation of specific bladder PAR receptors on MIF release, bladder pain, micturition and histological changes.MethodsMIF release was measured in vitro after exposing immortalized human urothelial cells (UROtsa) to PAR1 or PAR4 activating peptides (AP). Female C57BL/6 mice received intravesical PAR1- or PAR4-AP for one hour to determine: 1) bladder MIF release in vivo within one hour; 2) abdominal hypersensitivity (allodynia) to von Frey filament stimulation 24 hours after treatment; 3) micturition parameters 24 hours after treatment; 4) histological changes in the bladder as a result of treatment; 5) changes in expression of bladder MIF and MIF receptors using real-time RT-PCR; 6) changes in urothelial MIF and MIF receptor, CXCR4, protein levels using quantitative immunofluorescence; 7) effect of MIF or CXCR4 antagonism.ResultsPAR1- or PAR4-AP triggered MIF release from both human urothelial cells in vitro and mouse urothelium in vivo. Twenty-four hours after intravesical PAR1- or PAR4-AP, we observed abdominal hypersensitivity in mice without changes in micturition or bladder histology. PAR4-AP was more effective and also increased expression of bladder MIF and urothelium MIF receptor, CXCR4. Bladder CXCR4 localized to the urothelium. Antagonizing MIF with ISO-1 eliminated PAR4- and reduced PAR1-induced hypersensitivity, while antagonizing CXCR4 with AMD3100 only partially prevented PAR4-induced hypersensitivity.ConclusionsBladder PAR activation elicits urothelial MIF release and urothelial MIF receptor signaling at least partly through CXCR4 to result in abdominal hypersensitivity without overt bladder inflammation. PAR-induced bladder pain may represent an interesting pre-clinical model of Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS) where pain occurs without apparent bladder injury or pathology. MIF is potentially a novel therapeutic target for bladder pain in IC/PBS patients.     

TPT1 Supports Proliferation of Neural Stem/Progenitor Cells and Brain Tumor Initiating Cells Regulated by Macrophage Migration Inhibitory Factor (MIF)

Neurochemical Research - One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and...     

巨噬细胞迁移抑制因子拮抗剂对子宫内膜异位症的影响

目的：探讨巨噬细胞迁移抑制因子拮抗剂（ISO-1）对子宫内膜异位症的影响。方法：以裸鼠为研究对象,构建子宫内膜异位元症动物模型,应用巨噬细胞迁移抑制因子拮抗剂进行干预,观察子宫内膜异位症小鼠的成活率和体重变化;采用RT-PCR检测基质金属蛋白酶-2（MMP-2）,基质金属蛋白酶抑制剂-2（TIMP-2）,血管内皮细胞生长因子（VEGF）,TNF-αmRNA的表达,ELISA检测TNF-α蛋白的表达。结果：ISO-1对子宫内膜异位症小鼠的存活率无明显影响,但可增加其体重（P〈0.05）。ISO-1减少子宫内膜异位症小鼠受损组织中MMP-2、VEGF、TNF-α的表达（P〈0.05）,但对TIMP-2的表达无明显影响。结论：巨噬细胞迁移抑制因子被特异性阻断后,可明显抑制受损组织的重构、血管生成和炎症,最终影响子宫内膜异位症的组织生长及进一步恶化,这可能是临床治疗子宫内膜异位症的新策略。     

Charge Heterogeneity of Bovine Brain Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is known as a ubiquitous pluripotent cytokine originally identified for its capacity to inhibit the random migration of macrophages in vitro. It is recognized as an important regulator of the immunological, neuroendocrine and enzymatic processes. MIF is widely expressed in brain, but its role in the nervous system is not yet understood. In the course of the study of the primary structure of bovine brain MIF we have previously identified a number of MIF-related proteins having identical N-terminal sequences. In this paper we report the results of isoelectric focusing of MIF isolated to a homogeneous state from bovine brain that revealed MIF charge heterogeneity. We have detected isoelectric forms of MIF with pI values of 6.9, 7.0, 7.3, and 7.8. The diverse actions of MIF within the immuno-neuroendocrine system is suggested to be a result of its occurrence in different isoforms and oligomerization states.     

Homotrimeric Macrophage Migration Inhibitory Factor (MIF) Drives Inflammatory Responses in the Corneal Epithelium by Promoting Caveolin-rich Platform Assembly in Response to Infection

Acute inflammation that arises during Pseudomonas aeruginosa-induced ocular infection can trigger tissue damage resulting in long term impairment of visual function, suggesting that the appropriate treatment strategy should include the use of anti-inflammatory agents in addition to antibiotics. We recently identified a potential target for modulation during ocular infection, macrophage migration inhibitory factor (MIF). MIF deficiency protected mice from inflammatory-mediated corneal damage resulting from acute bacterial keratitis. To gain a better understanding of the molecular mechanisms of MIF activity, we analyzed the oligomeric states and functional properties of MIF during infection. We found that in human primary corneal cells infected with P. aeruginosa, MIF is primarily in a homotrimeric state. Homotrimeric MIF levels correlated with the severity of infection in the corneas of infected mice, suggesting that the MIF homotrimers were the functionally active form of MIF. During infection, human primary corneal cells released more IL-8 when treated with recombinant, locked MIF trimers than when treated with lower MIF oligomers. MIF promoted P. aeruginosa–induced IL-8 responses via the formation of caveolin-1-rich “signaling hubs” in the corneal cells that led to elevated MAPK p42/p44 activation and sustained inflammatory signaling. These findings suggest that inhibiting homotrimerization of MIF or the functional activities of MIF homotrimers could have therapeutic benefits during ocular inflammation.