Recombinant Macrophage Migration Inhibitory Factor Research Articles

Macrophage migration inhibitory factor (MIF) knockout preserves cardiac homeostasis through alleviating Akt-mediated myocardial autophagy suppression in high-fat diet-induced obesity.

BackgroundMacrophage migration inhibitory factor (MIF) plays a role in the development of obesity and diabetes. However, whether MIF plays a role in fat diet-induced obesity and associated cardiac anomalies still remains unknown. The aim of this study was to examine the impact of MIF knockout on high fat diet-induced obesity, obesity-associated cardiac anomalies and the underlying mechanisms involved with a focus on Akt-mediated autophagy.MethodsAdult male wild-type (WT) and MIF knockout (MIF−/−) mice were placed on 45% high fat diet for 5 months. Oxygen consumption, CO2 production, respiratory exchange ratio (RER), locomotor activity, and heat generation were measured using energy calorimeter. Echocardiographic, cardiomyocyte mechanical and intracellular Ca2+ properties were assessed. Apoptosis was examined using TUNEL staining and western blot analysis. Akt signaling pathway and autophagy markers were evaluated. Cardiomyocytes isolated from WT and MIF−/− mice were treated with recombinant mouse MIF (rmMIF).ResultsHigh fat diet feeding elicited increased body weight gain, insulin resistance, and caloric disturbance in WT and MIF−/− mice. High fat diet induced unfavorable geometric, contractile and histological changes in the heart, the effects of which were alleviated by MIF knockout. In addition, fat diet-induced cardiac anomalies were associated with Akt activation and autophagy suppression, which were nullified by MIF deficiency. In cardiomyocytes from WT mice, autophagy was inhibited by exogenous rmMIF through Akt activation. In addition, MIF knockout rescued palmitic acid-induced suppression of cardiomyocyte autophagy, the effect of which was nullified by rmMIF.ConclusionsThese results indicate that MIF knockout preserved obesity-associated cardiac anomalies without affecting fat diet-induced obesity, probably through restoring myocardial autophagy in an Akt-dependent manner. Our findings provide new insights for the role of MIF in obesity and associated cardiac anomalies.

Virtual Screening of Some Active Human Macrophage Migration Inhibitory Factor Antagonists

Macrophage migration inhibitory factor (MIF) is an autocrine- and paracrine-acting cytokine that is involved in several inflammatory, autoimmune, infectious, and oncogenic diseases. Clinical data have shown that inhibition of MIF, especially its tautomerase activity, with small compounds has been beneficial in some disease models. A virtual screening (VS) experiment is conducted for searching some active compounds to inhibit the tautomerase activity of MIF from the ZINC database. By using an x-ray–determined structure OXIM-11 as the query and an in-house developed two-dimensional scaffold comparing method designated as Sfilter, we have screened out some 1500 compounds for ranking by our previously published docking method ADDock. After further ranking by ADDock on 119 compounds screened, we have decided to choose 17 of them for measuring their inhibitory activity IC50 against the MIF tautomerase experimentally. The IC50’s are measured using both human monocytic THP-1 cell lysate and purified recombinant human MIF protein. We have found that the IC50’s measured for three searched compounds (namely, ZINC02693801, ZINC00141102, and ZINC12368346) are better than that determined for ISO-1, a known MIF tautomerase inhibitor and standard used throughout our VS experiment. Moreover, the scaffolds of most of our active compounds searched are also quite different from those searched and published by others previously.

Both host and parasite MIF molecules bind to chicken macrophages via CD74 surface receptor

Macrophage migration inhibitory factor (MIF) is recognized as a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. Our group has identified both chicken and Eimeria MIFs, and characterized their function in enhancing innate immune responses during inflammation. In this study, we report that chicken CD74 (ChCD74), a type II transmembrane protein, functions as a macrophage surface receptor that binds to MIF molecules. First, to examine the binding of MIF to chicken monocytes/macrophages, fresh isolated chicken peripheral blood mononuclear cells (PBMCs) were stimulated with rChIFN-γ and then incubated with recombinant chicken MIF (rChMIF). Immunofluorescence staining with anti-ChMIF followed by flow cytometry revealed the binding of MIF to stimulated PBMCs. To verify that ChCD74 acts as a surface receptor for MIF molecules, full-length ChCD74p41 was cloned, expressed and its recombinant protein (rChCD74p41) transiently over-expressed with green fluorescent protein in chicken fibroblast DF-1 cells. Fluorescence analysis revealed a higher population of cells double positive for CD74p41 and rChMIF, indicating the binding of rChMIF to DF-1 cells via rChCD74p41. Using a similar approach, it was found that Eimeria MIF (EMIF), which is secreted by Eimeria sp. during infection, bound to chicken macrophages via ChCD74p41 as a surface receptor. Together, this study provides conclusive evidence that both host and parasite MIF molecules bind to chicken macrophages via the surface receptor ChCD74.

The critical role of macrophage migration inhibitory factor in insulin activity

Macrophage migration inhibitory factor (MIF) is a molecule with plethora of functions such as regulation of immune response, hormone-like, enzymatic and chaperone-like activity. Further, MIF is a major participant in glucose homeostasis since it is an autocrine stimulator of insulin secretion. MIF absence in male knockout mice (MIF-KO) results in development of glucose intolerance, while sensitivity to insulin is fully preserved. Since our results confirm that beta cells from MIF-KO mice express, produce and secrete insulin similarly to beta cells of their wild type (WT) counterparts C57BL/6 mice, we hypothesize that MIF-KO-derived insulin is less active. Indeed, insulin from MIF-KO islets is unable to significantly induce glucose uptake into hepatocytes and to efficiently promote insulin-triggered Akt phosphorylation determined by immunoblot. However, MIF’s tautomerase function is not crucial for insulin biosynthesis since MIF inhibitors had no impact on WT insulin activity. Importantly, MIF recognition by anti-MIF antibody (ELISA) after in vitro co-incubation with purified insulin was significantly lower suggesting that insulin covers MIF immunodominant epitope. In addition, MIF binds insulin within beta cell as confirmed by co-immunoprecipitation. WT and MIF-KO-derived insulin exhibited different cleavage patterns suggesting different protein conformations. Finally, pre-incubation of recombinant MIF with insulin promotes formation of insulin hexamers. These results imply that MIF probably enables proper insulin folding what results in insulin full activity. This newly discovered feature of the cytokine MIF could be potentially important for commercially produced insulin, for increasing its stability and/or bioavailability.

Macrophage Migration Inhibitory Factor Inhibits the Migration of Cartilage End Plate-Derived Stem Cells by Reacting CD74

Introduction Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that regulates inflammatory reactions and the pathophysiology of many inflammatory diseases. Intervertebral disc (IVD) degeneration is characterized by an inflammatory reaction, but the potential role of MIF in IVD degeneration has not yet been determined. Recent studies have shown that MIF and its receptor, CD74, are involved in regulating the migration of human mesenchymal stem cells (MSCs). Thus, MIF might impair the ability of mesenchymal stem cells (MSCs) to home to injured tissues. Our previous studies indicated that cartilage endplate-derived stem cells (CESCs) as a type of MSCs exist in human degenerate IVDs. Here, we investigate the role of MIF in regulating the migration of CESCs. Material and Method CESCs were isolated and identified. Results We have shown that MIF was distributed in human degenerate IVD tissues and was subject to regulation by the proinflammatory cytokine tumor necrosis factor-α. Furthermore, in-vitro cell migration assays revealed that nucleus pulposus (NP) cells inhibited the migration of CESCs in a number-dependent manner, and enzyme-linked immunosorbent assays revealed that the amount of MIF in conditioned medium was significantly increased as a function of increasing cell number. In addition, recombinant human MIF inhibited the migration of CESCs in a dose-dependent manner. CESCs migration was restored when an antagonist of MIF, (S, R)-3(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), was added. Finally, a CD74 activating antibody (CD74Ab) was used to examine the effect of CD74 on CESCs motility and inhibited the migration of CESCs in a dose-dependent manner. Conclusion We have identified and characterized a novel regulatory mechanism governing cell migration during IVD degeneration. The results will benefit understanding of another possible mechanism for IVD degeneration, and might provide a new method to repair degenerate IVD by enhancing CESCs migration to degenerated NP tissues to exert their regenerative effects. Disclosure of Interest None declared

Macrophage migration inhibitory factor increases vascular permeability through autophagy formation (832.3)

Vascular hyperpermeability is an early pathogenic change in acute inflammatory shock. The high mortality rate of shock patients suggest our understanding of the molecular mechanisms to cause vascular hyperpermeability is not enough. Macrophage migration inhibitory factor (MIF) is a pro‐inflammatory cytokine that plays important roles in many different forms of shock. In the past we found recombinant MIF can induce vascular leakage in mice and induce autophagy in cells. However, little is known whether autophagy is involved in MIF‐induced vascular hyperpermeability. Here, we demonstrated that the permeability of human endothelial cell line HMEC‐1 cells was increased in the presence of MIF in a dose‐and time‐dependent manner. In addition, the permeability of HMEC‐1 cell increased by MIF was inhibited in the presence of MIF inhibitor (ISO‐1) as well as ROS scavenger N‐acetyl‐L‐cysteine (NAC) or PI3K inhibitor (3‐MA), indicating autophagy formation is involved in MIF‐induced hyperpermeability of HMEC‐1 cells. Furthermore, western blot analysis of HMEC‐1 cell lysate confirmed that MIF promoted LC3 conversion, together with activation of autophagy upstream signal pathway. Taken together, these results suggest, in addition to block MIF, blocking autophagy formation by its inhibitors may provide potential therapeutic approaches against shock.

Macrophage migration inhibitory factor diminishes muscle glucose transport induced by insulin and AICAR in a muscle type-dependent manner

Skeletal muscle is a primary organ that uses blood glucose. Insulin- and 5′AMP-activated protein kinase (AMPK)-regulated intracellular signaling pathways are known as major mechanisms that regulate muscle glucose transport. It has been reported that macrophage migration inhibitory factor (MIF) is secreted from adipose tissue and heart, and affects these two pathways. In this study, we examined whether MIF is a myokine that is secreted from skeletal muscles and affects muscle glucose transport induced by these two pathways. We found that MIF is expressed in several different types of skeletal muscle. Its secretion was also confirmed in C2C12 myotubes, a skeletal muscle cell line. Next, the extensor digitorum longus (EDL) and soleus muscles were isolated from mice and treated with recombinant MIF in an in vitro muscle incubation system. MIF itself did not have any effect on glucose transport in both types of muscles. However, glucose transport induced by a submaximal dose of insulin was diminished by co-incubation with MIF in the soleus muscle. MIF also diminished glucose transport induced by a maximal dose of 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), an AMPK activator, in the EDL muscle. These results suggest that MIF is a negative regulator of insulin- and AICAR-induced glucose transport in skeletal muscle. Since MIF secretion from C2C12 myotubes to the culture medium decreased during contraction evoked by electrical stimulations, MIF may be involved in the mechanisms underlying exercise-induced sensitization of glucose transport in skeletal muscle.

Human single-chain variable fragment antibody inhibits macrophage migration inhibitory factor tautomerase activity

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, secreted from a variety of immune cells, that regulates innate and adaptive immune responses. Elevation of MIF levels in plasma correlates with the severity of inflammatory diseases in humans. Inhibition of MIF or its tautomerase activity ameliorates disease severity by reducing inflammatory responses. In this study, the human single-chain variable fragment (HuScFv) antibody specific to MIF was selected from the human antibody phage display library by using purified recombinant full-length human MIF (rMIF) as the target antigen. Monoclonal HuScFv was produced from phage-transformed bacteria and tested for their binding activities to rMIF by indirect enzyme-linked immunosorbent assay as well as to native MIF by western blot analysis and immunofluorescence assay. The HuScFv with highest binding signal to rMIF also inhibited the tautomerase activities of both rMIF and native MIF in human monoblastic leukemia (U937) cells in a dose-dependent manner. Mimotope searching and molecular docking concordantly demonstrated that the HuScFv interacted with Lys32 and Ile64 in the MIF tautomerase active site. To the best of our knowledge, this is the first study to focus on MIF-specific fully-human antibody fragment with a tautomerase-inhibitory effect that has potential to be developed as anti-inflammatory biomolecules for human use.

Susceptibility to Toxoplasma gondii proliferation in BeWo human trophoblast cells is dose-dependent of macrophage migration inhibitory factor (MIF), via ERK1/2 phosphorylation and prostaglandin E2 production

IntroductionMacrophage migration inhibitory factor (MIF) participates in the immune response to Toxoplasma gondii, triggers ERK1/2 and prostaglandin E2 (PGE2) activation, but there is limited information on these mechanisms in human trophoblast. The present study aimed to verify the role of MIF in the ERK1/2 phosphorylation and PGE2 production, as well as its effect on the susceptibility to T. gondii in BeWo cells. MethodsBeWo cells were treated with increasing concentrations of recombinant MIF (rMIF) and/or T. gondii-soluble tachyzoite antigen (STAg) and analyzed for ERK1/2 phosphorylation and PGE2 production by Western blotting and ELISA, respectively. Cells were also treated with increasing concentrations of rMIF, rPGE2, or ERK1/2 inhibitor and tested for T. gondii proliferation. The supernatants of cells treated with rPGE2 were assayed for cytokine production by ELISA or CBA. ResultsERK1/2 phosphorylation and PGE2 production increased when the cells were treated with low MIF concentrations while the parasitism control occurred only at high MIF concentrations. STAg was unable to change ERK1/2 phosphorylation or PGE2 release. BeWo cells demonstrated increased T. gondii proliferation and reduced production of pro-inflammatory cytokines when treated with PGE2, while PD98059 diminished the parasite proliferation. DiscussionThe intracellular mechanisms triggered by MIF are dose-dependent in BeWo cells, and PGE2 is an important factor for the persistence of T. gondii at the maternal fetal interface. ConclusionMIF was unable to control T. gondii infection in BeWo cells at low concentrations since ERK1/2 and PGE2 expression were activated, demonstrating a critical effect of these mediators favoring parasite proliferation.

Abstract A181: Oxidized macrophage migration inhibitory factor (oxMIF) is a previously unrecognized, disease-related isoform of MIF and a potential new drug target in cancer.

Abstract Background: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with pleiotropic biologic effects and which is pathologically associated with several types of cancer. MIF is markedly different from other cytokines in that it is constitutively expressed, is stored in the cytoplasm and is present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention suffers from the drawback of targeting a ubiquitous protein. Here we report the discovery of a previously unrecognized conformational isoform of MIF that can be mimicked in vitro by mild oxidation of recombinant MIF and has therefore been designated as oxidized MIF (oxMIF). Ex vivo studies show that oxMIF is produced in patients with cancer. Highly selective, fully human monoclonal antibodies that specifically target oxMIF, but do not bind the ubiquitous reduced MIF isoform (redMIF), were shown to inhibit downstream signaling pathways associated with tumor proliferation and progression in vitro and in vivo. Methods: New ELISA methods have been established that allow the detection of oxMIF and redMIF in plasma samples acquired from commercial providers. The effect of highly selective oxMIF-specific antibodies on human cancer cells was explored in vitro and in mouse xenograft models using the cell lines PC-3 (prostate cancer), IGROV-1 (ovarian cancer) and COLO-357 (pancreatic cancer). Results: The baseline level of MIF detected in the plasma of healthy subjects (median 5123 pg/ml, n=69) was as expected according to the literature. However, our analysis revealed that this represents the ubiquitous reduced isoform of MIF, redMIF. OxMIF was, in general, not detected in the plasma from healthy controls (median 0 pg/mL, n=69). By contrast, significantly increased concentrations of oxMIF were detected in plasma from patients with breast cancer (644 pg/mL, p<0.05, n=15), ovarian cancer (3483 pg/mL, p<0.0007, n=42) and prostate cancer (2445 pg/mL, p<0.0005, n=14). Animal studies and in vitro studies revealed that novel human antibodies that selectively target oxMIF (i) inhibit proliferation by reducing phosphorylation of ERK1/2 and AKT, and promote apoptosis by activating caspase 3, (ii) inhibit angiogenesis and metastasis by reduction of VEGF expression and blood vessel density within the tumor, and (iii) reduce cancer-associated inflammation of the tumor by downregulating the production of proinflammatory cytokines. Conclusion: Our studies demonstrate that the oxMIF isoform is associated with cancer. OxMIF is a potential marker for disease activity or disease progression and is a promising new drug target for the treatment of cancer. A phase 1 clinical study of a novel human antibody that selectively targets oxMIF is currently ongoing in patients with solid malignancies (ClinicalTrials.gov identifier: NCT01765790). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A181. Citation Format: Michael Thiele, Patrice Douillard, Dirk Voelkel, Thorsten Hagemann, Michael Freissmuth, Hartmut Ehrlich, Friedrich Scheiflinger, Randolf J. Kerschbaumer. Oxidized macrophage migration inhibitory factor (oxMIF) is a previously unrecognized, disease-related isoform of MIF and a potential new drug target in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A181.

Macrophage migration inhibitory factor counter‐regulates dexamethasone‐induced annexin 1 expression and influences the release of eicosanoids in murine macrophages

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine and glucocorticoid (GC) counter-regulator, has emerged as an important modulator of inflammatory responses. However, the molecular mechanisms of MIF counter-regulation of GC still remain incomplete. In the present study, we investigated whether MIF mediated the counter-regulation of the anti-inflammatory effect of GC by affecting annexin 1 in RAW264.7 macrophages. We found that stimulation of RAW264.7 macrophages with lipopolysaccharide (LPS) resulted in down-regulation of annexin 1, while GC dexamethasone (Dex) or Dex plus LPS led to significant up-regulation of annexin 1 expression. RNA interference-mediated knockdown of intracellular MIF increased annexin 1 expression with or without incubation of Dex, whereas Dex-induced annexin 1 expression was counter-regulated by the exogenous application of recombinant MIF. Moreover, recombinant MIF counter-regulated, in a dose-dependent manner, inhibition of cytosolic phospholipase A2α (cPLA2α) activation and prostaglandin E2 (PGE2 ) and leukotriene B4 (LTB4 ) release by Dex in RAW264.7 macrophages stimulated with LPS. Endogenous depletion of MIF enhanced the effects of Dex, reflected by further decease of cPLA2α expression and lower PGE2 and LTB4 release in RAW264.7 macrophages. Based on these data, we suggest that MIF counter-regulates Dex-induced annexin 1 expression, further influencing the activation of cPLA2α and the release of eicosanoids. These findings will add new insights into the mechanisms of MIF counter-regulation of GC.

Biochemical and Immunological Characterization of Toxoplasma gondii Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 μmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.

Abstract 2579: Silibinin inhibits tumor associated macrophages-induced lung cancer cell growth and invasiveness by down-regulating macrophage migration inhibitory factor .

Abstract Lung cancer is the leading cause of cancer related deaths worldwide. According to ACS, 226,160 new lung cancer cases and 160,340 associated deaths are estimated in the United States for 2012. The alarming statistics demand additional preventive and therapeutic strategies to reduce lung cancer burden. One of the novel and rationalized approaches to control lung cancer is to target tumor microenvironment (TM) along with cancer cells. Tumor associated macrophages (TAMs) are important component of TM, and play a pivotal role in the growth and progression of lung cancer. Clinical studies have shown a positive correlation between TAMs and poor prognosis in lung cancer, confirming TAMs as an important drug target. Earlier, we have reported that natural flavonolignan silibinin feeding inhibits urethane-induced lung tumorigenesis by targeting TAMs in the TM. Here, we developed cell culture models to study inhibitory effects and mechanism of silibinin action against macrophages-induced growth and invasiveness in lung cancer cells. We exposed U937 and THP-1 monocytes to phorbol 12-myristate 13-acetate (PMA) to generate M2-type macrophages that closely resemble TAMs. Next, PMA-induced U937 and THP-1 macrophages were treated with DMSO (control) or silibinin (25-100 μM) for 24-72 h; then fresh serum free media was added and at definite time-points control conditioned media (CCM) and silibinin-treated conditioned media (SBCM) were collected. Thereafter, lung cancer A549 cells were exposed to CCM or SBCM and cell proliferation was studied. Results showed that A549 cells proliferation was increased by CCM (from both U937 and THP-1 macrophages) but inhibited by SBCM. Exogenous addition of silibinin (25-100 μM) in CCM also inhibited the growth of A549 cells. Also, A549 cells co-cultured with macrophages showed a ∼3 fold increase in invasiveness, while silibinin (100 μM) treatment of U937 and THP-1 macrophages inhibited the A549 cells invasiveness by 50% (p<0.001) and 55% (p<0.001), respectively. Cytokine array analyses showed that silibinin decreased the level of 15 cytokines (MIF, MCP1, VEGF, HGF, TGFβ, and TNFα etc) and increased the level of 3 cytokines (ENA78, IL12 and MIP-1β) in U937 macrophages. Among the several silibinin-modulated cytokines, we focused on MIF (macrophage migration inhibitory factor) as this cytokine is overexpressed in lung tumors and implicated in all stages of the neoplasia. We confirmed cytokine array results by ELISA that showed 62% and 50% decrease in MIF in SBCM from U937 and THP-1 macrophages, respectively. Importantly, supplementation of recombinant MIF (70 pg) in SBCM reversed SBCM-caused growth inhibition of A549 cells. Similar results were also observed in lung cancer cell line H322. Taken together, these results suggest that silibinin could effectively target TAMs in the TM and inhibit lung tumor growth and invasiveness. Citation Format: Kumaraguruparan Ramasamy, Gagan Deep, Alpna Tyagi, Chapla Agarwal, Rajesh Agarwal. Silibinin inhibits tumor associated macrophages-induced lung cancer cell growth and invasiveness by down-regulating macrophage migration inhibitory factor . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2013-2579

Release of macrophage migration inhibitory factor by neuroendocrine-differentiated LNCaP cells sustains the proliferation and survival of prostate cancer cells

The acquisition of neuroendocrine (NE) characteristics by prostate cancer (PCa) cells is closely related to tumour progression and hormone resistance. The mechanisms by which NE cells influence PCa growth and progression are not fully understood. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in oncogenic processes, and MIF serum levels correlate with aggressiveness of PCa. Here, we investigated the regulation and the functional consequences of MIF expression during NE transdifferentiation of PCa cells. NE differentiation (NED) of LNCaP cells, initiated either by increasing intracellular levels of cAMP or by culturing cells in an androgen-depleted medium, was associated with markedly increased MIF release. Yet, intracellular MIF protein and mRNA levels and MIF gene promoter activity decreased during NED of LNCaP cells, suggesting that NED favours MIF release despite decreasing MIF synthesis. Adenoviral-mediated forced MIF expression in NE-differentiated LNCaP cells increased cell proliferation without affecting the expression of NE markers. Addition of exogenous recombinant MIF to LNCaP and PC-3 cells stimulated the AKT and ERK1/2 signalling pathways, the expression of genes involved in PCa, as well as proliferation and resistance to paclitaxel and thapsigargin-induced apoptosis. Altogether, these data provide evidence that increased MIF release during NED in PCa may facilitate cancer progression or recurrence, especially following androgen deprivation. Thus, MIF could represent an attractive target for PCa therapy.

Macrophage migration inhibitory factor acts as a neurotrophin in the developing inner ear

This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system 'inflammatory' cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development.

Macrophage migration inhibitory factor (MIF) enhances palmitic acid- and glucose-induced murine beta cell dysfunction and destruction in vitro

Although several reports suggest a potentially deleterious role of macrophage migration inhibitory factor (MIF) in type 2 diabetes (T2D) pathology, it is still unclear how this pro-inflammatory cytokine acts on pancreatic beta cells. The aim of the present study was to evaluate MIF effects on murine beta cells in the in vitro settings mimicking T2D-associated conditions. Results indicate that recombinant MIF further increased apoptosis of pancreatic islets or MIN6 cells upon exposure to palmitic acid or glucose. This was accompanied by upregulation of several pro-apoptotic molecules. Furthermore, MIF potentiated nutrient-induced islet cell dysfunction, as revealed by lower glucose oxidation rate, ATP content, and depolarized mitochondrial membrane. The final outcome was potentiation of mitochondrial apoptotic pathway. The observed upregulation of nutrient-induced islet cell dysfunction and apoptosis by MIF implicates that silencing MIF may be beneficial for maintaining integrity of endocrine pancreas in obesity-associated T2D.

Macrophage Migration Inhibitory Factor Deficiency Is Associated With Impaired Killing of Gram-Negative Bacteria by Macrophages and Increased Susceptibility to Klebsiella pneumoniae Sepsis

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIF's regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

Expression of macrophage migration inhibitory factor by osteoblastic cells: Protection against cadmium toxicity

Exposition to cadmium (Cd) has been linked to bone metabolism alterations and occurrence of osteoporosis. Despite its known renal toxicity which indirectly disrupts bone metabolism through impairment of vitamin D synthesis, increasing evidence argues for the direct action of Cd on bone-forming osteoblasts. Indeed, accumulation of Cd in osteoblasts and metal-induced cell death has been documented but little is known about the intracellular mechanisms of protection against this stress. In this work, we investigated the protection afforded by thiol-containing proteins against Cd cytotoxicity in MC3T3 osteoblastic cells. Viability of MC3T3 cells was reduced by Cd in a concentration-dependent manner with a LC50 of 7.6±1.1μM. Depletion of glutathione by l-buthionine sulphoximine (BSO) increased cell sensitivity to Cd cytotoxicity, suggesting the involvement of thiol-containing peptides as a mechanism of protection. Accordingly, Cd was shown to promote progressive depletion of reduced thiol content and to stimulate the production of reactive oxygen species (ROS). Interestingly, low non cytotoxic concentrations of Cd increased the gene expression of macrophage migration inhibitory factor (MIF), also a thiol-containing protein. Inhibition of the transcription factor NFκB prevented Cd-dependent upregulation of MIF expression and consequently, increased Cd cytotoxicity in osteoblasts. Moreover, MIF deficient mouse osteoblasts were more sensitive to Cd cytotoxicity than the corresponding control cells. By gel-filtration chromatography, we demonstrated that MIF acts as a thiol-containing protein and thereby promotes Cd complexation. In accordance with its binding ability, addition of recombinant MIF to the culture medium reduced Cd cytotoxicity. Overall, upregulation of MIF expression by Cd may protect against the cytotoxicity of this metal in the osteoblasts.

Overexpression of macrophage migration inhibitory factor in adenoid cystic carcinoma: correlation with enhanced metastatic potential

Adenoid cystic carcinoma (ACC) is a malignant tumor frequently arising in salivary glands with poor long-term prognosis due to high rates of local recurrences and distant metastases. Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine and has recently emerged as a pro-tumorigenic factor in various cancers. This study is designed to investigate the expression status and functional significance of MIF in ACC. Immunohistochemical staining was performed to evaluate the expression levels of MIF, HIF-1α, MMP-9, p53, and p-JNK in ACC tissues. In vitro, ACC-2 cells were exposed to recombinant human MIF (rMIF) or ISO-1 (an inhibitor of MIF) at different concentrations and times, followed by the detection of cell growth, viability, migration, and invasion, as well as the expression levels of several cellular signals. The immunohistochemical results demonstrated the overexpression of MIF in ACC tissues as well as its association with the distant metastasis. Further analyses showed a significant correlation between the staining of MIF and p-JNK. Moreover, the in vitro studies revealed that the treatment for ACC cells with ISO-1 significantly attenuated cell migratory and invasive capacity, as opposed to the limited promotive effects of rMIF. More importantly, MIF inhibition could cause the activation of JNK, correlating with the immunohistochemical findings on ACC tissues. The results suggest that MIF is likely to be an important player in the pathogenesis of ACC and may promote cancer metastasis, which possibly involves JNK inactivation. Further investigation of MIF-mediated molecular events may provide novel insights into the treatment for ACC.

Mechanism of macrophage migration inhibitory factor‐induced decrease of T‐type Ca2+ channel current in atrium‐derived cells

The T-type Ca(2+) current (I(Ca,T)) plays an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate the role of macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, in the regulation of T-type Ca(2+) channels (TCCs) in atrial myocytes. We used the whole-cell voltage-clamp technique and biochemical assays to study the regulation and expression of I(Ca,T) in atrial myocytes. Gene levels of the α1G and α1H subunit of TCCs were decreased in human atrial tissue of patients with AF. In cultured atrium-derived myocytes (HL-1 cells), mouse recombinant MIF (20 or 40 nm, 24 h) suppressed peak I(Ca,T) in a concentration-dependent manner, impaired the voltage-dependent activation of I(Ca,T) and downregulated TCC α1G and α1H mRNA. The Src inhibitors genistein and PP1 significantly enhanced I(Ca,T). The reduction of I(Ca,T) and TCC subunit mRNA induced by recombinant MIF could be reversed by genistein and PP1. The TCC α1G associated with Src in HL-1 cells and mouse cardiomycytes. Macrophage migration inhibitory factor is involved in the pathogenesis of AF, probably by decreasing the T-type calcium current in atrium-derived myocytes through impairment of channel function and activation of c-Src kinases, representing a potential pathogenic mechanism in atrial fibrillation.