Neuroendocrine properties of macrophage migration inhibitory factor (MIF)

The cytokine macrophage migration inhibitory factor (MIF) is produced by neuroendocrine and immune tissues and possesses several features that allow it to be characterized as a neuroendocrine mediator. Its pro-inflammatory action and its pathogenic role in inflammatory diseases, such as septic shock, arthritis and other diseases, have clearly been demonstrated and may be based in part on neuroendocrine mechanisms. Macrophage migration inhibitory factor possesses glucocorticoid-antagonist properties within the immune system and participates in the regulation of several endocrine circuits. This review summarizes the current state of MIF research and focuses on MIF expression and function in nervous and endocrine tissues.     

Cutting edge: Deficiency of macrophage migration inhibitory factor impairs murine airway allergic responses

Increased levels of macrophage migration inhibitory factor (MIF) in serum, sputum, and bronchioalveolar lavage fluid (BALF) from asthmatic patients and time/dose-dependent expression of MIF in eosinophils in response to phorbol myristate acetate suggest the participation of MIF in airway inflammation. In this study, we examined inflammation in OVA-sensitized mouse lungs in wild-type and MIF-deficient mice (MIF(-/-)). We report increased MIF in the lung and BALF of sensitized wild-type mice. MIF(-/-) mice demonstrated significant reductions in serum IgE and alveolar inflammatory cell recruitment. Reduced Th1/Th2 cytokines and chemokines also were detected in serum or BALF from MIF(-/-) mice. Importantly, alveolar macrophages and mast cells, but not dendritic cells or splenocytes, from MIF(-/-) mice demonstrated impaired CD4+ T cell activation, and the reconstitution of wild-type mast cells in MIF(-/-) mice restored the phenotype of OVA-induced airway inflammation, revealing a novel and essential role of mast cell-derived MIF in experimentally induced airway allergic diseases.     

Loss of autophagy enhances MIF/macrophage migration inhibitory factor release by macrophages

MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]) is a pro-inflammatory cytokine expressed in multiple cells types, including macrophages. MIF plays a pathogenic role in a number of inflammatory diseases and has been linked to tumor progression in some cancers. Previous work has demonstrated that loss of autophagy in macrophages enhances secretion of IL1 family cytokines. Here, we demonstrate that loss of autophagy, by pharmacological inhibition or siRNA silencing of Atg5, enhances MIF secretion by monocytes and macrophages. We further demonstrate that this is dependent on mitochondrial reactive oxygen species (ROS). Induction of autophagy with MTOR inhibitors had no effect on MIF secretion, but amino acid starvation increased secretion. This was unaffected by Atg5 siRNA but was again dependent on mitochondrial ROS. Our data demonstrate that autophagic regulation of mitochondrial ROS plays a pivotal role in the regulation of inflammatory cytokine secretion in macrophages, with potential implications for the pathogenesis of inflammatory diseases and cancers.     

Receptor agonists of macrophage migration inhibitory factor

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also promotes AMPK activation with potential benefits for response to myocardial infarction and ischemia-reperfusion. Structure-based molecular design has led to the discovery of not only antagonists, but also the first agonists of MIF-CD74 binding. The compounds contain a triazole core that is readily assembled via Cu-catalyzed click chemistry. The agonist and antagonist behaviors were confirmed via study of MIF-dependent ERK1/2 phosphorylation in human fibroblasts.     

Chaperone-like activity of macrophage migration inhibitory factor

Macrophage migration inhibitory factor is a ubiquitous multifunctional cytokine having diverse immunological and neuroendocrine properties. Although this protein is known to be released into the circulation from the secretory granules of anterior pituitary or directly from immune cells as a consequence of stress, its participation in heat stress-induced aggregation of proteins has not yet been reported. We provide here the first evidence that the macrophage migration inhibitory factor possesses chaperone-like properties. It was shown to exist in the form of a mixture of low and high molecular weight oligomers. At heat stress temperatures the large oligomers dissociate into monomers that bind and stabilize thermally denatured malate dehydrogenase and glycogen phosphorylase b and thus prevent aggregation of the model proteins. Similar chaperone-like effects were also observed in the presence of partially purified brain extract containing besides the macrophage migration inhibitory factor a number of ubiquitous hydrophobic low molecular weight proteins identified by N-terminal microsequence analysis. Being highly stable and hydrophobic, the macrophage migration inhibitory factor in combination with other proteins of similar properties may comprise a family of constitutively expressed "small chaperones" that counteract the early onset of stress, around physiological conditions, when heat shock proteins are not abundant.     

巨噬细胞迁移抑制因子基因多态性与结直肠癌风险研究

目的研究巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)基因单核苷酸多态性(SNPs;rs755662,rs11548059,rs1049829,rs1803976)与结直肠癌发生风险的关系。方法收集共计192例结直肠癌患者(CRC)和256名正常对照者外周血样本,以聚合酶链反应和Taqman探针分析方法,检测MIF基因单核苷酸多态性;以Logistic回归模型计算不同基因型与结直肠癌发生风险的关系。结果 rs755662基因型的出现频率在CRC组和正常对照组之间差异有统计学意义(P=0.011),而在rs11548059、rs1049829和rs1803976位点则差异无统计学意义(P=0.660、P=0.700和P=0.959)。此外,rs755662还分别与早期发病(年龄≤50岁,P=0.026)、分期(Ⅳ期,P=0.038)以及分化(P=0.040)有关。与正常对照组比,rs755662与Ⅲ期和Ⅳ期显著相关(P值分别为0.034和0.003)。结论 MIF基因5′-UTR区域rs755662(G/C)单核苷酸多态性与结直肠癌的易感性、患者发病年龄和分期有关。     

Steroid receptor coactivator 3 regulates autophagy in breast cancer cells through macrophage migration inhibitory factor

SRC-3/AIB1 (steroid receptor coactivator 3/amplified in breast cancer 1) is an authentic oncogene that contributes to the development of drug resistance and poor disease-free survival in cancer patients. Autophagy is also an important cell death mechanism that has tumor suppressor function. In this study, we identified macrophage migration inhibitory factor (MIF) as a novel target gene of SRC-3 and demonstrated its importance in cell survival. Specifically, we showed that MIF is a strong suppressor of autophagic cell death. We further showed that suppression of MIF, in turn, induced autophagic cell death, enhanced chemosensitivity and inhibited tumorigenesis in a xenograft mouse tumorigenesis model. Our study demonstrated that regulation of MIF expression and suppression of autophagic cell death is a potent mechanism by which SRC-3 contributes to increased chemoresistance and tumorigenicity.     

Regulation of human lung adenocarcinoma cell migration and invasion by macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is expressed and secreted in response to mitogens and integrin-dependent cell adhesion. Once released, autocrine MIF promotes the activation of RhoA GTPase leading to cell cycle progression in rodent fibroblasts. We now report that small interfering RNA-mediated knockdown of MIF and MIF small molecule antagonism results in a greater than 90% loss of both the migratory and invasive potential of human lung adenocarcinoma cells. Correlating with these phenotypes is a substantial reduction in steady state as well as serum-induced effector binding activity of the Rho GTPase family member, Rac1, in MIF-deficient cells. Conversely, MIF overexpression by adenovirus in human lung adenocarcinoma cells induces a dramatic enhancement of cell migration, and co-expression of a dominant interfering mutant of Rac1 (Rac1(N17)) completely abrogates this effect. Finally, our results indicate that MIF depletion results in defective partitioning of Rac1 to caveolin-containing membrane microdomains, raising the possibility that MIF promotes Rac1 activity and subsequent tumor cell motility through lipid raft stabilization.     

The expression of macrophage migration inhibitory factor in the non-small cell lung cancer

ObjectiveThe objective was to investigate the expression of macrophage migration inhibitory factor (MIF) in non-small cell lung cancer (NSCLC), as well as the effects of macrophage MIF on tumor cells.MethodsThe human NSCLC cell strains H358 and H524 were selected as research objects. The Real-Time Polymerase Chain Reaction (RT-PCR) and Western Blot were utilized to detect the expression levels of MIF in human NSCLC cell strains. The lentiviral plasmid was utilized for MIF-mRNA interference. The expression levels of MIF before and after transfection were compared. The cell strains were cultured and proliferated for cell count and comparison.ResultsH358 showed MIF high expression while H524 showed MIF low expression. Once the H358 cells were constructed as silent MIF expression, compared with the original H358 cells, the difference was statistically significant. Once the H524 cells were constructed as high MIF expression, compared with original H524 cells, the difference was statistically significant. Being cultured for respective 3, 5, and 7 days, the transfected H358 cells showed a significant decrease in proliferative activity compared with original H358 cells, while the transfected H524 cells showed a significant increase in proliferative activity compared with original H524 cells.ConclusionMIF has high expression in H358 cells while low expression in H524 cells. The expression of MIF could enhance the proliferative activity of NSCLC tumor cells.     

Impairment of macrophage migration inhibitory factor synthesis and macrophage migration in protein-malnourished mice

Weanling CD2F1 mice were fed isocaloric diets that were protein sufficient (PS; containing 27% casein) or protein deficient (PD; containing 8% casein). Weight measurements demonstrated that the growth of PD mice was significantly impaired, thus indicating that the PD diet induced protein malnutrition. The cellular immune responsiveness of these mice was assessed from Day 21 to Day 49 of the diet using, as indicators, in vitro production of migration inhibitory factor (MIF) by splenic lymphocytes and MIF responsiveness of peritoneal macrophages. PD lymphocytes, when stimulated with the polyclonal activator concanavalin A, produced significantly less MIF than did PS lymphocytes. The amount of MIF produced by PD lymphocytes, however, increased throughout the study, possibly indicating delayed maturation of MIF synthetic capacity in PD mice. Normal CD2F1 mouse macrophages were used for these assays. MIF responsiveness of PD and PS macrophages was not significantly different when assayed using MIF produced by normal CD2F1 mouse lymphocytes. As compared to that of PS macrophages, the migratory ability of PD macrophages decreased progressively throughout the study. This impaired migratory ability did not interfere with MIF responsiveness of PD macrophages.     

Benzisothiazolones as modulators of macrophage migration inhibitory factor

Substituted N-phenylbenzisothiazolones have been investigated as inhibitors of the tautomerase activity of the proinflammatory cytokine MIF (macrophage migration inhibitory factor). Numerous compounds were found to possess antagonist activity in the low micromolar range with the most potent being the 6-hydroxy analog 1w. Compound 1w and the p-cyano analog 1c were also shown to exhibit significant inhibition of the binding of MIF to its transmembrane receptor CD74. Consistently, both compounds were also found to retard the MIF-dependent phosphorylation of ERK1/2 in human synovial fibroblasts.     

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.     

High expression of macrophage migration inhibitory factor in human melanoma cells and its role in tumor cell growth and angiogenesis.

Macrophage migration inhibitory factor (MIF) is known to function as a cytokine, hormone, and glucocorticoid-induced immunoregulator. In this study, we reported for the first time that human melanocytes and melanoma cells express MIF mRNA and produce MIF protein. Immunohistochemical analysis demonstrated that MIF was mostly localized in the cytoplasm of melanocytes and G361 cells, a widely available human melanoma cell line. In particular, strong positive staining was observed at the dendrites of these cells. Expression of MIF mRNA and production of MIF protein were much higher in human melanoma cells such as G361, A375, and L32 than in normal cultured melanocytes. To assess the role of MIF overexpression in melanoma cells, G361 cells were transfected with an antisense human MIF plasmid. The results demonstrated that the cell growth rate of the transfected cells was markedly suppressed, suggesting that MIF participates in the mechanism of proliferation of melanoma cells. To further evaluate the function of MIF, we employed the Boyden chamber method to examine the effect on tumor cell migration and found that MIF enhanced the migration of G361 cells in a dose-dependent manner. Furthermore, we administered anti-MIF antibody into tumor (G361 cells in a Millipore chamber)-bearing mice to assess the effect on tumor-associated angiogenesis. The anti-MIF antibody significantly suppressed tumor-induced angiogenesis. Taken together, these results indicated that it is likely that MIF may function as a novel growth factor that stimulates incessant growth and invasion of melanoma concomitant with neovascularization.     

Regulation of the CTL response by macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) has been shown to be a pivotal cytokine that mediates host inflammatory and immune responses. Recently, immunoneutralization of MIF has been found to inhibit tumor growth in mice; however, the contributing mechanisms underlying this effect have not been well defined. We investigated whether MIF plays a regulatory role in the expression of CTL activity. In a mouse model of the CTL response using the OVA-transfected tumor cell line EL4 (EG.7), we found that cultures of splenocytes obtained from EG.7-primed mice secrete high levels of MIF following Ag stimulation in vitro. Notably, parallel splenocyte cultures treated with neutralizing anti-MIF mAb showed a significant increase in the CTL response directed against EG.7 cells compared with control mAb-treated cultures. This effect was accompanied by elevated expression of IFN-gamma. Histological examination of the EG. 7 tumors from anti-MIF-treated animals showed a prominent increase in both CD4(+) and CD8(+) T cells as well as apoptotic tumor cells, consistent with the observed augmentation of CTL activity in vivo by anti-MIF. This increased CTL activity was associated with enhanced expression of the common gamma(c)-chain of the IL-2R that mediates CD8(+) T cell survival. Finally, CD8(+) T lymphocytes obtained from the spleens of anti-MIF-treated EG.7 tumor-bearing mice, when transferred into recipient tumor-bearing mice, showed increased accumulation in the tumor tissue. These data provide the first evidence of an important role for MIF in the regulation and trafficking of anti-tumor T lymphocytes in vivo.     

Novel anti-inflammatory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in various infectious and non-infectious diseases. We have investigated the mechanism of anti-inflammatory activity of epoxyazadiradione, a limonoid purified from neem (Azadirachta indica) fruits, against MIF. Epoxyazadiradione inhibited the tautomerase activity of MIF of both human (huMIF) and malaria parasites (Plasmodium falciparum (PfMIF) and Plasmodium yoelii (PyMIF)) non-competitively in a reversible fashion (K(i), 2.11-5.23 μm). Epoxyazadiradione also significantly inhibited MIF (huMIF, PyMIF, and PfMIF)-mediated proinflammatory activities in RAW 264.7 cells. It prevented MIF-induced macrophage chemotactic migration, NF-κB translocation to the nucleus, up-regulation of inducible nitric-oxide synthase, and nitric oxide production in RAW 264.7 cells. Epoxyazadiradione not only exhibited anti-inflammatory activity in vitro but also in vivo. We tested the anti-inflammatory activity of epoxyazadiradione in vivo after co-administering LPS and MIF in mice to mimic the disease state of sepsis or bacterial infection. Epoxyazadiradione prevented the release of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α when LPS and PyMIF were co-administered to BALB/c mice. The molecular basis of interaction of epoxyazadiradione with MIFs was explored with the help of computational chemistry tools and a biological knowledgebase. Docking simulation indicated that the binding was highly specific and allosteric in nature. The well known MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) inhibited huMIF but not MIF of parasitic origin. In contrast, epoxyazadiradione inhibited both huMIF and plasmodial MIF, thus bearing an immense therapeutic potential against proinflammatory reactions induced by MIF of both malaria parasites and human.     

Interferon-tau stimulates secretion of macrophage migration inhibitory factor from bovine endometrial epithelial cells

During early pregnancy in ruminants, the embryo not only prevents prostaglandin F2alpha release, but it also modifies protein synthesis in the endometrium. This is accomplished by the secretion of interferon-tau (IFN-tau) from the embryo. The objective of this study was to identify and characterize specific proteins secreted from endometrial epithelial cells in response to IFN-tau that could be important for endometrial function and/or embryo development. The epithelial cells were prepared and cultured to confluence and then incubated with or without 100 ng/ml IFN-tau. At the end of the incubation, the proteins in the medium were analyzed by two-dimensional PAGE. The result showed that two major protein spots were induced by IFN-tau. One has a molecular mass of approximately 12 kDa and an isoelectric point (pI) of 6.7; the other has a molecular mass of 76 kDa and pI of 4.8. Protein sequence analysis showed that the 12-kDa protein contained a partial amino acid sequence that corresponded to macrophage migration inhibitory factor (MIF). To determine whether MIF is expressed in endometrial cells, isolated stromal or epithelial cells were incubated with or without 100 ng/ml IFN-tau for 0, 3, 6, 12, 24, and 48 h. After incubation, the MIF protein in cells was examined by Western blotting analysis, and the steady-state mRNA for MIF was examined by Northern analysis. Results showed that MIF protein and mRNA were present in the epithelial cells but not the stromal cells. The presence of MIF in the luminal epithelium of endometrial tissue was confirmed by immunohistochemistry. However, there was no effect of IFN-tau on MIF expression in the epithelial cells. The concentration of MIF in the medium was quantified by Western blotting analysis to determine if IFN-tau altered MIF protein secretion from the epithelial cells. The results showed that IFN-tau significantly stimulated the secretion of MIF protein from the cells. These data show that MIF is expressed in the epithelial, but not the stromal, cells of the endometrium and that MIF secretion from the epithelial cells is stimulated by IFN-tau. It is therefore likely that MIF plays a role in early embryo development, and further characterization of MIF expression and its regulation in the endometrium will add significantly to our understanding of early embryo-uterine interactions.