Macrophage Migration Inhibitory Factor Proteins Research Articles

1615-P: The Peripheral and Central Cross Talk Mediated by Adipose MIF—A Novel Mechanism of Hyperphagia following Olanzapine Treatment

Olanzapine is one of the first-line antipsychotic medications but it usually induces metabolic side effects, characterized by obesity. We previously indicated that olanzapine leads to hyperphagia through the effects of a proinflammatory cytokine, macrophage migration inhibitory factor (MIF) in the hypothalamus. However, whether olanzapine's upregulated peripheral MIF could affect the central mechanism is currently unknown. Our present study found that olanzapine increases ectopic expression of dopamine receptor 1 (DRD1) in adipocytes which relatively upregulates MIF expression and release through a PKA/CREB signaling pathway. In global MIF knockout mice, MIF accumulation appears in the hypothalamus following peripheral injection of recombinant MIF proteins, suggesting that peripheral MIF indeed travels to the hypothalamus. Mif Lung Tg mice (MIF overexpression in the lung) with increased circulating MIF levels had hyperphagia and activation of the CaMKKβ/AMPK/AgRP signaling pathway in the hypothalamus. Overall, our study for the first time proved that adipose-derived MIF is a crucial factor in regulating metabolic side effects induced by olanzapine. Disclosure X.Chen: None. Y.Huang: None. Y.Qi: None. L.Chen: None. L.Li: None. P.Gao: None. D.Cui: None. D.Qi: None. Funding National Sciences and Engineering Research Council of Canada (RGPIN-2017-04542); Canadian Institutes of Health Research (PJT-156116); China Scholarship Council (202106230212)

Detection of a glutathionyl-carbonylated group (GS–CO–) on D-dopachrome tautomerase with preferential binding of GS–CO– to MIF proteins in rat livers damaged by carbon tetrachloride

Liver damage has been induced in animal experiments using carbon tetrachloride (CCl4), a potent hepatotoxin. CCl4 is activated by cytochrome P450 2E1, which results in the formation of various metabolites including phosgene. Although D-dopachrome tautomerase (DDT) is abundant in the liver, its role currently remains unclear. The biological activity of DDT, for which the N-terminal proline is a key site, has been detected in various tissues. We herein incidentally detected a 333 Da modification to the N-terminal proline of DDT in rat livers damaged by CCl4. We identified that this modification as glutathionyl carbonylated group, which was formed by condensation of phosgene and reduced glutathione (GSH). We examined other glutathionyl-carbonylated proteins using two dimensional-polyacrylamide gel electrophoresis, mass spectrometry, and Western blotting for GSH, and detected only one glutathionyl-carbonylated protein, macrophage migration inhibitory factor (MIF). DDT belongs to the MIF family of proteins, and amino acid sequence identity between DDT and MIF is 33%. We concluded that MIF family proteins are major targets for glutathionyl carbonylation.

D-dopachrome tautomerase in cardiovascular and inflammatory diseases-A new kid on the block or just another MIF?

Macrophage migration inhibitory factor (MIF) as well as its more recently described structural homolog D-dopachrome tautomerase (D-DT), now also termed MIF-2, are atypical cytokines and chemokines with key roles in host immunity. They also have an important pathogenic role in acute and chronic inflammatory conditions, cardiovascular diseases, lung diseases, adipose tissue inflammation, and cancer. Although our mechanistic understanding of MIF-2 is relatively limited compared to the extensive body of evidence available for MIF, emerging data suggests that MIF-2 is not only a functional phenocopy of MIF, but may have differential or even oppositional activities, depending on the disease and context. In this review, we summarize and discuss the similarities and differences between MIF and MIF-2, with a focus on their structures, receptors, signaling pathways, and their roles in diseases. While mainly covering the roles of the MIF homologs in cardiovascular, inflammatory, autoimmune, and metabolic diseases, we also discuss their involvement in cancer, sepsis, and chronic obstructive lung disease (COPD). A particular emphasis is laid upon potential mechanistic explanations for synergistic or cooperative activities of the MIF homologs in cancer, myocardial diseases, and COPD as opposed to emerging disparate or antagonistic activities in adipose tissue inflammation, metabolic diseases, and atherosclerosis. Lastly, we discuss potential future opportunities of jointly targeting MIF and MIF-2 in certain diseases, whereas precision targeting of only one homolog might be preferable in other conditions. Together, this article provides an update of the mechanisms and future therapeutic avenues of human MIF proteins with a focus on their emerging, surprisingly disparate activities, suggesting that MIF-2 displays a variety of activities that are distinct from those of MIF.

A structurally preserved allosteric site in the MIF superfamily affects enzymatic activity and CD74 activation in D‐dopachrome tautomerase

The human macrophage migration inhibitory factor (MIF) superfamily consists of MIF and D‐dopachrome tautomerase (also called MIF‐2). Both MIF proteins have critical innate and adaptive immunomodulatory functions in regulating differentiation, expression of pro‐inflammatory cytokines, and activation and recruitment of immune cells. They are broadly and constitutively expressed, but elevated levels of MIF family proteins are upregulated in various inflammatory diseases such as asthma, acute respiratory distress syndrome (ARDS) and arthritis, and antibody neutralization of MIF decreased inflammatory symptoms in murine models. While their potential as drug targets has led to the creation of a few commercial inhibitors of MIF, the efficacy of targeting MIF proteins as a therapeutic is currently obstructed by a lack of molecular level detail about their structures and dynamics and how those properties impact disease‐state activity.Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. The MIF homolog DDT (MIF‐2) has low sequence identity with MIF (35%) though they overlap in multiple functions, including activation of the CD74 receptor, and share a nearly identical tertiary structure. This prompted the question of whether this dynamic regulatory network is conserved within the superfamily. We identify a comparable allosteric network in MIF‐2, connecting the catalytic site to CD74‐activating residues, showing with solution nuclear magnetic resonance (NMR) spectroscopy that dynamic communication is preserved in MIF‐2 despite differences in primary sequence. Disruption of the allosteric relay by mutagenesis also attenuates MIF‐2 enzymatic activity in vitro and biological CD74 signaling in vivo, highlighting amino acids in this pathway as trigger residues for potential functional control of the protein.

A cross‐kingdom view on the immunomodulatory role of MIF/D‐DT proteins in mammalian and plant Pseudomonas infections

AbstractGram‐negative Pseudomonas bacteria are largely harmless saprotrophs, but some species can be potent pathogens of both plants and mammals. Macrophage migration‐inhibitory factor (MIF) and its homologue D‐dopachrome tautomerase (D‐DT, also referred to as MIF‐2) are multifunctional proteins that in addition to their intracellular functions also serve as extracellular signalling molecules (cytokines) in orchestrating mammalian immune responses. It recently emerged that plants also possess MIF‐like proteins, termed MIF/D‐DT‐like (MDL) proteins. We here provide a comparative cross‐kingdom view on the immunomodulatory role of MIF and MDL proteins during Pseudomon as infections in mammals and plants. Although in both kingdoms the lack of MIF/MDL proteins is associated with a reduction in bacterial load and disease symptoms, the underlying molecular principles seem to be different. We provide a perspective for future research activities to unravel additional commonalities and differences in the MIF/MDL‐mediated adjustment of antibacterial immune activities.

Downregulation of macrophage migration inhibitory factor attenuates NLRP3 inflammasome mediated pyroptosis in sepsis-induced AKI

Sepsis-induced AKI (acute kidney injury) is considered an inflammation-related disease with high mortality. LPS-induced (Lipopolysaccharide) TLR4-NFκB pathway activation plays an important role in sepsis-induced AKI. Pyroptosis closely associated with inflammation response includes inflammasome formation, caspase1 activation and GSDMD N-terminal fragment cleavage that leads to cell membrane rupture and cell death, which may be related to the pathogenesis of sepsis-induced AKI. MIF (Macrophage migration inhibitory factor), associated with inflammation response, has been proved as a biomarker of sepsis, and perhaps regulate pyroptosis in sepsis-induced AKI. In this study, we focus on investigating the mechanism of MIF promoting pyroptosis in sepsis-induced AKI. MIF and pyroptosis-related proteins were up-regulated in kidney tissue of mice with CLP (cecum ligation puncture) surgery and in LPS-injured human kidney-2 (HK-2) cells. NLRP3 was down-regulated following the suppression of MIF topoisomerase activity by ISO-1 in kidney tissue of CLP mice. Knockdown of MIF alleviated NLRP3 inflammasome mediated pyroptosis in LPS-injured HK-2 cells. Meanwhile, we noted that phosphorylation of p65 was down-regulated by knockdown of MIF. Up-regulation of NLRP3 in response to LPS stimulation could be reversed by JSH-23, an inhibitor of NFκB pathway, but MIF was not affected. In conclusion, up-regulation of MIF in sepsis-induced AKI shows a renal damaged effect that aggravates NLRP3 inflammasome mediated cell pyroptosis through promoting phosphorylation of p65. This study demonstrated a novel mechanism of MIF regulating NLRP3 inflammasome mediated pyroptosis in sepsis-induced AKI.

Co-Expression and Functional Interaction between CXCR4 and CD74 in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by progressive accumulation of small, mature B-lymphocytes aberrantly expressing CD5. One of the crucial factors in B-CLL development is the decreased apoptosis of malignant B-Cells.The chemokine receptor CXCR4 and the MHC class II invariant chain (Ii) CD74, are expressed on CLL B-cells and play a role in cells' migration and survival. CXCL12, the cognate ligand of CXCR4, promotes homing of CLL cells into the bone marrow (BM) and survival within the niche. Furthermore, CXCR4 is activated by macrophage migration inhibitory factor (MIF), a chemokine-like inflammatory cytokine and non-cognate ligand of CXCR4. MIF and its homolog MIF2 are bona fide ligands of CD74.CD74 is highly expressed on CLL cells and expression is correlated with the adversely prognostic factor ZAP 70. Interestingly, MIF plays a role in the survival of CLL cells by binding to CD74. Furthermore, increased MIF levels are found in the plasma of CLL patients and genetic Mif deficiency leads to delayed development of CLL in the Eµ-TCL1 mouse model. Finally, the aim of our study was to investigate the expression and interaction between CXCR4 and CD74 as well as their potential contribution to B-Cell survival in B-CLL.An initial cohort of 350 patients with different backgrounds of hematological malignancies was analyzed for CXCR4 and CD74 expression on mononuclear cells. Interestingly, the highest coexpression of both receptors was observed in B-CLL (n=10) by flow cytometry (FCM), where cells were stained for CD3+, CD14+ and CD19+/CD5+ and compared to cells from healthy donors (n=20). For in vivo and in vitro studies cell lines and a mice model were chosen, which express both receptors CXCR4 and CD74 on the surface of B-Cells.The human CLL line JVM3 and primary CLL cells from aged leukemic Eμ-TCL1 mice were used to study the cross-talk of CXCR4 and CD74 in regulation of CLL survival after incubation with rhMIF, rhCXCL12, or rhMIF-2 in conjunction with Bcl2-inhibitor treatment (Venetoclax, 30µM) or pharmacological blockade of CXCR4 and/or CD74 (AMD3100 30µM, neutralizing CD74 antibody LN2 5µg/ml). Potential direct CXCR4/CD74 interactions were studied by confocal laser scanning microscopy (CLSM) and coimmunoprecipitation (CoIP). Chemotaxis towards CXCL12, MIF and MIF-2 were analyzed to understand homing behavior, which contributes to B-Cell survival.CXCR4 and CD74 were found significantly coexpressed on B-cells from CLL patients compared to controls (p< 0.0001), suggesting a functional interaction between these receptors. Colocalization was confirmed by CLSM and CoIP detected receptor complexes between CD74 and CXCR4 on B- Cells from the JVM3 and OSU CLL cell lineFunctionally, inhibition of either receptor substantially impaired survival rates that did not differ from a simultaneous co-blockade of CD74 and CXCR4, indicating a crosstalk and/or merging of downstream signaling pathways. Apoptosis assays indicated that inhibition of CXCR4 and/or CD74 triggered substantial cell death (78,17 +/- 1,84%). Of note, single vs. double blockade experiments confirmed that CD74 and CXCR4 functionally interact to control CLL survival. Inversely, pre-incubation of JVM3 cells with CXCL12, MIF and MIF-2 decreased apoptosis by 43, 36 % (MIF) and 51, 50 % (CXCL12) compared to the control.The ligands MIF, MIF2 and CXCL12 promoted chemotactic migration of JVM3 B-cells that contributes to the B-Cell survival mechanism.Together, our results suggest an important role of MIF proteins in the survival and homing of malignant CLL cells that is mediated through a functional interaction between the CXCR4 and CD74 survival receptors. In conclusion, high levels of MIF attract B-Cells with the coexpression of CXCR4 and CD74, which build a complex after binding to their ligands and activate survival pathways to inhibit cell death. The MIF/CXCR4/CD74 axis could represent a novel target for therapeutic intervention. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Koschmieder:Roche: Other: Clinical Trial participation; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding.

Revisiting the secretion mechanism(s) of macrophage migration inhibitory factor-welcome to the "UPS club".

Revisiting the secretion mechanism(s) of macrophage migration inhibitory factor-welcome to the "UPS club".

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Interaction between parasite-encoded JAB1/CSN5 and macrophage migration inhibitory factor proteins attenuates its proinflammatory function

Multiple protozoans produce homologs of the cytokine MIF which play a role in immune evasion, invasion and pathogenesis. However, how parasite-encoded MIF activity is controlled remains poorly understood. Cytokine activity can be inhibited by intracellular binding partners that are released in the extracellular space during cell death. We investigated the presence of an endogenous parasite protein that was capable of interacting and interfering with MIF activity. A screen for protein-protein interaction was performed using immunoaffinity purification of amebic cell lysate with specific anti-Entamoeba histolytica MIF (EhMIF) antibody followed by mass spectrometry analysis, which revealed an E. histolytica-produced JAB1 protein (EhJAB1) as a potential binding partner. JAB1 was found to be highly conserved in protozoans. Direct interaction between the EhMIF and EhJAB1 was confirmed by several independent approaches with GST pull-down, co-immunoprecipitation, and Biolayer interferometry (BLI) assays. Furthermore, the C-terminal region outside the functional JAMM deneddylase motif was required for EhMIF binding, which was consistent with the top in silico predictions. In addition, EhJAB1 binding blocked EhMIF-induced IL-8 production by human epithelial cells. We report the initial characterization of a parasite-encoded JAB1 and uncover a new binding partner for a protozoan-produced MIF protein, acting as a possible negative regulator of EhMIF.

TLR7 and TLR8 agonist resiquimod (R848) differently regulates MIF expression in cells and organs

Since its first description in 1966, macrophage migration inhibitory factor (MIF) was found to play a critical role in inflammatory and immune responses as well as in disease pathogenesis especially in tumor pathogenesis and cancer progression. MIF is expressed in different cell types and is associated with many disease severity and tumor pathogenesis. Here, we investigated the influence of TLR7 and TLR8 agonist resiquimod (R848), an immune response inducer used as a prophylactic agent for several infectious diseases as well as anticancer agents and vaccine adjuvant on MIF expression in cells and organs. Humans, mice and rats cell lines from different tissues (blood, retinal, nasopharynx, brain and liver) and C57BL/6J mice organs (brain, liver and spleen) were used for this investigation. In vitro, R848 induced MIF gene overexpression except in brain and liver cells. Furthermore, it enhanced cells ability to release soluble MIF and differently regulated mRNA expression of MIF-related receptors (CD74, CXCR4, CXCR2 and CD44). Its influence on MIF gene expression and MIF proteins release was more consistent in cancer cells. In vivo, a strong positive expression of MIF was observed in different regions in brain and spleen in response to R848 treatment; however in liver, increased MIF expression was observed in hepatocytes only. On the other hand, R848 treatment had induced a slight enhancement of MIF concentration in the plasma of C57BL/6J mice. Taken together, these data suggest that R848 differently regulates MIF mRNA expression depending on organ types and could influence MIF concentration in cellular microenvironment.

MIF Promoted Cardiovascular Angiogenesis via Erk/Mapk Pathway

As the pivotal part of cardiovascular angiogenesis, endothelial cells dysfunction is the leading cause of cardiovascular diseases. Macrophage migration inhibitory factor (MIF) is a tumor growth factor with important roles in cervical tumor formation, invasion, progression and metastasis. However, there was no report on effect of MIF on endothelial cells is unclear, and it is still unknown whether MIF is associated with angiogenesis of endothelial cells. Our study was focused on the effect of MIF and PD98059 on endothelial cells HUVEC cell line, so as to investigate the influence of MIF on expression of vascular endothelial growth factor (VEGF). We also explored whether MIF will influence angiogenesis of endothelial cells via ERK/MAPK pathways. Endothelial cells HUVEC cells were conventionally cultured, and Western blot were used to detect the expression of MIF, ERK1 and VEGF proteins after HUVEC cells were intervened by MTT and PD98059. Inter-group difference was statistically assessed. Positive expressions of MIF, ERK1 and VEGF were observed in HUVEC cells. Proliferation activity in MIF group gradually increased after 24 h, 48 h or 72 h treatment (P 0.05). Expressions of ERK1 and VEGF are involved in the process of endothelial cells HUVEC cell line. MIF is correlated with increased cell proliferation and promoted cardiovascular angiogenesis via ERK/MAPK pathway

A Secreted MIF Cytokine Enables Aphid Feeding and Represses Plant Immune Responses

Aphids attack virtually all plant species and cause serious crop damages in agriculture. Despite their dramatic impact on food production, little is known about the molecular processes that allow aphids to exploit their host plants. To date, few aphid salivary proteins have been identified that are essential for aphid feeding, and their nature and function remain largely unknown. Here, we show that a macrophage migration inhibitory factor (MIF) is secreted in aphid saliva. In vertebrates, MIFs are important pro-inflammatory cytokines regulating immune responses. MIF proteins are also secreted by parasites of vertebrates, including nematodes, ticks, and protozoa, and participate in the modulation of host immune responses. The finding that a plant parasite secretes a MIF protein prompted us to question the role of the cytokine in the plant-aphid interaction. We show here that expression of MIF genes is crucial for aphid survival, fecundity, and feeding on a host plant. The ectopic expression of aphid MIFs in leaf tissues inhibits major plant immune responses, such as the expression of defense-related genes, callose deposition, and hypersensitive cell death. Functional complementation analyses in vivo allowed demonstrating that MIF1 is the member of the MIF protein family that allows aphids to exploit their host plants. To our knowledge, this is the first report of a cytokine that is secreted by a parasite to modulate plant immune responses. Our findings suggest a so-far unsuspected conservation of infection strategies among parasites of animal and plant species.

Exendin-4 ameliorates oxidized-LDL-induced inhibition of macrophage migration in vitro via the NF-κB pathway

To investigate the effects of the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 on oxidized low-density lipoprotein (ox-LDL)-induced inhibition of macrophage migration and the mechanisms underlying the effects of exendin-4. Primary peritoneal macrophages were extracted from the peritoneal cavity of mice treated with 3% thioglycollate (2 mL, ip). Migration of the macrophages was examined using a cell migration assay. Macrophage migration-related factors including leptin-like ox-LDL receptor (LOX-1), cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin-1 (IL-1)β, matrix metalloproteinase-2 (MMP-2), intercellular adhesion molecule (ICAM)-1 and macrophage migration inhibitory factor (MIF) were measured using semi-quantitative RT-PCR. Expression of MIF and ICAM-1 proteins was examined with ELISA. Gelatin zymography was used to evaluate the activity of MMP-9. Activation of the NF-κB pathway was determined by confocal laser scanning microscopy. Treatment of the macrophages with ox-LDL (50 μg/mL) markedly suppressed the macrophage migration. Furthermore, ox-LDL treatment substantially increased the expression of the macrophage migration-related factors, the activity of MMP-9 and the translocation of the NF-κB p65 subunit. These effects of ox-LDL were significantly ameliorated by pretreatment with the specific NF-κB inhibitor ammonium pyrrolidine dithiocarbamate (100 μmol/L). These effects of ox-LDL were also significantly ameliorated by pretreatment with exendin-4 (25 and 50 nmol/L). Exendin-4 ameliorates the inhibition of ox-LDL on macrophage migration in vitro, via suppressing ox-LDL-induced expression of ICAM-1 and MIF, which is probably mediated by the NF-κB pathway.

Crystal structure of a macrophage migration inhibitory factor from Giardia lamblia

Macrophage migration inhibitory factor (MIF) is a eukaryotic cytokine that affects a broad spectrum of immune responses and its activation/inactivation is associated with numerous diseases. During protozoan infections MIF is not only expressed by the host, but, has also been observed to be expressed by some parasites and released into the host. To better understand the biological role of parasitic MIF proteins, the crystal structure of the MIF protein from Giardia lamblia (Gl-MIF), the etiological agent responsible for giardiasis, has been determined at 2.30 Å resolution. The 114-residue protein adopts an α/β fold consisting of a four-stranded β-sheet with two anti-parallel α-helices packed against a face of the β-sheet. An additional short β-strand aligns anti-parallel to β4 of the β-sheet in the adjacent protein unit to help stabilize a trimer, the biologically relevant unit observed in all solved MIF crystal structures to date, and form a discontinuous β-barrel. The structure of Gl-MIF is compared to the MIF structures from humans (Hs-MIF) and three Plasmodium species (falciparum, berghei, and yoelii). The structure of all five MIF proteins are generally similar with the exception of a channel that runs through the center of each trimer complex. Relative to Hs-MIF, there are differences in solvent accessibility and electrostatic potential distribution in the channel of Gl-MIF and the Plasmodium-MIFs due primarily to two "gate-keeper" residues in the parasitic MIFs. For the Plasmodium MIFs the gate-keeper residues are at positions 44 (Y --> R) and 100 (V --> D) and for Gl-MIF it is at position 100 (V --> R). If these gate-keeper residues have a biological function and contribute to the progression of parasitemia they may also form the basis for structure-based drug design targeting parasitic MIF proteins.

Ribosomal Protein S19 Interacts with Macrophage Migration Inhibitory Factor and Attenuates Its Pro-inflammatory Function

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (K(D) = 1.3 x 10(-6) m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF.

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.

INCREASING EXPRESSION OF GST-pi MIF, AND ID1 GENES IN CHEMORESISTANT PROSTATE CANCER CELLS

The differential expression of genes and related proteins of multidrug resistance in chemoresistant prostate cancer cell lines were elucidated in this study. RNA extracted from doxorubicin-resistant rat prostate cancer (PCa) cells (AT3/ADR1000) and native PCa cells was hybridized to expression arrays containing cDNAs from 588 known genes. Differential expression of selected genes was confirmed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Protein contents were measured by fluorescent flow cytometry and immunoblotting. Localization of selected proteins in cells was observed by immunocytochemical staining. Up-regulation of eleven genes and down-regulation of one single gene were displayed in the chemoresistant prostate cancer cells. Overexpression of mRNAs in macrophage migration inhibitory factor (MIF), DNA binding protein inhibitor 1 (ID1), and glutathione S-transferase-pi (GST-pi) were confirmed by gene-specific RT-PCR. Protein over-expression of GST-pi, MIF, and ID1 in resistant cells were 3.3-, 1.5-, and 1.5-fold to native cells, respectively. Immunocytochemistry revealed that GST-pi, MIF, and ID1 were present primarily in the cytoplasm of tumor cells, but ID1 also could be found in the nucleus. AT3/ADR1000 drug-resistant PCa cells displayed significantly increased expression of GST-pi, MIF, and ID1 proteins when compared with native PCa cells. It indicates these genes may play a role in drug resistance of prostate cancer.

Macrophage migration inhibitory factor (MIF) in chinese amphioxus as a molecular marker of immune evolution during the transition of invertebrate/vertebrate

Macrophage migration inhibitory factor (MIF) is an important cytokine related to host defenses and autoimmune diseases. Here, we reported two full-length cDNA clones isolated from Chinese amphioxus ( Branchiostoma belcheri tsingtaunese). Amino acid sequences analysis and structure prediction of these two molecules, called Bbt-MIF-I and Bbt-MIF-II, respectively, indicated that several conservative domains existed in the two amphioxus MIFs and their sequences were highly homologous to their counterparts of other species. Intriguingly, the Bbt-MIFs gene is present in multi-copy per haploid genome, which is very unusual compared with vertebrate's MIF gene given the known genome duplication theory. The genomic copy number, expression pattern of MIF gene and phylogenetic analysis of MIF proteins all suggested that a leap forward happened for MIF gene during the evolution from invertebrate to vertebrate. Considering the crucial role of MIF in innate immunity, MIF might serve as one of key molecular markers of evolution of immune system.

Macrophage migration inhibitory factor (MIF) of jawed and jawless fishes: implications for its evolutionary origin

The macrophage migration inhibitory factor (MIF) is a cytokine produced by T lymphocytes and macrophages in response to inflammatory stimuli. We sequenced MIF cDNA clones of two jawless fishes, the sea lamprey ( Petromyzon marinus) and the North Atlantic hagfish ( Myxine glutinosa), as well as of the jawed (cichlid) fish Paralabidochromis chilotes. The fish MIF-encoding genes have the same exon–intron organization as the mammalian MIF genes and are present in one copy per haploid genome. Secondary and tertiary structure predictions suggest that the fish MIF proteins have a topology characteristic of the entire MIF-family of proteins. Phylogenetic analysis separates the known nematode members of the family into two groups, one having a sister group relationship with the mammalian d-dopachrome tautomerase (DDT) proteins and the other being related to vertebrate MIFs. It also reveals a high degree of convergent evolution among the members of the family. Finally, it suggests that the divergence of MIF and DDT occurred before the emergence of nematodes in metazoan evolution.