Purification of thymic macrophage migration inhibitory factor (MIF)

Aqueous extracts of the thymus of animals which had been challenged immunologically have been shown to contain MIF activity. This MIF could be purified by precipitation with 70% ethanol, concentrated by ultrafiltration between 30,000 and 50,000 daltons, isoelectrically focused at pH 6.8–7.1, and electrophoresed on preparative acrylamide gels. The resulting product is electrophoretically homogeneous at pH 4.3 in polyacrylamide-gel electrophoresis and SDS-gel electrophoresis. It has a molecular weight of 36,000 daltons. It is trypsin- and neuraminidase-labile and is thermostable. It degrades and reassembles in electrophoresis at pH 7.0. It is not chemotactic for macrophages but apparently activates them phagocytically. It has no proteolytic activity.Supported in part by a contract from the Office of Nival Research N00014-71-C-0203.     

Increased density of macrophage migration inhibitory factor (MIF) in tuberculosis granuloma

Granulomas, the pathologic hallmarks of tuberculosis, are composed of tightly numerous immune cells that respond to a variety of persistent stimuli during pathogen-host interaction. The granuloma is essential for host containment of mycobacterial infection, however, the mechanism of host and pathogen determinants to recruit immune cells at the site of inflammation and the formation of granulomas remains elusive until now. Macrophage migration inhibitory factor (MIF), a cytokine produced by many cell types, modulates cellular and humoral immune responses and promote lymphocytes migration to the site of infection. In this study, we evaluate the expression of MIF in tuberculous granulomas by three different models of diseases: mouse, human tissues and zebrafish. The overall results demonstrated that the expression of MIF positive signals markedly increased in the tissues which have been infected with mycobacterium, whereas a few presence of MIF in the PBS-treated animals (means the control group). In the mycobacterial-infected animals, the MIF positives distributed extensively within the granuloma especially in the multinucleated giant cells. Thus, three independent lines of evidence support the hypothesis that MIF may be an important player in aggregate immune cells to the granuloma microenvironments in these animal models of tuberculosis.     

Mechanisms of macrophage migration inhibitory factor (MIF)-dependent tumor microenvironmental adaptation

Since its activity was first reported in the mid-1960s, macrophage migration inhibitory factor (MIF) has gone from a cytokine activity modulating monocyte motility to a pleiotropic regulator of a vast array of cellular and biological processes. Studies in recent years suggest that MIF contributes to malignant disease progression on several different levels. Both circulating and intracellular MIF protein levels are elevated in cancer patients and MIF expression reportedly correlates with stage, metastatic spread and disease-free survival. Additionally, MIF expression positively correlates with angiogenic growth factor expression, microvessel density and tumor-associated neovascularization. Not coincidentally, MIF has recently been shown to contribute to tumoral hypoxic adaptation by promoting hypoxia-induced HIF-1α stabilization. Intriguingly, hypoxia is a strong regulator of MIF expression and secretion, suggesting that hypoxia-induced MIF acts as an amplifying factor for both hypoxia and normoxia-associated angiogenic growth factor expression in human malignancies. Combined, these findings suggest that MIF overexpression contributes to tumoral hypoxic adaptation and, by extension, therapeutic responsiveness and disease prognosis. This review summarizes recent literature on the contributions of MIF to tumor-associated angiogenic growth factor expression, neovascularization and hypoxic adaptation. We also will review recent efforts aimed at identifying and employing small-molecule antagonists of MIF as a novel approach to cancer therapeutics.     

Molecular cloning and identification of macrophage migration inhibitory factor (MIF) in teleost fish

Macrophage migration inhibitory factor (MIF) is one of the first cytokines to be identified, which have been emerged to be an important mediator of the innate and adaptive immune system. Although MIF was well characterized in several mammal species, there was still little report in fish. In present study, we cloned the MIF gene from Tetraodon nigroviridis, and identified other six MIF genes from other teleost fishes, Fundulus heteroclitu, Oncorhynchus mykiss, Ictalurus punctatus, Danio rerio, Salmo salar and Haplochromis chilotes. The results showed that the fish MIF genes with the same organization as the mammalians consist of three exons and two introns. Tetraodon MIF gene located within a 1091bp genomic fragment of chromosome 1, transcribed into a 500bp mRNA including 14bp 5' untranslated region (UTR), 348bp ORF and 138bp 3'-UTR. Tetraodon MIF with 115aa has a calculated molecular mass of 12.5kDa and a theoretical pI of 6.81. The deduced amino-acid sequences of the teleost fish MIFs showed 64.1-73.5% sequence identity to mammalian MIFs, 61.5-70.1% to avian MIFs, 55.6-62.4% to amphibian MIFs, 74.4-97.4% among the teleost fishes. Phylogenetic analysis separates the teleost fish MIFs into an exclusive group. Genomic Southern blotting analyses suggest that Tetraodon has one copy of the MIF gene. RT-PCR and real-time PCR analyses reveal that Tetraodon MIF (TnMIF) mRNA was constitutively expressed in 10 selected tissues and induced by lipopolysaccharide (LPS) strikingly in head kidney and spleen. The bioactivity of recombinant TnMIF was tested by macrophage migration inhibition (MMI) assay. The result of MMI assay showed that the recombinant TnMIF inhibited the macrophage cells migration at rate of 35% (P<0.04). These results indicated that MIFs in fish may be involved in immune responses.     

Increased stability of macrophage migration inhibitory factor (MIF) in DU-145 prostate cancer cells.

Macrophage migration inhibitory factor (MIF) has been localized to the glandular epithelium of the prostate and stimulates the in vitro growth of prostate epithelial cells. [35S]Methionine labeling of MIF protein was used to determine if prostate cells synthesize and secrete this cytokine. The results demonstrated that the DU-145 prostate cancer cells secrete about twice the amount of a more stable protein compared with normal prostate epithelial cells. To investigate if differences in MIF mRNA levels account for the differences in MIF protein secreted by these cells, mRNA stability was analyzed by [3H]uridine incorporation. Following a 12-h pulse, DU-145 cells were found to contain four times the amount of [3H]uridine-labeled MIF mRNA, and this message exhibited a longer half-life than the message found in normal cells (33 h and 19 h, respectively). Nuclear run-on experiments confirmed that the MIF gene is transcribed at a greater rate (1.8-fold) in the DU-145 prostate cancer cells. This study documents, for the first time, that human prostate epithelial cells synthesize and secrete this cytokine. These results indicate that the increased levels of MIF found in prostate cancer cells is likely due to the increased protein and mRNA stability as exhibited by DU-145 cells.     

Analysis of macrophage migration inhibitory factor (MIF) in patients with idiopathic pulmonary fibrosis

By proteomic approach we previously characterised bronchoalveolar lavage (BAL) protein profiles of patients with idiopathic pulmonary fibrosis (IPF), sarcoidosis and systemic sclerosis. Among differently expressed proteins we identified macrophage migration inhibitory factor (MIF), a multi-function pleiotropic cytokine.This study was performed to validate our findings by a further proteomic approach and ELISA in a larger population of patients and controls. MIF expression in lung tissue was also evaluated by immunohistochemistry.MIF was identified in all 2-DE gels of IPF patients and it was significantly increased compared to controls (p < 0.05). This result was confirmed by ELISA: MIF concentrations were significantly higher in IPF patients than controls (p < 0.001) and were directly correlated with neutrophil percentages (p = 0.0095). Immunohistochemical analysis revealed enhanced expression in bronchiolar epithelium, alveolar epithelium, and fibroblastic foci.In conclusion, MIF is a pleiotropic cytokine that could be involved in the pathogenesis of IPF, being particularly abundant in BAL of these patients and mainly expressed in the areas of active fibrosis.     

Production of leucocyte inhibitory factor (LIF) and macrophage inhibitory factor (MIF) by PHA-stimulated lymphocytes.

Supernatants from human mononuclear cells cultured with PHA inhibited the migration of both human polymorphonuclear leucocytes and guinea-pig peritoneal exudate cells, but not human mononuclear cells. Using ultrafiltration it was shown that these supernatants contained two inhibiting factors, the one with a molecular weight of 15,000-50,000 inhibited only guinea-pig peritoneal exudate cells (MIF), whereas the fraction containing molecules of a size between 50,000 and 75,000 specifically inhibited the migration of polymorphonuclear leucocytes (LIF). The polymorphonuclear leucocyte inhibiting activity was heat labile. It is suggested that the leucocyte migration inhibition test is dependent upon the production of a lymphokine (LIF) which acts specifically on polymorphonuclear leucocytes causing their inhibition of migration.     

Macrophage migration inhibitory factor (MIF) in bronchial asthma

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine favouring the secretion of TNFalpha and IL-8 and counteracts anti-inflammatory effects of corticosteroids. Airways inflammation is a central feature of bronchial asthma and is characterized by the accumulation of eosinophils. OBJECTIVE: The aim of this study was to investigate whether MIF is related to asthma symptoms and eosinophil accumulation in the airways. METHODS: Serum MIF levels were measured by an enzyme-linked immunosorbent assay in 44 healthy subjects and 44 asthmatics. Levels of MIF in induced sputum were measured in 10 healthy subjects and 15 asthmatics. Levels of eosinophil cationic protein (ECP) in induced sputum were measured by a radioimmunosorbent assay. Fluorescence double immunostaining was conducted to examine cellular source and localization of MIF. RESULTS: Serum MIF levels were significantly increased in asthmatic patients compared with age and sex-matched control subjects. Symptomatic patients had a higher MIF level than asymptomatic patients. Induced sputum obtained from asthmatics contained higher levels of MIF than those from control subjects. MIF levels in induced sputum were correlated with ECP levels in induced sputum. MIF was colocalized with eosinophil peroxidase staining in the cytoplasm of sputum cells. CONCLUSION: Increased MIF levels are associated with asthma symptoms and one of the cellular sources of MIF in the airways are eosinophils.     

Mouse bone marrow-cultured macrophage as indicator cells for mouse and human migration inhibitory factor (MIF).

Macrophages cultured from mouse bone marrow served as excellent indicator cells for mouse as well as for human migration inhibitory factor (MIF) preparations. The tests were performed in the capillary system and showed high reproducibility. Using mouse or human MIF-containing supernatants, we found an inhibition of 53% compared with the controls. Similar results were obtained if concanavalin A-stimulated human peripheral blood lymphocytes were mixed with mouse test macrophages in a ratio of 1:5 or 1:10.     

Receptor binding and cellular uptake studies of macrophage migration inhibitory factor (MIF): use of biologically active labeled MIF derivatives.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine for which a receptor has not been identified. That MIF has intracellular functions has been suggested by its enzymatic activity and constitutive expression profile. The discovery of functional MIF-c-Jun activation domain binding protein 1 (JAB1) binding has confirmed this notion and indicated that nonreceptor-based signaling mechanisms are important for MIF function. Here, we have generated and tested several biologically active labeled MIF derivatives to further define target protein binding by MIF and its cellular uptake characteristics. (35)S-MIF, biotinylated MIF, and fluoresceinated MIF were demonstrated to exhibit full biologic activity. Neither by applying a standard iodinated MIF preparation nor by using the biologically active (35)S-MIF derivative in receptor-binding studies were we able to measure any receptor-binding activity on numerous cells, confirming that uptake of MIF into target cells and MIF signaling can occur by receptor-independent pathways. When MIF derivatives were applied in cellular uptake studies, MIF was found to be endocytosed into both immune and nonimmune cells and targeted to the cytosol and lysosomes. The entry of MIF was temperature and energy dependent and was inhibited by monodansylcadaverine but not by ouabain. Endocytosed biotin-MIF bound JAB1 not only in macrophages, as shown previously, but also in nonimmune cells. A tagged MIF construct, MIF-enhanced green fluorescent protein (EGFP), was shown to be a valuable tool, as EGFP constructs of critical MIF cysteine mutants exhibited identical cellular localization properties to those of wild-type MIF (wtMIF). Our results indicate that MIF membrane receptors are not widely expressed, if at all, and suggest that the cellular uptake of MIF occurs by nonreceptor-mediated endocytosis rather than penetration. All the derivatives investigated, except for iodinated MIF, represent valuable tools for further MIF target protein and cellular studies.     

Elevated macrophage migration inhibitory factor (MIF) levels in the urine of patients with focal glomerular sclerosis

The pathogenesis of focal glomerular sclerosis (FGS) is poorly understood. Macrophage migration inhibitory factor (MIF) is a potent pro-inflammatory cytokine released from T cells and macrophages, and is a key molecule in inflammation. To examine further the possible role of MIF in FGS, we measured MIF levels in the urine. The purpose of the present study was to evaluate the involvement of MIF in FGS. Urine samples were obtained from 20 FGS patients. The disease controls included 40 patients with minimal-change nephrotic syndrome (MCNS) and membranous nephropathy (MN). A group of healthy subjects also served as controls. Biopsies were performed in all patients prior to entry to the study. The samples were assayed for MIF protein by a sandwich enzyme-linked immunosorbent assay (ELISA). The levels of MIF in the urine of FGS patients were significantly higher than those of the normal controls and patients with MCNS and MN. In contrast, the levels of urinary MIF (uMIF) in patients with MCNS and MN did not differ significantly from normal values. In the present study, attention also focused on the relationship between uMIF levels and pathological features. Among the patients with FGS, uMIF levels were significantly correlated with the grade of mesangial matrix increase and that of interstitial fibrosis. There was also a significant correlation between uMIF levels and the number of both intraglomerular and interstitial macrophages. Although the underlying mechanisms remain to be determined, our study presents evidence that urinary excretion of MIF is increased in FGS patients with active renal lesions.     

Role of macrophage migration inhibitory factor (MIF) in murine antigen-induced arthritis: interaction with glucocorticoids

(MIF) is a broad-spectrum proinflammatory cytokine implicated in human rheumatoid arthritis. The synthesis of MIF by synovial cells is stimulated by glucocorticoids, and previous studies suggest that MIF antagonizes the anti-inflammatory effects of glucocorticoids. This has not been established in a model of arthritis. We wished to test the hypothesis that MIF can act to reverse the anti-inflammatory effects of glucocorticoids in murine antigen-induced arthritis (AIA). Cutaneous DTH reactions and AIA were induced by intradermal injection and intra-articular injection, respectively, of methylated bovine serum albumin in presensitized mice. Animals were treated with anti-MIF MoAbs, recombinant MIF, and/or dexamethasone (DEX). Skin thickness of DTH reactions was measured with callipers and arthritis severity was measured by blinded quantitative histological assessment of synovial cellularity. Cutaneous DTH to the disease-initiating antigen was significantly inhibited by anti-MIF MoAb treatment (P < 0.001). AIA was also significantly inhibited by anti-MIF MoAb (P < 0.02). DEX treatment induced a dose-dependent inhibition of AIA, which was significant at 0.2 mg/kg (P < 0.05). MIF treatment reversed the effect of therapeutic DEX on AIA (P < 0.001). DEX also significantly inhibited DTH reactions (P < 0.05) but rMIF had no effect on this effect of DEX. DTH and AIA are MIF-dependent models of inflammation and arthritis. The reversal of glucocorticoid suppression of AIA by MIF supports the concept that MIF is a counter-regulator of glucocorticoid control of synovial inflammation. Although DTH was observed to be MIF-dependent and glucocorticoid-sensitive, rMIF had no reversing effect on the suppression of DTH by glucocorticoids. This suggests that inflammatory processes in specific tissues may respond differently to MIF in the presence of glucocorticoids.     

Effect of macrophage migration inhibitory factor (MIF) in human placental explants infected with Toxoplasma gondii depends on gestational age

Because macrophage migration inhibitory factor (MIF) is a key cytokine in pregnancy and has a role in inflammatory response and pathogen defense, the objective of the present study was to investigate the effects of MIF in first- and third-trimester human placental explants infected with Toxoplasma gondii. Explants were treated with recombinant MIF, IL-12, interferon-γ, transforming growth factor-β1, or IL-10, followed by infection with T. gondii RH strain tachyzoites. Supernatants of cultured explants were assessed for MIF production. Explants were processed for morphologic analysis, immunohistochemistry, and real-time PCR analysis. Comparison of infected and stimulated explants versus noninfected control explants demonstrated a significant increase in MIF release in first-trimester but not third-trimester explants. Tissue parasitism was higher in third- than in first-trimester explants. Moreover, T. gondii DNA content was lower in first-trimester explants treated with MIF compared with untreated explants. However, in third-trimester explants, MIF stimulus decreased T. gondii DNA content only at the highest concentration of the cytokine. In addition, high expression of MIF receptor was observed in first-trimester placental explants, whereas MIF receptor expression was low in third-trimester explants. In conclusion, MIF was up-regulated and demonstrated to be important for control of T. gondii infection in first-trimester explants, whereas lack of MIF up-regulation in third-trimester placentas may be involved in higher susceptibility to infection at this gestational age.     

Studies on guinea-pig macrophage migration inhibitory factor (MIF). I. Glycoprotein nature and net charge.

Guinea-pig macrophage migration inhibitory factor (MIF), obtained by the stimulation of sensitized lymph node cells with tuberculin PPD, was characterized as a glycoprotein by the following criteria: (a) its activity is destroyed by 0.02 M sodium periodate; (b) when MIF-containing culture fluids are subjected to precipitation by perchloric acid (final concentration 1 M), the inhibitory activity is recovered in the supernatant; and (c) MIF binds to Sepharose-linked concanavalin A and can be eluted with methyl-alpha-D-glucopyranoside. When MIF-containing culture supernatants are fractionated by isoelectrofocussing, migration inhibitory activity is recovered in a fraction with an isoelectric point of 4.4--4.6.     

Molecular and functional characterization of macrophage migration inhibitory factor (MIF) homolog of human from lymphatic filarial parasite Wuchereria bancrofti

The ability of nematode parasites to survive in a highly complex immune system involves diverse strategies including production of a variety of host immune modulators. Various parasite-associated surface antigens or excretory and secretory products may possibly play a role in the host–parasite interactions and successful survival of parasite in their respective host. One among these molecules is a human cytokine homolog, macrophage migration inhibitory factor-1 (MIF-1) in various parasites. We identified a homolog of this cytokine from human lymphatic filarial parasite, Wuchereria bancrofti, expression cloned and investigated its molecular characteristics and catalytic properties. We also assessed the humoral reactivity of the recombinant MIF-1 of W. bancrofti (rWb-MIF-1) against sera belonging to different categories of individuals viz. microfilaremic, chronic patients, endemic normal, and non-endemic normal. Our results showed that the complete coding sequence of W. bancrofti is 1,078?bp, comprising two introns and three exons: first and second introns being 577 and 153?bp long, while the three exons I, II, and III being 108, 173, and 67?bp long, respectively. The rWb-MIF-1 was overexpressed in a salt-inducible host, Escherichia coli GJ 1158, and its functional activity was determined by dopachrome tautomerase and insulin reduction assays. The results of both the assays showed that the purified protein is functionally active and hence folded appropriately. The rWb-MIF-1 protein did not show elevation of specific IgG4 antibodies in microfilaremic cases, a hallmark in case of lymphatic filariasis, while it showed IgE reactivity in some of these cases (five out of ten).