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.     

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.     

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.     

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.     

Trauma patients with positive cultures have higher levels of circulating macrophage migration inhibitory factor (MIF)

Macrophage migration inhibitory factor (MIF) is a pituitary "stress" hormone that plays a critical role in the host immune response. The aims of the study were to determine whether MIF was detectable in the circulation of trauma patients, to assess whether MIF levels were associated with injury severity, days post injury, infection, and to examine concentrations of other pro-inflammatory cytokines in circulation. We collected plasma samples from 35 trauma (multiple injury) patients and 18 healthy controls. Concentrations of MIF, TNF-alpha, IL-1beta, and IL-6 were measured by ELISA. Average MIF concentration in plasma of trauma patients was 14 fold higher than that of healthy controls (19,439+/-2,615 pg/ml in trauma vs 1,337+/-286 pg/ml in control; p=0.0002). There was no correlation between MIF values and injury severity score or days post injury. Average level of IL-6 in trauma patients was 587+/-85 pg/ml but was not correlated with MIF concentration. TNF-alpha and IL-1beta were not detectable in trauma patients or healthy controls. Higher MIF levels were associated with positive cultures (blood, urine, sputum, wound). These data suggest that MIF may be a possible indicator of infection in trauma patients.     

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.     

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.     

巨噬细胞移动抑制因子在自身免疫性和炎症性疾病中的关键作用

巨细胞移动抑制因子（MIF）是一种在体外能抑制巨噬细胞移动的物质。现已发现MIF在．类风湿性关节炎（RA）、动脉粥样硬化（AS）和系统性红斑狼疮（SLE）等急性和慢性免疫炎症情况中具有关键的调节作用。这些疾病的共同特征是损伤部位有免疫细胞的浸润和活化。在这些疾病中起作用的炎症过程相互重叠,并观察到AS是RA、SLE的一种主要伴随疾病,这使MIF可以成为治疗这些疾病的一个极有希望的候选靶点。MIF和糖皮质激素（GC）之间独特的关系突显了MIF可以作为一种“类固醇减量”疗法治疗这些疾病的一个潜在性靶点。     

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.     

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.     

巨噬细胞移动抑制因子在前列腺癌中的表达及其临床意义探讨

目的:探讨前列腺癌巨噬细胞移动抑制因子(MIF)的表达及其临床意义。方法:采用ELISA法检测36例前列腺癌、32例良性前列腺增生(BPH)及20例健康成年男性的血清MIF水平,并做统计学分析。结果:MIF在前列腺癌组血清中的水平明显高于BPH组和正常对照组,差异有统计学意义(P<0.05)。血清MIF水平与临床分期和Gleason评分明显相关(P>0.05)。结论:MIF在前列腺癌、侵袭过程中起着重要的作用,可能作为前列腺癌早期诊断和预测疾病进展的生物标记物之一。     

Elevated levels of serum macrophage migration inhibitory factor in patients with pulmonary tuberculosis

Macrophage migration inhibitory factor (MIF) was originally described as a T-cell-derived cytokine that inhibits the random migration of macrophages and promotes the delayed-type hypersensitivity reaction. MIF plays an important role in the regulation of the Th1/Th2 balance in inflammatory response. This study investigated serum levels of circulating MIF in patients with pulmonary tuberculosis. The levels of MIF in sera were measured by enzyme-linked immunosorbent assay in 34 patients with pulmonary tuberculosis (16 males and 18 females) and 30 healthy controls (15 males and 15 females). The mean levels of circulating MIF values were significantly higher in those with pulmonary tuberculosis (19.84 +/- 11.27 ng/ml; P < 0.0001) than in the healthy controls (4.38 +/- 1.34 ng/ml). Circulating MIF values significantly correlated with circulating interferon-gamma values (r = 0.537, P < 0.0001). Thus, MIF may play an important role in immune responses to human infection with Mycobacterium tuberculosis.     

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.     

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.     

Different influence of macrophage migration inhibitory factor (MIF) in signal transduction pathway of various T cell subsets

It has been shown that macrophage migration inhibitory factor (MIF) modulates not only macrophage functions, but also T cell functions. However, detailed analysis of the MIF function on responses of various T cell subpopulations remained to be elucidated. In this report, using a neutralizing anti-MIF monoclonal antibody (mAb) we examined MIF functions on various T cell lineages. It was shown that anti-MIF mAb inhibited antigen-specific responses of both IFN-gamma producing and IL-4 producing T cells. The inhibition appeared to be related to blockade of the signal pathway via T cell receptor (TCR) but not that via IL-2 receptor (IL-2R). However, the anti-MIF mAb showed no inhibitory effect on NK-T cell responses stimulated through TCR. These results suggest that MIF is involved in the signal pathway via TCR in mainstream T cells but not in NK-T cells.