Production of macrophage migration inhibitory factor(s) (MIF) by virus-transformed cells

Rodent cell lines transformed by SV40, polyoma virus and Rous sarcoma virus cultured in vitro release material into the culture medium which inhibits the migration of guinea pig macrophages. Similar macrophage migration inhibitory factors (MIF) were not detected in cell-free supernatants harvested from untransformed cell cultures. Comparison of the MIF produced by virus-transformed cells with MIF derived from peripheral lymphocytes stimulated in vitro by phytohemagglutinin (PHA) revealed that they had similar molecular weights (25 000), heat stability and were both inhibited by α-fucose and lotus agglutinin. Incubation of MIF-containing cell-free supernatants from transformed cells with pancreatic trypsin inhibitor, soybean trypsin inhibitor and diisopropylfluorophosphate eliminated the MIF activity. The possible identity of the MIF released by transformed cells as a protease is discussed with reference to a potential role in modifying the surface properties of transformed cells.     

Macrophage migration inhibitory factor (MIF) produced by a human T cell hybridoma clone

A human T cell hybridoma clone, F5, producing high levels of macrophage migration inhibitory factor (MIF) was established by the emetine-actinomycin D selection method. This clone produced two species of MIF which were separated on a Phenyl Sepharose column. We purified MIF-2 (the more hydrophobic species of the two) to homogeneity from the conditioned medium of stimulated F5 cells by a series of steps that included hydrophobic chromatography, ion-exchange chromatography. Ricinus communis lectin affinity chromatography, and high-performance liquid chromatography on anion exchange and reverse-phase columns. Purified MIF was digested with endoproteinase Lys-C and Asp-N. The amino acid sequences of the generated peptides were determined. No sequence similarity with any other protein was found. The molecular weight of MIF-2 was estimated to be 45 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitates with anti-peptide antibodies. These results show that F5MIF-2 is a novel cytokine.     

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.     

Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF

Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.     

Macrophage migration inhibitory factor (MIF)--its role in catecholamine metabolism.

Macrophage migration inhibitory factor (MIF) was originally identified several decades ago as a lymphokine-derived protein that inhibited monocyte migration. Recently, it has been reported that MIF has D-dopachrome tautomerase, phenylpyruvate tautomerase and thiol protein oxidoreductase activities, although the physiological significance of those activities is not yet clear. Here we show that MIF is able to catalyze the conversion of dopaminechrome and norepinephrinechrome, toxic quinone products of the neurotransmitters dopamine and norepinephrine, respectively, to indole derivatives that may serve as precursors to neuromelanin. Since MIF is highly expressed in human brain, these observations raise the possibility that MIF participates in a detoxification pathway for catecholamine products and could therefore have an important role for neural tissues. The potential role of MIF in the formation of neuromelanin from catecholamines is also an extremely interesting possibility.     

A rapid method for measuring the production of human migration inhibitory factor (MIF)

A rapid method for assaying Migration Inhibitory Factor (MIF) prepared from human peripheral venous blood is described. Using increased concentrations of human peripheral lymphocytes, MIF could be demonstrated in supernatant at the end of a 24 hr culture period. Both tuberculin and histoplasmin sensitivity were investigated. In persons with normal cell-mediated immunity, there was good correlation between skin test sensitivity and MIF production by this method.     

Inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity by dopachrome analogs

A macrophage migration inhibitory factor (MIF) dopachrome tautomerization assay was employed for identification of MIF inhibitors. One group of dopachrome analogs was identified which inhibits MIF tautomerase activity. In particular, the analogs with a leaving group at beta position displayed activity at a concentration of tenfold less than that of the MIF-substrate. These findings could lead to a better understanding of MIF biological activities and the development of agents for the treatment of MIF related diseases.     

Macrophage migration inhibitory factor (MIF) contributes to the development of allergic rhinitis

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose expression has been found to be critical to the generation of antigen-specific immune responses. Recent studies suggested that MIF played a role in the initiation and maintenance of allergic diseases. To elucidate MIF's role in the pathogenesis of allergic rhinitis (AR), we sensitized MIF-deficient gene knockout (KO) mice and wild-type (WT) mice intraperitoneally with ovalbumin (OVA) and compared their clinical symptoms and allergic responses after intranasal challenge. Antigen-induced nasal symptoms were significantly reduced in MIF KO mice compared to WT mice. Histological examination of nasal mucosa showed that the number of infiltrating eosinophils in MIF KO mice was significantly lower than that in WT mice (P < 0.05). The concentration of TNF-alpha in nasal mucosa was also significantly lower in MIF KO mice than in WT mice (P < 0.05). We have demonstrated that the absence of MIF affects several aspects of experimental AR. One mechanism by which these effects might be mediated is by down regulating TNF-alpha. The block of allergic inflammation in MIF KO mice suggests that MIF may play a role in the allergic response.     

Characterization of catalytic centre mutants of macrophage migration inhibitory factor (MIF) and comparison to Cys81Ser MIF.

Macrophage migration inhibitory factor (MIF) displays both cytokine and enzyme activities, but its molecular mode of action is still unclear. MIF contains three cysteine residues and we showed recently that the conserved Cys57-Ala-Leu-Cys60 (CALC) motif is critical for the oxidoreductase and macrophage-activating activities of MIF. Here we probed further the role of this catalytic centre by expression, purification, and characterization of the cysteine-->serine mutants Cys60Ser, Cys57Ser/Cys60Ser, and Cys81Ser of human MIF and of mutants Ala58Gly/Leu59Pro and Ala58Gly/Leu59His, containing a thioredoxin (Trx)-like and protein disulphide isomerase (PDI)-like dipeptide, respectively. The catalytic centre mutants formed inclusion bodies and the resultant mutant proteins Cys57Ser/Cys60Ser, Ala58Gly/Leu59Pro, and Als58Gly/Leu59His were only soluble in organic solvent or 6 m GdmHCl when reconstituted at concentrations above 1 microgram.mL-1. This made it necessary to devise new purification methods. By contrast, mutant Cys81Ser was soluble. Effects of pH, solvent, and ionic strength conditions on the conformation of the mutants were analysed by far-UV CD spectropolarimetry and mutant stability was examined by denaturant-induced unfolding. The mutants, except for mutant Cys81Ser, showed a close conformational similarity to wild-type (wt) MIF, and stabilization of the mutants was due mainly to acid pH conditions. Intramolecular disulphide bond formation at the CALC region was confirmed by near-UV CD of mutant Cys60Ser. Mutant Cys81Ser was not involved in disulphide bond formation, yet had decreased stability. Analysis in the oxidoreductase and a MIF-specific cytokine assay revealed that only substitution of the active site residues led to inactivation of MIF. Mutant Cys60Ser had no enzyme and markedly reduced cytokine activity, whereas mutant Cys81Ser was active in both tests. The Trx-like variant showed significant enzyme activity but was less active than wtMIF; PDI-like MIF was enzymatically inactive. However, both variants had full cytokine activity. Together with the low but nonzero cytokine activity of mutant Cys60Ser, this indicated that the cytokine activity of MIF may not be tightly regulated by redox effects or that a distinguishable receptor mechanism exists. This study provides evidence for a role of the CALC motif in the oxidoreductase and cytokine activities of MIF, and suggests that Cys81 could mediate conformational effects. Availability and characterization of the mutants should greatly aid in the further elucidation of the mechanism of action of the unusual cytokine MIF.     

Tumor-derived macrophage migration inhibitory factor (MIF) inhibits T lymphocyte activation

Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine that is considered a pro-inflammatory cytokine. However, our studies show that MIF, when produced in super-physiological levels by a murine neuroblastoma cell line (Neuro-2a) exceeding those normally seen during an immune response, inhibits cytokine-, CD3-, and allo-induced T-cell activation. MIF is also able to inhibit T cells that have already received an activation signal. The T-cell inhibitory effects of culture supernatants from neuroblastoma cells were reversed when the cells were transfected with dicer-generated si-RNA to MIF. When T cells were activated in vitro by co-culture with interleukin (IL)-2 and IL-15 and analyzed for cytokine production in the presence or absence of MIF-containing culture supernatant, inhibition of T-cell proliferation and induced cell death were observed even as the treated T cells produced high levels of interferon-gamma (IFN-gamma). The inhibitory effects of MIF were partially reversed when lymphocytes from IFN-gamma knockout mice were tested. We propose that the high levels of MIF produced by neuroblastoma cause activation induced T-cell death through an IFN-gamma pathway and may eliminate activated T cells from the tumor microenvironment and thus contribute to escape from immune surveillance.     

Partial characterization of murine migration inhibitory factor (MIF).

These studies describe the production of murine migration inhibitory factor (MIF)3 in sufficient quantities to allow its partial characterization by physiochemical and enzymatic methods. MIF was obtained from murine spleen cell cultures (C57BL/6 strain) stimulated with concanavalin A (Con A). Characterization of murine MIF was performed using Sephadex G-100 gel chromatography, isopycnic centrifugation in a CsCl density gradient, polyacrylamide disc electrophoresis, heat stability, and enzymatic treatment. MIF-containing and control fractions were assayed on normal C57BL/6 peritoneal exudate cells by using a microcapillary tube assay. Peak MIF activity was found in a Sephadex G-100 fraction containing molecules the size of albumin and slightly smaller, molecular weight 67,000 to 48,000. Murine MIF was stable to heating at 56 degrees C for 30 min but lost its activity at 80 degrees C for 30 min. Incubation of G-100 fractions containing MIF with water insoluble chymotrypsin destroyed the activity of MIF, indicating its protein nature. CsCl density gradient centrifugation revealed that murine MIF had a buoyand density greater than protein, consistent with its being a glycoprotein. Further, when subjected to disc electrophoresis on polyacylamide gels, murine MIF migrated in a region cathodal to albumin. Thus, mitogen stimulation of murine spleen cells produced MIF in quantities which allowed its partial characterization and purification, and its comparison with human and guinea pig MIF; this makes it feasible to analyze the role of murine MIF in cellular immunity and in its relationship to lymphocyte mediators which regulate humoral immune responses.     

Histamine-induced suppressor factor (HSF): effect on migration inhibitory factor (MIF) production and proliferation.

Histamine added in vitro to cultures of sensitized lymphocytes suppresses antigen-induced production of migration inhibitory factor (MIF) and proliferation by these cells. Recent studies have suggested that lymphocytes bearing histamine type-2 receptors play a regulatory role in these in vitro responses. The present studies were undertaken to determine if suppressor function by cells having histamine receptors was mediated through a soluble product. It was found that lymph node cells from nonimmune or immune strain 2 guinea pigs elaborate a nondialyzable factor into the culture supernatant when incubated with 10(-3) to 10(-5) M histamine (histamine-induced suppressor factor of HSF). HSF, when cocultured with sensitized lymphocytes, suppressed their MIF and proliferative responses to antigen. HSF was made by lymphocytes but not macrophages. Its production could be blocked by an H2 receptor antagonist (burimamide) but not an H1 receptor antagonist (chlorpheniramine). Furthermore, the inhibitory effect of HSF was reversible as lymphocytes washed free of the factor after 24 hr and recultured with fresh medium and antigen were able to produce MIF. Gel filtration by Sephadex G-100 chromatography indicated that HSF had an approximate m.w. of 23,000 to 40,000. These results suggest that the release of histamine at the sites of immediate hypersensitivity reactions, possibly by generating HSF activity, may play a regulatory role in the subsequent development of cellular-immune reactions at the same site.     

Association between high expression macrophage migration inhibitory factor (MIF) alleles and West Nile virus encephalitis

Infection with mosquito-borne West Nile virus (WNV) is usually asymptomatic but can lead to severe WNV encephalitis. The innate cytokine, macrophage migration inhibitory factor (MIF), is elevated in patients with WNV encephalitis and promotes viral neuroinvasion and mortality in animal models. In a case-control study, we examined functional polymorphisms in the MIF locus in a cohort of 454 North American patients with neuroinvasive WNV disease and found patients homozygous for high-expression MIF alleles to be >20-fold (p = 0.008) more likely to have WNV encephalitis. These data indicate that MIF is an important determinant of severity of WNV neuropathogenesis and may be a therapeutic target.     

Macrophage migration inhibitory factor (MIF) promotes fibroblast migration in scratch-wounded monolayers in vitro

MIF was recently redefined as an inflammatory cytokine, which functions as a critical mediator of diseases such as septic shock, rheumatoid arthritis, atherosclerosis, and cancer. MIF also regulates wound healing processes. Given that fibroblast migration is a central event in wound healing and that MIF was recently demonstrated to promote leukocyte migration through an interaction with G-protein-coupled receptors, we investigated the effect of MIF on fibroblast migration in wounded monolayers in vitro. Transient but not permanent exposure of primary mouse or human fibroblasts with MIF significantly promoted wound closure, a response that encompassed both a proliferative and a pro-migratory component. Importantly, MIF-induced fibroblast activation was accompanied by an induction of calcium signalling, whereas chronic exposure with MIF down-regulated the calcium transient, suggesting receptor desensitization as the underlying mechanism.     

Pathogenic bacteria and TNF do not induce production of macrophage migration inhibitory factor (MIF) by human monocytes

Elevated serum macrophage migration inhibitory factor (MIF) is associated with severe sepsis, but it is not clear whether bacteria stimulate synthesis of MIF by blood leukocytes directly or via induction of TNF. Here we assess production of MIF mRNA and protein by blood leukocytes from healthy human subjects (n = 28) following exposure to bacteria commonly associated with sepsis (Escherichia coli and Streptococcus pneumoniae). Bacteria did not increase levels of MIF mRNA or secreted protein. CD14⁺ monocytes were the main cell type producing MIF before and after stimulation. Exposure of leukocytes to TNF did not induce MIF. Hence elevated levels of serum MIF observed in sepsis may not reflect MIF produced by blood leukocytes stimulated directly by bacteria or TNF.     

The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction.

BACKGROUND: Recent studies of melanin biosynthesis have uncovered an unusual enzymatic activity which converts the non-naturally occurring D-isomer of 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) into 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The aim of the present investigation was to isolate and characterize the enzyme catalyzing this tautomerization reaction. MATERIALS AND METHODS: After we performed a tissue survey of D-dopachrome tautomerase activity, 10 bovine lenses were homogenized and used as a source of enzyme. A soluble fraction was obtained by high-speed centrifugation and subjected to successive FPLC chromatography on Phenyl-sepharose, Mono S cation-exchange, and Superdex gel-filtration. The isolated enzyme was electrophoresed, blotted onto PVDF membrane, and the N terminus analyzed by gas phase micro-sequencing. RESULTS: The protein catalyzing the conversion of D-dopachrome to DHICA was purified to homogeneity in 14% yield and showed a molecular weight of 12 kD when analyzed by SDS-PAGE. The first 27 amino acid residues of this protein were sequenced and found to be identical with those of bovine macrophage migration inhibitory factor (MIF). The catalytic activity of native MIF was confirmed by studies of purified recombinant human MIF, which showed the same tautomerase activity. While L-dopachrome was not a substrate for this reaction, the methyl esters of the L- and D-isomers were found to be better substrates for MIF than D-dopachrome. CONCLUSIONS: MIF has been described recently to be an anterior pituitary hormone and to be released from immune cells stimulated by low concentrations of glucocorticoids. Once secreted, MIF acts to control, or counter-regulate, the immunosuppressive effects of glucocorticoids on the immune system. Although the tested substrate, D-dopachrome, does not occur naturally, the observation that MIF has tautomerase activity suggests that MIF may mediate its biological effects by an enzymatic reaction. These data also offer a potential approach for the design of small molecule pharmacological inhibitors of MIF that may modulate its potent immunoregulatory effects in vivo.     

Macrophage migration inhibitory factor (MIF) acetylation protects neurons from ischemic injury

Ischemia-induced neuronal death leads to serious lifelong neurological deficits in ischemic stroke patients. Histone deacetylase 6 (HDAC6) is a promising target for neuroprotection in many neurological disorders, including ischemic stroke. However, the mechanism by which HDAC6 inhibition protects neurons after ischemic stroke remains unclear. Here, we discovered that genetic ablation or pharmacological inhibition of HDAC6 reduced brain injury after ischemic stroke by increasing macrophage migration inhibitory factor (MIF) acetylation. Mass spectrum analysis and biochemical results revealed that HDAC6 inhibitor or aspirin treatment promoted MIF acetylation on the K78 residue. MIF K78 acetylation suppressed the interaction between MIF and AIF, which impaired MIF translocation to the nucleus in ischemic cortical neurons. Moreover, neuronal DNA fragmentation and neuronal death were impaired in the cortex after ischemia in MIF K78Q mutant mice. Our results indicate that the neuroprotective effect of HDAC6 inhibition and aspirin treatment results from MIF K78 acetylation; thus, MIF K78 acetylation may be a therapeutic target for ischemic stroke and other neurological diseases.Subject terms: Cell death in the nervous system, Stroke