Macrophage migration inhibitory factor (MIF) is induced by cytotoxic drugs and is involved in immune escape and migration in childhood rhabdomyosarcoma.

Macrophage migration inhibitory factor (MIF) is known to be involved in oncogenic transformation, tumour progression, and immunosuppression and is overexpressed in many solid tumours, including paediatric rhabdomyosarcoma (RMS). We investigated the function of MIF in RMS during treatment with cytotoxic drugs. RMS cell lines were analysed by flow cytometry, immunofluorescence staining, and ELISA. We demonstrated the overexpression of MIF in RMS cells and the enhanced expression and secretion after treatment with cytotoxic agents. Migration assays of RMS cells revealed that inhibitors of MIF (ISO-1, Ant.III 4-IPP, Ant.V, sulforaphane (SF)) and blocking antibodies caused reduced migration, indicating a role for MIF in metastatic invasion. Additionally, we investigated the function of MIF in immune escape. The development of a population containing immunosuppressive myeloid-derived suppressor cells was promoted by incubation in conditioned medium of RMS cells comprising MIF and was reversed by MIF inhibitors but not by antibodies. Although most inhibitors may restore immune activity, Ant.III and 10 µM SF disturbed T cell proliferation in a CFSE assay, whereas T cell proliferation was not reduced by 3 µM SF, ISO-1 or antibodies. However, the inhibition of MIF by blocking antibodies did not increase the killing activity of allogenic PBMCs co-cultured with RMS cells. Our results reveal that MIF may be involved in an immune escape mechanism and demonstrate the involvement of MIF in immunogenic cell death during treatment with cytotoxic drugs. Targeting MIF may contribute to the restoration of immune sensitivity and the control of migration and metastatic invasion.

Susceptibility to Toxoplasma gondii proliferation in BeWo human trophoblast cells is dose-dependent of macrophage migration inhibitory factor (MIF), via ERK1/2 phosphorylation and prostaglandin E2 production.

INTRODUCTION: Macrophage migration inhibitory factor (MIF) participates in the immune response to Toxoplasma gondii, triggers ERK1/2 and prostaglandin E2 (PGE2) activation, but there is limited information on these mechanisms in human trophoblast. The present study aimed to verify the role of MIF in the ERK1/2 phosphorylation and PGE2 production, as well as its effect on the susceptibility to T. gondii in BeWo cells. METHODS: BeWo cells were treated with increasing concentrations of recombinant MIF (rMIF) and/or T. gondii-soluble tachyzoite antigen (STAg) and analyzed for ERK1/2 phosphorylation and PGE2 production by Western blotting and ELISA, respectively. Cells were also treated with increasing concentrations of rMIF, rPGE2, or ERK1/2 inhibitor and tested for T. gondii proliferation. The supernatants of cells treated with rPGE2 were assayed for cytokine production by ELISA or CBA. RESULTS: ERK1/2 phosphorylation and PGE2 production increased when the cells were treated with low MIF concentrations while the parasitism control occurred only at high MIF concentrations. STAg was unable to change ERK1/2 phosphorylation or PGE2 release. BeWo cells demonstrated increased T. gondii proliferation and reduced production of pro-inflammatory cytokines when treated with PGE2, while PD98059 diminished the parasite proliferation. DISCUSSION: The intracellular mechanisms triggered by MIF are dose-dependent in BeWo cells, and PGE2 is an important factor for the persistence of T. gondii at the maternal fetal interface. CONCLUSION: MIF was unable to control T. gondii infection in BeWo cells at low concentrations since ERK1/2 and PGE2 expression were activated, demonstrating a critical effect of these mediators favoring parasite proliferation.

Macrophage migration inhibitory factor deficiency augments doxorubicin-induced cardiomyopathy.

BACKGROUND: Recent evidence has depicted a role of macrophage migration inhibitory factor (MIF) in cardiac homeostasis under pathological conditions. This study was designed to evaluate the role of MIF in doxorubicin-induced cardiomyopathy and the underlying mechanism involved with a focus on autophagy. METHODS AND RESULTS: Wild-type (WT) and MIF knockout (MIF(-/-)) mice were given saline or doxorubicin (20 mg/kg cumulative, i.p.). A cohort of WT and MIF(-/-) mice was given rapamycin (6 mg/kg, i.p.) with or without bafilomycin A1 (BafA1, 3 µmol/kg per day, i.p.) for 1 week prior to doxorubicin challenge. To consolidate a role for MIF in the maintenance of cardiac homeostasis following doxorubicin challenge, recombinant mouse MIF (rmMIF) was given to MIF(-/-) mice challenged with or without doxorubicin. Echocardiographic, cardiomyocyte function, and intracellular Ca(2+) handling were evaluated. Autophagy and apoptosis were examined. Mitochondrial morphology and function were examined using transmission electron microscopy, JC-1 staining, MitoSOX Red fluorescence, and mitochondrial respiration complex assay. DHE staining was used to evaluate reactive oxygen species (ROS) generation. MIF knockout exacerbated doxorubicin-induced mortality and cardiomyopathy (compromised fractional shortening, cardiomyocyte and mitochondrial function, apoptosis, and ROS generation). These detrimental effects of doxorubicin were accompanied by defective autophagolysosome formation, the effect of which was exacerbated by MIF knockout. Rapamycin pretreatment rescued doxorubicin-induced cardiomyopathy in WT and MIF(-/-) mice. Blocking autophagolysosome formation using BafA1 negated the cardioprotective effect of rapamycin and rmMIF. CONCLUSIONS: Our data suggest that MIF serves as an indispensable cardioprotective factor against doxorubicin-induced cardiomyopathy with an underlying mechanism through facilitating autophagolysosome formation.

Macrophage migration inhibitory factor mediates the antidepressant actions of voluntary exercise.

Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif(-/-) mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain.

[Multidirectional effect of MIF and sodium polyprenyl phosphate on the course of experimental flavivirus infection in mice].

AIM: Study of macrophage migration inhibiting factor (MIF) effect after intracerebral administration on the course of experimental infection induced in mice by tick borne encephalitis virus (TEV), and study of sodium polyprenyl phosphate (PPP) and/or antibodies against MIF on the course of this infection against the background of MIF administration. MATERIALS AND METHODS: Phosprenil preparation was used as a source of PPP. PPP was administered intracerebrally. MIF--human recombinant (R&D, USA), mice--Balb/c line. RESULTS: In the sera of mice infected with TEV, MIF production stimulation was detected at days 8 through 10 after the infection--against the background of clinical signs presentation of tick borne encephalitis (TE). Administration of PPP to infected mice, on the contrary, resulted in MIF production suppression at the specified period. After administration of 20 ng of MIF to mice, lethality increased by 40% and average life span decreased by 2.3 days. Thus, MIF at high doses caused an increase of infection course severity, induced by TEV in mice, and administration of 60 microg of PPP resulted in the protection from infection in 100% of cases. Intracerebral administrationto mice of antibodies against MIF resulted in a decrease of lethality indicator up to 26% as compared with control and an increase of averagelife span by 5.5 days. During simultaneous administration into the brain of infected mice of MIF, PPP and antibodies against MIF, prevention of MIF-induced increase of TE course severity was registered. CONCLUSION: The data obtained allow to conclude that MIF may serve as an indicator of TE course severity, and possible prognostic indicator of meningo-encephalitic form development in humans.

Role of macrophage migration inhibitory factor in glucocorticoid release and glucocorticoid receptor function in rats.

Experimental evidence indicates that macrophage migration inhibitory factor (MIF) plays a pathological role in the development of glucocorticoid resistance in sepsis patients. However, the impact of MIF on glucocorticoid release and the functions of the glucocorticoid receptor (GR) have not been studied extensively. In this study, exogenous administration of recombinant mouse macrophage migration inhibitory factor (rMIF), at a dose close to the levels found in the blood of sepsis patients, significantly enhanced serum corticosterone concentration but neither altered circulatory function nor stimulated nitric oxide (NO) release in rats. Incubation of cultured rat cardiomyocytes with 10 ng/ml and 20 ng/ml of rMIF did not cause changes in GR protein expression compared to control values but significantly decreased the expression of heat shock protein 90. GR specific binding activity was significantly inhibited by incubation of rat hepatocytes with 20 ng/ml rMIF alone or with 100 µmol/L L-NAME (a NO synthase inhibitor), and no significant difference was found between the two groups. These results suggest that elevated MIF levels in sepsis patients, at least in part, play an important role in the development of glucocorticoid resistance and that the NO signaling pathway is not involved in this pathological process.

Amelioration of intestinal colitis by macrophage migration inhibitory factor isolated from intestinal parasites through toll-like receptor 2.

In a previous study, we cloned type II MIFs (As-MIF) from Anisakis simplex 3rd stage larva and expressed a recombinant protein that suppressed allergic airway inflammation via regulatory T (CD4(+) CD25(+) Foxp3(+) T; T(reg) )-cell recruitment. In this study, in an effort to evaluate the function of rAs-MIF on another immune disease, we induced intestinal inflammation in mice using dextran sodium sulphate (DSS) with or without the application of rAs-MIF treatment to the mice. As a consequence, weight losses were recovered, and the value of disease activity index (DAI) was reduced by rAs-MIF treatment during the experimental period. The levels of TGF-ß and IL-10 in the spleens and mesenteric lymph nodes (MLN) from the rAs-MIF-treated mice were higher, but the levels of IFN-γ, IL-6 and IL-13 were lower than those of the mice treated with DSS but not with rAs-MIF. Additionally, the T(reg) cells observed were greatly increased in the MLNs of the rAs-MIF-treated mice than those of mice not treated with rAs-MIF. The results of our in vitro experiments showed that the elevated IL-10 production induced by rAs-MIF was generated via toll-like receptor 2. In conclusion, rAs-MIF appears to ameliorate DSS-induced colitis and may prove useful as a therapeutic agent for the treatment of intestinal inflammatory disease.

[Experimental hemorrhagic stroke: the study of neuropeptides (MIF, selank) in the intraperitoneal injection].

The aim of this study was to examine Taftsin derivates--macrophage inhibitory factor (MIF, Thr-Lys-Pro) and heptapeptide selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) in the model of experimental intracerebral hemorrhage in rats. The double autologous blood injection in the basal nucleus was used as a model of intracerebral hemorrhage. Animals ware randomly divided into three groups--the control group (n = 5) was treated with saline, the second group (n = 5) was injected with MIF in dose 150 mkg/ kg/day, the third group (n = 5) received Selank in dose 300 mkg/kg/day. Intraperitoneal injection of peptides was used. Body weight assessment, neurological examination and brain MRI were performed in 24, 72 hours and 10 days after the hematoma formation. The effect of neuropeptides on the functional restoration in animals, in the absence of the effect on hematoma volume and perifocal edema, was found. The significant reduction of perifocal edema and hematoma volume was observed in the 10th day after the hematoma formation in all experimental groups (p < 0.05). Only the control group of animals showed the significant (p < 0.05) weight loss in the 3rd day after the operation. The rate of neurological deficit was different: the significant improvement assessed with Menzes and limb placing test scales was seen only in the groups treated with neuropeptides in the 10th day.

Modulation of microglial/macrophage activation by macrophage inhibitory factor (TKP) or tuftsin (TKPR) attenuates the disease course of experimental autoimmune encephalomyelitis.

BACKGROUND: Myelin Oligodendrocyte Glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used mouse model for multiple sclerosis (MS). During the of progression of EAE, microglia, the immunocompetent cells of the brain, become activated and accumulate around demyelinated lesions. Microglial activation is mediated by the extracellular protease tissue Plasminogen Activator (tPA), and mice lacking tPA display altered EAE progression. In this study, we have used pharmacological inhibitors and stimulators of microglial/macrophage activation to examine the temporal requirement for microglial activation in EAE progression and to determine whether such approaches might potentially be of therapeutic value. RESULTS: Intervention using the tripeptide macrophage/microglia inhibitory factor MIF (TKP) and the tetrapeptide macrophage/microglial stimulator tuftsin (TKPR) attenuated EAE symptoms and revealed that the timing of macrophage/microglial activation is critical for the clinical outcome of EAE. We show that the disease progression can potentially be manipulated favorably at early stages by altering the timing of microglial activation, which in turn alters the systemic immune response to favor upregulation of T helper cell 2 genes that promote recovery from EAE. CONCLUSION: Preventative and therapeutic modulation of macrophage/microglial activity significantly alters the outcome of EAE at symptomatic stages. Specific molecular targets have been identified that represent potential avenues of exploration for the treatment and prevention of MS.

A critical role for the host mediator macrophage migration inhibitory factor in the pathogenesis of malarial anemia.

The pathogenesis of malarial anemia is multifactorial, and the mechanisms responsible for its high mortality are poorly understood. Studies indicate that host mediators produced during malaria infection may suppress erythroid progenitor development (Miller, K.L., J.C. Schooley, K.L. Smith, B. Kullgren, L.J. Mahlmann, and P.H. Silverman. 1989. Exp. Hematol. 17:379-385; Yap, G.S., and M.M. Stevenson. 1991. Ann. NY Acad. Sci. 628:279-281). We describe an intrinsic role for macrophage migration inhibitory factor (MIF) in the development of the anemic complications and bone marrow suppression that are associated with malaria infection. At concentrations found in the circulation of malaria-infected patients, MIF suppressed erythropoietin-dependent erythroid colony formation. MIF synergized with tumor necrosis factor and gamma interferon, which are known antagonists of hematopoiesis, even when these cytokines were present in subinhibitory concentrations. MIF inhibited erythroid differentiation and hemoglobin production, and it antagonized the pattern of mitogen-activated protein kinase phosphorylation that normally occurs during erythroid progenitor differentiation. Infection of MIF knockout mice with Plasmodium chabaudi resulted in less severe anemia, improved erythroid progenitor development, and increased survival compared with wild-type controls. We also found that human mononuclear cells carrying highly expressed MIF alleles produced more MIF when stimulated with the malarial product hemozoin compared with cells carrying low expression MIF alleles. These data suggest that polymorphisms at the MIF locus may influence the levels of MIF produced in the innate response to malaria infection and the likelihood of anemic complications.

Macrophage migration inhibitory factor within the alveolar spaces induces changes in the heart during late experimental sepsis.

Respiratory dysfunction during sepsis is common. However, although lung function can often be adequately supported, death frequently results from cardiovascular collapse. Despite intense investigation, the mechanism underlying the myocardial dysfunction of sepsis remains unclear. Macrophage migration inhibitory factor (MIF), an important cytokine released in sepsis and the acute respiratory distress syndrome, is a known cardiac depressant. We hypothesized that MIF released from the lung results in myocardial dysfunction during sepsis. In murine models of polymicrobial sepsis, we demonstrate a significant increase in the lungs of total and lavagable MIF between 20 and 30 h post induction of sepsis. At 30 h post sepsis, the lungs released MIF into the pulmonary circulation, increasing the plasma concentration by up to 51% in a single pass. Exogenous MIF, instilled into the lungs, increased alveolar keratinocyte-derived chemokine (KC), Macrophage inflammatory protein-2 (MIP2), and tumor necrosis factor alpha (TNFalpha) at 3 h, and plasma KC and MIP2 at 6 h postinstillation. This was associated with an increase in p38 mitogen-activated protein kinase and c-Jun N-terminal kinase phosphorylation. Because changes in mitogen-activated protein kinase activation can lead to myocardial depression, these data suggest that MIF released from the lungs may be responsible, at least in part, for the cardiac dysfunction seen in the late stages of sepsis.

[Efficiency of treatment of uveitis with the drug superlymph].

Comparative analysis of the results of combination treatment using superlymph and the conventional therapy in 17 and 52 patients, respectively, demonstrated that the combination therapy showed a marked clinical and immunological effect. The use of superlymph in local therapy, unlike the conventional etiopathogenetic treatment, resulted in an earlier decrease and arrest of an inflammatory reaction, in recovery of visual functions (for 2-4 day). Superlymph was found to be well tolerated and to cause no subjective discomfort or complications. Its immunomodulating effect was to normalize the count of T-theophylline-sensitive lymphocytes and, accordingly, the immunoregulation index, the serum concentrations of serum IgG, IgA, particularly IgE, and the phagocytic function of neutrophils. Superlymph exerted a pronounced effect on the systemic and local cytokine status, by decreasing the hypersecretion of cytokines, the regulators of inflammation. The clinical and immunological effects and good tolerability of superlymph permit it to recommend for its wide use in therapy of inflammatory eye diseases.

Macrophage migration inhibitory factor is released from pituitary folliculo-stellate-like cells by endotoxin and dexamethasone and attenuates the steroid-induced inhibition of interleukin 6 release.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by peripheral immune cells and also by endocrine cells in the anterior pituitary gland. MIF exerts its proinflammatory actions in the host-defense system by blocking the inhibitory effects of glucocorticoids on the release of other proinflammatory cytokines (e.g. IL-1, IL-6, TNFalpha). Reports that pituitary folliculo-stellate (FS) cells share many characteristics with immune cells led us to propose that these cells may serve as an additional source of MIF in the pituitary and that pituitary-derived MIF may act in an autocrine or paracrine manner to modulate endotoxin-induced cytokine release from FS cells. In the present study we addressed this hypothesis by using 1) immunohistochemistry to localize MIF in primary pituitary tissue and 2) well-characterized FS (TtT/GF), corticotroph (AtT20), and macrophage/monocyte (RAW 264.7) cell lines to explore the effects of CRH, endotoxin, and dexamethasone on MIF release and to examine the effects of MIF on IL-6 release. Our immunohistochemical study showed that MIF is expressed in abundance in S100-positive FS cells and also in other pituitary cell types. All three cell lines expressed MIF protein and responded to endotoxin (10-1000 ng/ml, 24 h) and dexamethasone (100 pM to 10 nM, 24 h) with concentration-dependent increases in MIF release. CRH (10-100 nM) also stimulated MIF release from AtT20 cells but, unlike endotoxin and dexamethasone, it had no effect on MIF release from TtT/GF or RAW cells. Recombinant MIF did not affect the basal release of IL-6 from TtT/GF cells; however, it effectively reversed the inhibitory effects of dexamethasone (1 nM) on the endotoxin-induced release of IL-6 from these cells. The results suggest that the FS cells are both a source of and a target for MIF and raise the possibility that MIF serves as a paracrine/autocrine factor in the pituitary gland that contributes to the protective neuroendocrine response to endotoxin.

Protective effect on Leishmania major infection of migration inhibitory factor, TNF-alpha, and IFN-gamma administered orally via attenuated Salmonella typhimurium.

The genes encoding murine macrophage migration inhibitory factor (MIF), IL-2, IFN-gamma or TNF-alpha were cloned individually into an expression plasmid under the control of the inducible promoter nirB and transfected into the aroA- aroD- deletion mutant strain of Salmonella typhimurium (BRD509). These S. typhimurium derivatives (henceforward called constructs and termed GIDMIF, GIDIL2, GIDIFN and GIDTNF) expressed their respective cytokines in vitro under anaerobic conditions and stably colonized BALB/c mice up to 14 days after oral administration. The highly susceptible BALB/c mice that had received the constructs orally and that had been subsequently infected via the footpad with Leishmania major, developed significantly reduced disease compared with control mice administered the untransfected Salmonella strain (BRD509). Importantly, a combination of GIDMIF, GIDIFN, and GIDTNF administered orally after L. major infection was able to significantly limit lesion development and reduced parasite loads by up to three orders of magnitude. Spleen and lymph node cells of mice administered this combination expressed markedly higher levels of inducible nitric oxide synthase (iNOS) compared with those from mice receiving an equivalent dose of the control strain of Salmonella (BRD509). These data therefore demonstrate the feasibility of therapeutic treatment in an infectious disease model using cytokines delivered by attenuated Salmonella. The protective effect observed correlates with the induction of inducible nitric oxide synthase in vivo.

Defective monocyte leukotaxis in sarcoidosis: possible relationship to a plasma factor.

Monocyte leukotactic function was studied in 25 untreated patients with histologically confirmed sarcoidosis. Monocyte leukotactic responses were significantly depressed (P less than 0.001), most strikingly in patients with stage I disease; however, the severity of the leukotactic defect did not correlate with duration, activity, or extrathoracic dissemination of disease. Preincubation of normal monocytes in sarcoid plasma, but not normal plasma, resulted in partial inhibition of leukotactic responsiveness. The leukotactic inhibition was not reversed by washing the cells after preincubation or by subsequent exposure to normal plasma. The inhibitory activity, which was found in all sarcoid plasma samples, was nondialyzable, was heat stable, and could be localized to the 25 to 35 per cent saturated ammonium sulfate fraction of plasma. Lesser amounts of similar inhibitory activity were detected in comparable fractions of normal plasma. A highly significant correlation between monocyte leukotactic responses and plasma leukotactic inhibitory activity was found in patients with sarcoidosis, suggesting an important in vivo modulatory nole for this substance.