Dissociation of human interferon-gamma-like activity from migration-inhibition factor

Supernatants harvested from concanavalin A-stimulated human peripheral mononuclear cells after 24 hr of incubation contain one interferon species similar to human interferon-gamma (IFN-γ) with a pI of 4.6–5.3 (first day pH 5 IFN-γ). In contrast, during the subsequent 24 hr of incubation two species with properties of IFN-γ are produced with pI of 3.6–4.0 (second day pH 4 IFN-γ) and 4.6–5.6 (second day pH 5 IFN-γ), respectively. First day pH 5 IFN-γ and second day pH 5 IFN-γ have been found to differ on the basis of trypsin sensitivity. This pattern of polymorphism is similar to the pattern previously described for human migration-inhibitory factor (MIF) which can be separated into first day pH 5 MIF, second day pH 3 MIF, and second day pH 5 MIF. However, IFN-γ-like species can be differentiated from MIF biochemically and antigenically. Fractions with second day pH 4 IFN-γ have no MIF activity and fractions with second day pH 3 MIF contain no IFN activity. In addition, first and second day pH 5 MIF, which also contain IFN-γ activity, can be separated from the latter by precipitation as well as neutralization with polyclonal and monoclonal anti-human MIF antibodies.     

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.     

Regulating effect of bone marrow cells on human T-lymphocyte function

A study was made of the regulatory effect of human bone marrow cells in two experimental systems: lymphocyte proliferation in response to PHA, and spontaneous and PHA-induced production of macrophage migration inhibition factor (MIF) by peripheral blood lymphocytes. It was shown that bone marrow cells inhibit the proliferative activity of stimulated peripheral blood lymphocytes and induced MIF production. The effect of bone marrow cells on spontaneous MIF production was found to be inconclusive.     

Glycolipid receptor for human migration inhibitory factor: fucose and sialic acid are important for the human monocyte response to migration inhibitory factor

The role of carbohydrate in the interaction of human migration inhibitory factor (MIF) with human peripheral blood monocytes was investigated by studying the effects of different exoglycosidases on the cellular response to MIF. When monocytes were pretreated with neuraminidase, an exoglycosidase specific for sialic acid, they became unresponsive to MIF. Other glycosidases, such as beta-galactosidase and alpha-mannosidase, were inactive in this respect. The effect of neuraminidase was reversible since the response to MIF was restored to normal levels after 24 hr. In parallel studies, a glycolipid-enriched extract from U937 cells, a human macrophage-like cell line, known to enhance the monocyte response to MIF, lost this activity when treated with neuraminidase and alpha-L-fucosidase, but not with beta-galactosidase. This suggests the importance of terminal sialic acid and fucose residues for the interaction between monocyte membrane glycolipids and MIF.     

Migration inhibitory factor (MIF) production by skin fibroblast cultures from patients with severe combined immunodeficiency

The supernatants of fibroblast cultures derived from skin biopsies of each of two patients with severe combined immunodeficiency were studied for the presence of migration inhibitory activity (MIF). The supernatants of both of these fibroblast cultures were found to contain inhibitory activity for the migration of cultured human lymphoid cells (PGLC-33H). This MIF activity was found to share chromatographic similarities with the MIF contained in the supernatants of a lymphoid cell line (PGLC-33H) and phytohemagglutinin (PHA) and tuberculin (PPD) stimulated human peripheral lymphocytes. These data suggest that MIF is not solely a lymphoid product and that severe combined immunodeficiency does not represent a gene deletion for MIF production.     

MIF-like activity of natural and recombinant human interferon-gamma and their neutralization by monoclonal antibody

By utilizing elutriation-purified human monocytes, we found that human interferon (IFN) inhibits monocyte migration in a manner similar to migration inhibitory factor (MIF) and does it without demonstrable cytotoxicity. We observed that human IFN-gamma is 10 to 300 times more potent in its MIF activity than is IFN-alpha and that monoclonal antibodies (MoAb) can be used to distinguish between them. Studies with recombinant IFN-gamma indicate that the migration inhibition seen with natural IFN-gamma is due to IFN-gamma itself and is not due to co-purification of another lymphokine with the natural IFN-gamma. Although interferons exhibit MIF activities, there are apparently other cytokines, without antiviral activity, that also have MIF activities. MIF from the lymphoblastoid cell line RPMI 1788 was not neutralized by MoAb to IFN. However, MIF activity in supernatant fluid from human peripheral blood lymphocyte cultures stimulated with Con A-Sepharose was completely neutralized with MoAb anti-IFN-gamma. These data indicate that MIF is really a family of cytokines that inhibit macrophage/monocyte migration and that the major portion of MIF activity associated with crude supernatant of mitogen-stimulated lymphocytes is due to IFN-gamma.     

New insights into the role and mechanism of macrophage migration inhibitory factor in steroid-resistant patients with systemic lupus erythematosus

Introduction

Glucocorticoid (GC) therapy remains important in improving the prognosis of patients with systemic lupus erythematosus (SLE). However, some patients do not achieve an effective response with GC treatment, creating an obstacle to the remission of SLE. Identification of the underlying mechanisms responsible for steroid resistance can be significant. Macrophage migration inhibitory factor (MIF) arouses our interest because of its reciprocal relationship with GCs. In the present study, we investigated for the first time whether MIF correlated with steroid resistance in SLE and explored potential mechanisms of action.

Methods

Sixty-two patients with SLE (40 steroid sensitive and 22 steroid resistant) and 21 normal controls were recruited. Serum levels of MIF were measured by ELISA. Cytosolic MIF and IκB expression in peripheral blood mononuclear cells (PBMCs) were determined by western blotting. The electrophoretic mobility shift assay was assessed by NF-κB in nuclear aliquots. Gene silencing was applied to reduce expression of MIF in PBMCs in steroid-resistant patients. PBMCs obtained from steroid-sensitive patients were treated with recombinant human MIF of different concentrations.

Results

MIF levels in serum and PBMCs were higher in steroid-resistant patients compared with steroid-sensitive patients and controls. In contrast to the steroid-sensitive group, NF-κB levels were significantly higher and IκB levels lower in steroid-resistant patients. After MIF gene silencing, IκB levels in cells from steroid-resistant patients were increased. In steroid-sensitive patients, a decrease in IκB levels and an increase in NF-κB expression from baseline were detected in PBMCs treated with a higher concentration of recombinant human MIF. Treatment with recombinant human MIF did not regulate expression of IκB and NF-κB in PBMCs from patients treated with an anti-MIF monoclonal antibody.

Conclusions

Our results indicated that MIF may play a role in the formation of steroid resistance in SLE by affecting the NF-κB/IκB signaling cascade. As a regulator of glucocorticoid sensitivity, MIF may be a potential target for steroid sparing.     

Glycolipid-dependent interaction between human migration-inhibitory factor and mononuclear phagocytes

It has been previously established in the guinea pig that the response of peritoneal macrophages to migration inhibitory factor (MIF) is enhanced by a macrophage glycolipid and that gangliosides reversibly bind MIF. This suggests that glycolipids function as cell surface receptors for MIF. In this report, it is demonstrated that the response of human peripheral blood monocytes to human MIF is augmented by preincubation of these cells with glycolipidenriched material extracted from the human macrophage-like cell line U937 or human peripheral blood monocytes and with a purified glycolipid from guinea pig peritoneal macrophages. In addition, a mixed ganglioside preparation from bovine brain shows the same effect. In contrast, the pure gangliosides, G_M1 and G_D1a, and glycolipids from the HL-60 cell line, which is a MIF-unresponsive cell line, were not able to enhance the response to human MIF. The specificity of enhancement by particular glycolipids could not be attributed to an increased uptake of only enhancing glycolipids since there was no significant difference between the association of monocytes with radioactive liposomes containing biologically active or inactive glycolipids. Pronase treatment did not affect the enhancing activity of the U937 glycolipidenriched material. Incubation of cells with glycolipids results in enhancement only if done at 37 °C and not at 4 °C. Therefore, the association of lipid with the monocyte surface appears to be dependent on temperature.Further evidence for the receptor nature of these enhancing glycolipids is provided by experiments involving affinity purification experiments. Coupling of bovine brain mixed gangliosides to agarose resulted in a matrix capable of reversibly binding MIF. G_D1a-agarose was inactive in this respect.     

Intervillous Macrophage Migration Inhibitory Factor Is Associated with Adverse Birth Outcomes in a Study Population in Central India

Macrophage migration inhibitory factor (MIF) is a pluripotent factor produced by a variety of cells. It plays an important biological role in the regulation of pregnancy and has been shown to influence malaria pathogenesis. In this study, the levels of MIF in the peripheral, cord and placental intervillous blood (IVB) plasma collected from women residing in a malaria endemic region of Central India was determined and its association with malaria in pregnancy and birth outcomes was investigated. MIF levels were significantly different in IVB, peripheral, and cord plasma, with IVB plasma having the highest MIF levels and peripheral plasma having the lowest. Placental malaria positive women had significantly higher IVB plasma MIF levels than placental malaria negative women, but this relationship was not seen in peripheral or cord plasma MIF levels. In addition, the odds of stillbirth and low birth weight deliveries for the uppermost placental MIF quartile (irrespective of placental malaria status) was significantly higher than that of the lowest placental MIF quartile, supporting the hypothesis that elevated concentrations of placental MIF may be associated with an increased risk of adverse birth outcome.     

Comparative assessment of the properties of a factor inhibiting macrophage migration and interferon]

The authors investigated some functional properties of interferon and the macrophages migration inhibitory factor (MIF) obtained by stimulation of human tonsil lymphocytes with the virus of Newcastle disease (NDV) or O-streptolysin. Both the interferon and MIF inhibited actively the migration of human tonsil cells, but differed by the anti-viral activity, and sensitivity to heating at 56 degrees C for 30 min. Stimulation of human tonsil lymphocytes with NDV leads to production of a more wide range of delayed hypersensitivity mediators than stimulation with O-streptolysin.     

High expression of macrophage migration inhibitory factor in human melanoma cells and its role in tumor cell growth and angiogenesis.

Macrophage migration inhibitory factor (MIF) is known to function as a cytokine, hormone, and glucocorticoid-induced immunoregulator. In this study, we reported for the first time that human melanocytes and melanoma cells express MIF mRNA and produce MIF protein. Immunohistochemical analysis demonstrated that MIF was mostly localized in the cytoplasm of melanocytes and G361 cells, a widely available human melanoma cell line. In particular, strong positive staining was observed at the dendrites of these cells. Expression of MIF mRNA and production of MIF protein were much higher in human melanoma cells such as G361, A375, and L32 than in normal cultured melanocytes. To assess the role of MIF overexpression in melanoma cells, G361 cells were transfected with an antisense human MIF plasmid. The results demonstrated that the cell growth rate of the transfected cells was markedly suppressed, suggesting that MIF participates in the mechanism of proliferation of melanoma cells. To further evaluate the function of MIF, we employed the Boyden chamber method to examine the effect on tumor cell migration and found that MIF enhanced the migration of G361 cells in a dose-dependent manner. Furthermore, we administered anti-MIF antibody into tumor (G361 cells in a Millipore chamber)-bearing mice to assess the effect on tumor-associated angiogenesis. The anti-MIF antibody significantly suppressed tumor-induced angiogenesis. Taken together, these results indicated that it is likely that MIF may function as a novel growth factor that stimulates incessant growth and invasion of melanoma concomitant with neovascularization.     

Macrophage migration inhibitory factor antagonizes hydrocortisone-induced increases in cytosolic IkappaBalpha

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine secreted by several cell types, including mononuclear and pituitary cells. It has also been shown to counteract cortisol-induced inhibition of inflammatory cytokine secretion. The purpose of this study was to determine whether MIF antagonized the effect of hydrocortisone on the NF-kappaB/IkappaB signal transduction pathway in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells. Physiological doses of hydrocortisone (50-200 ng/ml) diminished both the LPS-stimulated decrease in cytosolic IkappaBalpha levels and the subsequent increase in nuclear NF-kappaB DNA binding. In the presence of both LPS and hydrocortisone, 1 ng/ml of MIF antagonized the effects of hydrocortisone, resulting in decreased cytosolic IkappaBalpha levels (P < 0.05) and increased nuclear NF-kappaB DNA binding (P < 0.05). In the absence of hydrocortisone, MIF had no effect on LPS-induced decreases in IkappaBalpha. In the absence of LPS, MIF inhibited hydrocortisone-induced increases in IkappaBalpha (P = 0.03). Thus the mechanism by which MIF antagonizes the effect of hydrocortisone on the NF-kB/IkappaB signal transduction pathway is through inhibiting the ability of hydrocortisone to increase cytosolic IkappaBalpha.     

Enzymatically inactive macrophage migration inhibitory factor inhibits monocyte chemotaxis and random migration.

Macrophage migration inhibitory factor (MIF) is a cytokine that was first described as an inhibitor of the random migration of monocytes and macrophages and has since been proposed to have a number of immune and catalytic functions. One of the functions assigned to MIF is that of a tautomerase that interconverts the enol and keto forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate and converts D-dopachrome, a stereoisomer of naturally occurring L-dopachrome, to 5,6-dihydroxyindole-2-carboxylic acid. The physiological significance of the MIF enzymatic activity is unclear. The three-dimensional structure of MIF is strikingly similar to that of two microbial enzymes (4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase) that otherwise share little sequence identity with MIF. MIF and these two enzymes have an invariant N-terminal proline that serves as a catalytic base. Here we report a new biological function for MIF, as an inhibitor of monocyte chemoattractant protein 1- (MCP-1-) induced chemotaxis of human peripheral blood monocytes. We find that MIF inhibition of chemotaxis does not occur at the level of the CC chemokine receptor for MCP-1, CCR2, since MIF does not alter the binding of (125)I-MCP-1 to monocytes. The role of MIF enzymatic activity in inhibition of monocyte chemotaxis and random migration was studied with two MIF mutants in which the N-terminal proline was replaced with either a serine or a phenylalanine. Both mutants remain capable of inhibiting monocyte chemotaxis and random migration despite significantly reduced or no phenylpyruvate tautomerase activity. These data suggest that this enzymatic activity of MIF does not play a role in its migration inhibiting properties.     

Endogenous macrophage migration inhibitory factor modulates glucocorticoid sensitivity in macrophages via effects on MAP kinase phosphatase-1 and p38 MAP kinase

The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is induced by glucocorticoids (GCs), but it was not previously known if MIF regulates cellular sensitivity to GC. Here we show in GC and LPS-treated peritoneal macrophages derived from MIF-/- and wt mice that the absence of endogenous MIF is associated with increased sensitivity to GC of TNF release. This is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), concomitant decreased phosphorylation of p38 MAPK, but no effect of MIF on nuclear factor kappaB (NF-kappaB). These results demonstrate that MIF regulates GC sensitivity by phosphorylation of p38, and provides a cellular mechanism for this observation, indicating that MKP-1 is a central target of this regulation.     

Inhibition of macrophage migration by virus-induced interferon preparations.

It was found that a preparation of mouse L cell interferon induced by Newcastle disease virus (NDV) possessed not only interferon activity but also inhibitory activity upon migration of guinea pig peritoneal macrophages (MIF activity). These activities were also observed in a preparation of human leukocyte interferon induced by NDV. The interferon and MIF activities shared common characteristics in the dose response, time course of in vitro production, thermal stability, sensitivity to trypsin and periodate, and elution pattern in CM-Sephadex column chromatography. However, gel filtration pattern with Sephadex G-100 showed two separate peaks. Fractions collected from the first peak, corresponding to a molecular weight of about 45 000, had only the MIF activity, while those collected from the second peak, corresponding to a molecular weight of about 30 000, had both the interferon and MIF activities. A preparation of mouse brain interferon induced by Japanese encephalitis virus had a much weaker MIF activity than the L cell interferon, although these preparations were equal in interferon activity (5000 units/ml).     

Human lymphoid cell lines: Models for immunological analysis

Summary Investigations with human long term lymphoid cell lines have amply demonstrated the versatility of these tissue culture systems for the detection, definition, and solution of current problems in cell biology, biochemistry, genetics, and immunology. These systems are contributing much to our understanding of the multiple functions of lymphoid cells in the immune response. Human lymphoid cell lines produce, in large quantities, the putative extracellular mediators of cell-mediated immunity, including migration inhibitory factor (MIF), lymphotoxin, interferon, and a specific, reversible inhibitor of lymphocyte biosynthetic activity. The MIF released by human lymphoid cell lines is similar to that produced by phytomitogen- or antigen-stimulated human peripheral lymphocytes. Human lymphoid cells from lines producing MIF mimic the capillary migration patterns of guinea pig peritoneal macrophages, and are more sensitive than the guinea pig cells to human MIFs. Studies with these migrating cells indicate that MIF is not solely a lymphoid cell product, but is synthesized by a wide variety of activated cell types. Extracts of cultured human lymphoid cells inhibit the synthesis of RNA, protein, and DNA by established lymphoid cell lines and by phytomitogen-stimulated human peripheral lymphocytes, but have no inhibitory effects on human nonlymphoid cells. The reversible inhibition is produced with physiological quantities of extract, suggesting a functional immunoregulatory activity for this material in lymphocyte-mediated immunological reactions. Initial findings indicate that these mediators are multiple and distinct molecular species. The remarkable proliferative and synthetic potential of human lymphoid cell systems provides a most useful resource for the purification and characterization of these immunological substances. This invited paper was presented at the Hematopoietic Systems Sessions in Depth section of the 24th Annual Meeting of the Tissue Culture Association, Inc., Boston, June 4, 1973. The work was supported by Grants RO1-AI10422 and TO1-AI00445 from the National Institute of Allergy and Infectious Diseases and PO1-GM19443 from the National Institute of General Medical Sciences of the National Institute of Health, United States Public Health Service. Recipient of Research Career Development Award AI46371 from the National Institutes of Allergy and Infectious Diseases, National Institutes of Health.     

Production of migration-inhibitory factor by a human T-lymphoblast cell line

Migration-inhibitory-factor (MIF) activity was detected in culture supernatants of the human T-lymphoblast cell line Mo after stimulation with phytohemagglutinin and phorbol myristate acetate. MIF activity was not detected in unstimulated cultures reconstituted with phytohemagglutinin and phorbol myristate acetate. Conditioned medium from the cell line Mo was fractionated by Sephadex G-100 gel nitration. MIF-containing Sephadex fractions corresponding to a M_r, of 60,000 to 70,000 were further fractionated by isoelectrofocusing, resulting in a sharp peak of activity with a pI of 4.6 to 5.2. This MIF species constitutes a major form secreted by Mo cells; it adheres to Con A-Sepharose, is trypsin-resistant, and is denser than pure protein as determined by CsCl density gradient centrifugation. These are the same physicochemical characteristics previously established for second-day pH5-MIF from peripheral blood mononuclear cells (W. Y. Weiser et al., J. Immunol.126, 1958, 1981). In contrast, Sephadex fractions corresponding to larger molecules (M_r 70,000–90,000) contain at least two additional MIF species. These larger MIF forms have a pI of 3.0 to 3.5 and of 4.6 to 5.2 and lack affinity to Con A-Sepharose. Thus, the Mo T-cell line produces large quantities of at least three different species of human MIF.     

Novel derivatives of ISO-1 as potent inhibitors of MIF biological function

A series of novel 1,2,4-oxadiazole, phthalimide, amide and other derivatives of ISO-1 were synthesized and probed for inhibition of macrophage migration inhibitory factor (MIF) activity. Several compounds inhibited MIF enzymatic activity at levels better than ISO-1. Of note, compounds 7, 22, 23, 24, 25 and 27 inhibited the spontaneous secretion/release/recognition of MIF from freshly isolated human peripheral blood mononuclear cells and, more importantly, inhibited the MIF-induced production of interleukin-6 (IL-6) and/or interleukin-1β (IL-1β) significantly better than ISO-1.     

Production of migration inhibitory factor (MIF) by human leukocytes following exposure to Epstein-Barr virus.

The ability of Epstein-Barr virus (EBV) to induce a cell-mediated immune response (CMI) in cultures of human leukocytes was investigated. Partially purified EBV, obtained from culture fluids of AV-1 cells, was inactivated by uv-irradiation. Inactivated virus was mixed with peripheral leukocytes from Hodgkin's disease (HD), infectious mononucleosis (IM) and malignant lymphoma patients as well as from normal individuals in an in vitro culture system. Production of migration inhibitory factor (MIF), as measured by guinea pig macrophage migration inhibition (MMI), was utilized as an indicator of CMI response. Significant differences in MIF response were observed subsequent to exposure of the cells to EBV. Leukocytes from patients in each of the disease categories tested exhibited greater MIF production than did those from the normal controls. There were significant differences in MIF production by leukocytes from the malignant and non-malignant disease categories. Serum from each subject was examined for immunoglobulin specific for EBV capsid antigen (anti-VCA). Although the majority of individuals within the disease categories tested had elevated anti-EBV serum titers, no correlation could be made between elevated anti-VCA titer and levels of MIF production.