Impairment of macrophage migration inhibitory factor synthesis and macrophage migration in protein-malnourished mice

Weanling CD2F1 mice were fed isocaloric diets that were protein sufficient (PS; containing 27% casein) or protein deficient (PD; containing 8% casein). Weight measurements demonstrated that the growth of PD mice was significantly impaired, thus indicating that the PD diet induced protein malnutrition. The cellular immune responsiveness of these mice was assessed from Day 21 to Day 49 of the diet using, as indicators, in vitro production of migration inhibitory factor (MIF) by splenic lymphocytes and MIF responsiveness of peritoneal macrophages. PD lymphocytes, when stimulated with the polyclonal activator concanavalin A, produced significantly less MIF than did PS lymphocytes. The amount of MIF produced by PD lymphocytes, however, increased throughout the study, possibly indicating delayed maturation of MIF synthetic capacity in PD mice. Normal CD2F1 mouse macrophages were used for these assays. MIF responsiveness of PD and PS macrophages was not significantly different when assayed using MIF produced by normal CD2F1 mouse lymphocytes. As compared to that of PS macrophages, the migratory ability of PD macrophages decreased progressively throughout the study. This impaired migratory ability did not interfere with MIF responsiveness of PD macrophages.     

Receptor agonists of macrophage migration inhibitory factor

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also promotes AMPK activation with potential benefits for response to myocardial infarction and ischemia-reperfusion. Structure-based molecular design has led to the discovery of not only antagonists, but also the first agonists of MIF-CD74 binding. The compounds contain a triazole core that is readily assembled via Cu-catalyzed click chemistry. The agonist and antagonist behaviors were confirmed via study of MIF-dependent ERK1/2 phosphorylation in human fibroblasts.     

Chaperone-like activity of macrophage migration inhibitory factor

Macrophage migration inhibitory factor is a ubiquitous multifunctional cytokine having diverse immunological and neuroendocrine properties. Although this protein is known to be released into the circulation from the secretory granules of anterior pituitary or directly from immune cells as a consequence of stress, its participation in heat stress-induced aggregation of proteins has not yet been reported. We provide here the first evidence that the macrophage migration inhibitory factor possesses chaperone-like properties. It was shown to exist in the form of a mixture of low and high molecular weight oligomers. At heat stress temperatures the large oligomers dissociate into monomers that bind and stabilize thermally denatured malate dehydrogenase and glycogen phosphorylase b and thus prevent aggregation of the model proteins. Similar chaperone-like effects were also observed in the presence of partially purified brain extract containing besides the macrophage migration inhibitory factor a number of ubiquitous hydrophobic low molecular weight proteins identified by N-terminal microsequence analysis. Being highly stable and hydrophobic, the macrophage migration inhibitory factor in combination with other proteins of similar properties may comprise a family of constitutively expressed "small chaperones" that counteract the early onset of stress, around physiological conditions, when heat shock proteins are not abundant.     

Regulation of human lung adenocarcinoma cell migration and invasion by macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is expressed and secreted in response to mitogens and integrin-dependent cell adhesion. Once released, autocrine MIF promotes the activation of RhoA GTPase leading to cell cycle progression in rodent fibroblasts. We now report that small interfering RNA-mediated knockdown of MIF and MIF small molecule antagonism results in a greater than 90% loss of both the migratory and invasive potential of human lung adenocarcinoma cells. Correlating with these phenotypes is a substantial reduction in steady state as well as serum-induced effector binding activity of the Rho GTPase family member, Rac1, in MIF-deficient cells. Conversely, MIF overexpression by adenovirus in human lung adenocarcinoma cells induces a dramatic enhancement of cell migration, and co-expression of a dominant interfering mutant of Rac1 (Rac1(N17)) completely abrogates this effect. Finally, our results indicate that MIF depletion results in defective partitioning of Rac1 to caveolin-containing membrane microdomains, raising the possibility that MIF promotes Rac1 activity and subsequent tumor cell motility through lipid raft stabilization.     

Leukocyte migration in guinea pigs. II. Partial characterization of a leukocyte migration inhibitory factor distinct from macrophage migration inhibitory factor.

In this study we present data on the partial biological and biochemical characterization of guinea pig leukocyte migration inhibition factor (LIF) and migration inhibition factor (MIF). The results indicate that guinea pig LIF and MIF are distinct mediators of cellular immunity, in terms of indicator cells affected and molecular weight. This is in agreement with previous reports showing distinctions between human LIF and MIF. Partial characterization of guinea pig LIF suggested that it is a heat-stable protein of molecular weight 68,000–158,000 and does not contain terminal sialic acid groups.     

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.     

Benzisothiazolones as modulators of macrophage migration inhibitory factor

Substituted N-phenylbenzisothiazolones have been investigated as inhibitors of the tautomerase activity of the proinflammatory cytokine MIF (macrophage migration inhibitory factor). Numerous compounds were found to possess antagonist activity in the low micromolar range with the most potent being the 6-hydroxy analog 1w. Compound 1w and the p-cyano analog 1c were also shown to exhibit significant inhibition of the binding of MIF to its transmembrane receptor CD74. Consistently, both compounds were also found to retard the MIF-dependent phosphorylation of ERK1/2 in human synovial fibroblasts.     

Novel anti-inflammatory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in various infectious and non-infectious diseases. We have investigated the mechanism of anti-inflammatory activity of epoxyazadiradione, a limonoid purified from neem (Azadirachta indica) fruits, against MIF. Epoxyazadiradione inhibited the tautomerase activity of MIF of both human (huMIF) and malaria parasites (Plasmodium falciparum (PfMIF) and Plasmodium yoelii (PyMIF)) non-competitively in a reversible fashion (K(i), 2.11-5.23 μm). Epoxyazadiradione also significantly inhibited MIF (huMIF, PyMIF, and PfMIF)-mediated proinflammatory activities in RAW 264.7 cells. It prevented MIF-induced macrophage chemotactic migration, NF-κB translocation to the nucleus, up-regulation of inducible nitric-oxide synthase, and nitric oxide production in RAW 264.7 cells. Epoxyazadiradione not only exhibited anti-inflammatory activity in vitro but also in vivo. We tested the anti-inflammatory activity of epoxyazadiradione in vivo after co-administering LPS and MIF in mice to mimic the disease state of sepsis or bacterial infection. Epoxyazadiradione prevented the release of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α when LPS and PyMIF were co-administered to BALB/c mice. The molecular basis of interaction of epoxyazadiradione with MIFs was explored with the help of computational chemistry tools and a biological knowledgebase. Docking simulation indicated that the binding was highly specific and allosteric in nature. The well known MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) inhibited huMIF but not MIF of parasitic origin. In contrast, epoxyazadiradione inhibited both huMIF and plasmodial MIF, thus bearing an immense therapeutic potential against proinflammatory reactions induced by MIF of both malaria parasites and human.     

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.     

Expression of macrophage migration inhibitory factor in acute ischemic myocardial injury.

Macrophage migration inhibitory factor (MIF) is a key mediator in inflammatory or immune-mediated diseases, although its role in heart diseases is unknown. This study investigated the expression of MIF in the myocardium in the development of acute myocardial infarction (AMI). By use of immunohistochemistry, Western blotting, RT-PCR, and in situ hybridization, the gene and protein expression of MIF in the heart at 6 hr, 1 day, 3 days, 1 week, and 2 weeks after AMI was studied. In both normal and sham-operated rats, MIF mRNA and protein were expressed constitutively at low levels by the myocytes. By contrast, MIF mRNA was rapidly upregulated by the surviving myocytes in the infarcted region and, to a lesser extent, the non-infarcted region, accounting for a sevenfold increase at 6 hr after AMI (p<0.001). This was followed by a fourfold increase in MIF protein expression at day 1 after AMI (p<0.05). Macrophages were found accumulated in the infarcted region, being significant at day 1 (p<0.01) and progressive increased over the 2-week time course (p<0.01) in which MIF was found expressed in these cells. The results indicated that the infiltrating macrophages and myocytes were sources of MIF in the infarcted region. The latter cells became activated and involved in the amplification of inflammatory response in AMI. Therefore, upregulation of myocardial MIF may contribute to macrophage accumulation in the infarcted region and their pro-inflammatory role may participate in the myocyte damage seen in AMI.     

Histochemical analysis of macrophage migration inhibitory factor in psoriasis vulgaris

Psoriasis is a persistent cutaneous disease characterized by skin inflammation and infiltration of immunocytes such as lymphocytes and monocytes/macrophages, concomitant with abnormal epidermal hyperproliferation. We previously showed that the serum level of macrophage migration inhibitory factor (MIF) and its production by peripheral blood mononuclear cells of patients with psoriasis were closely correlated with the severity of clinical symptoms; however, the precise role of MIF in psoriatic epidermis remains to be clarified. The current study was carried out to elucidate the possible involvement of MIF in psoriasis, using immunohistochemistry and in situ hybridization. In contrast to elevated serum MIF in psoriasis, MIF-positive staining in the lesional psoriatic epidermis was significantly decreased, as demonstrated by immunohistochemical analysis using an anti-MIF antibody. Consistent with this finding, we found, by in situ hybridization, that MIF mRNA concomitantly decreased in the psoriatic lesions. Although the reason for the different MIF levels in the psoriatic epidermis and in the circulation remains unknown, it is hypothesized that MIF, a potential growth factor, might be decreased in psoriatic lesions to counterregulate the abnormal epidermal proliferation caused by dysregulation of cytokines and growth factors.     

Orthologs of macrophage migration inhibitory factor from parasitic nematodes

Chronic helminth infections are associated with modulation of host cellular immune responses, presumably to prolong parasite survival within the mammalian host. This phenomenon is attributed, at least in part, to the elaboration of parasite molecules, including orthologs of host cytokines and receptors, at the host-parasite interface. This review describes recent progress in the characterization of macrophage migration inhibitory factor (MIF) orthologs from parasitic nematodes. The roles of these molecules in parasite developmental biology and pathogenesis are discussed. Further knowledge of the species-specific activities and three-dimensional structures of human and parasitic nematode MIF molecules should make them ideal targets for drug- and/or vaccine-based strategies aimed at nematode disease control.     

Synthesis and bio-evaluation of human macrophage migration inhibitory factor inhibitor to develop anti-inflammatory agent

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is involved in the development of an array of inflammatory disorders including rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis and sepsis. The synthesis of MIF-inhibitor is a rationale approach to develop novel anti-inflammatory agent to treat multitude of inflammatory diseases. In this work, we have synthesized and evaluated MIF-inhibitory activity of a series of small molecules containing isoxazoline skeleton. Mode of binding of this inhibitor to human MIF (huMIF) was determined by docking studies. The synthesized molecules inhibit tautomerase activity of huMIF. The anti-inflammatory activity of the most active inhibitor, 4-((3-(4-hydroxy-3-methoxyphenyl)-4, 5-dihydroisoxazol-5-yl) methoxy) benzaldehyde (4b) was evaluated against huMIF-induced inflammation in a cellular model (RAW 264.7 cell). Compound 4b significantly inhibits huMIF-mediated NF-κB translocation to the nucleus, up-regulation of inducible nitric oxide synthase and nitric oxide production in RAW 264.7 cell which are the markers for inflammation. The compound 4b is not cytotoxic as evident from cell viability assay. Hence, the compound 4b has potential to be a novel anti-inflammatory agent.     

A Leishmania ortholog of macrophage migration inhibitory factor modulates host macrophage responses

Parasitic organisms have evolved specialized strategies to evade immune defense mechanisms. We describe herein an ortholog of the cytokine, macrophage migration inhibitory factor (MIF), which is produced by the obligate intracellular parasite, Leishmania major. The Leishmania MIF protein, Lm1740MIF, shows significant structural homology with human MIF as revealed by a high-resolution x-ray crystal structure (1.03 A). Differences between the two proteins in the N-terminal tautomerization site are evident, and we provide evidence for the selective, species-specific inhibition of MIF by small-molecule antagonists that target this site. Lm1740MIF shows significant binding interaction with the MIF receptor, CD74 (K(d) = 2.9 x 10(-8) M). Like its mammalian counterpart, Lm1740MIF induces ERK1/2 MAP kinase activation in a CD74-dependent manner and inhibits the activation-induced apoptosis of macrophages. The ability of Lm1740MIF to inhibit apoptosis may facilitate the persistence of Leishmania within the macrophage and contribute to its evasion from immune destruction.     

Growth inhibitory factor of human alveolar macrophage

The effect of human lung conditioned medium (HLCM) on the DNA synthesis in cultured human alveolar macrophages (HAM) was evaluated. The medium from human lung cultured for 3 days (3d-HLCM) promoted the DNA synthesis as well as the recombinant human GM-CSF does. On the other hand, that cultured for six days (6d-HLCM) did not promote the DNA synthesis but strongly suppressed GM-CSF induced DNA synthesis in HAM. This growth inhibitory effect was also observed when several macrophage like cell lines were cultured with 6d-HLCM. Partial purification of an inhibitory factor on gel permeation HPLC revealed two distinct peaks of activity with molecular weights of 38 kd and 110 kd.