Regulation of Toll-like receptor 4 expression in mouse colon by macrophage migration inhibitory factor

Recent studies have indicated that macrophage migration inhibitory factor (MIF) and Toll-like receptor (TLR) play an important role in the regulation of innate immune responses. In this study, we investigated the effect of MIF on the expression of TLR4, a receptor that recognizes lipopolysaccharide, in colon using MIF-deficient mice. TLR4 mRNA expression in the colon tissues was determined by northern blot analysis. Western blot analysis and immunohistochemistry in the colon tissues were performed to evaluate the expression of TLR4 protein. The expressions of TLR4 mRNA and protein were remarkably down-regulated in colon tissues of MIF-deficient mice compared with wild-type mice and up-regulated by treatment with recombinant MIF. Immunohistochemical study revealed the presence of TLR4–positive staining in mononuclear cells in the lamina propria and intraepithelial mononuclear cells as well as weak staining in epithelial cells and crypts in colon tissues of wild-type mice. In contrast, MIF-deficient mice did not show TLR4-positive staining in the colonic mucosa. In MIF-deficient mice injected with recombinant mouse MIF (rMIF), TLR4-positive staining cells were observed in colon tissues similar to the findings in wild-type mice. Administration of dextran sulfate sodium (DSS) up-regulated the expression of TLR4 in the colons of WT mice but not in those of MIF-deficient mice. Furthermore, pretreatment with rMIF up-regulated the expression of TLR4 in response to DSS in MIF-deficient mice. Our results suggest that MIF affects the expression of TLR4 in mouse colon under both normal and colitic conditions.An erratum to this article can be found at     

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.     

Re-examining the oligomerization state of macrophage migration inhibitory factor (MIF) in solution

The state of oligomerization of macrophage migration inhibitory factor (MIF, also known as glycosylation inhibiting factor, GIF) in solution has been variously reported as monomer, dimer, trimer, or mixtures of all three. Several crystal structures show MIF to be a trimer. Sedimentation velocity shows a recombinant human MIF sample is quite homogeneous, with 98% as a species with s(20,w)=3.07 S and D(20,w)=8.29 x 10(-7) cm(2)/s. Using the partial specific volume calculated from the amino acid composition these values imply a mass of 33.56 kDa, well above that of dimer, but also 9% below the trimer mass of 37.035 kDa. Sedimentation equilibrium data at loading concentrations from 0.01 to 1 mg/ml show unequivocally that the self-association is extremely tight. However, the apparent mass is 33.53 kDa [95% confidence 33.25-33.82], again 9% below that expected for 100% trimer. To examine the possibility this protein has an unusual partial specific volume, sedimentation equilibrium was also done in H(2)O/D(2)O mixtures, giving 0.765+/-0.017 ml/g rather than the calculated 0.735 ml/g. With this revised partial specific volume, the equilibrium and velocity data each give M=37.9+/-2.8 kDa, fully consistent with a strongly-associated trimeric quaternary structure.     

The expression of macrophage migration inhibitory factor in the non-small cell lung cancer

ObjectiveThe objective was to investigate the expression of macrophage migration inhibitory factor (MIF) in non-small cell lung cancer (NSCLC), as well as the effects of macrophage MIF on tumor cells.MethodsThe human NSCLC cell strains H358 and H524 were selected as research objects. The Real-Time Polymerase Chain Reaction (RT-PCR) and Western Blot were utilized to detect the expression levels of MIF in human NSCLC cell strains. The lentiviral plasmid was utilized for MIF-mRNA interference. The expression levels of MIF before and after transfection were compared. The cell strains were cultured and proliferated for cell count and comparison.ResultsH358 showed MIF high expression while H524 showed MIF low expression. Once the H358 cells were constructed as silent MIF expression, compared with the original H358 cells, the difference was statistically significant. Once the H524 cells were constructed as high MIF expression, compared with original H524 cells, the difference was statistically significant. Being cultured for respective 3, 5, and 7 days, the transfected H358 cells showed a significant decrease in proliferative activity compared with original H358 cells, while the transfected H524 cells showed a significant increase in proliferative activity compared with original H524 cells.ConclusionMIF has high expression in H358 cells while low expression in H524 cells. The expression of MIF could enhance the proliferative activity of NSCLC tumor cells.     

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.     

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.     

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.     

Dual regulation of macrophage migration inhibitory factor (MIF) expression in hypoxia by CREB and HIF-1

Macrophage migration inhibitory factor (MIF) is a well-described pro-inflammatory mediator that has also been implicated in the process of oncogenic transformation and tumor progression. However, despite the compelling evidence that MIF is overexpressed in, and contributes to, the pathology of inflammatory and malignant diseases the mechanisms that contribute to exaggerated expression of MIF have been poorly described. Here we show that hypoxia, and specifically HIF-1alpha, is a potent and rapid inducer of MIF expression. In addition, we demonstrate that hypoxia-induced MIF expression is dependent upon a HRE in the 5'UTR of the MIF gene but is further modulated by CREB expression. We propose a model where hypoxia-induced MIF expression is driven by HIF-1 but amplified by hypoxia-induced degradation of CREB. Given the importance of MIF in inflammatory and malignant diseases these data reveal a HIF-1-mediated pathway as a potential therapeutic target for suppression of MIF expression in hypoxic tissues.     

High expression of macrophage migration inhibitory factor in the synovial tissues of rheumatoid joints

Macrophage migration inhibitory factor (MIF) plays an important role in inflammation and immunity via autocrine/paracrine and endocrine routes. We examined the presence of MIF in the synovial fluids of rheumatoid arthritis (RA) patients. The content of MIF in the synovial fluid was quantitated by enzyme-linked immunosorbent assay which revealed that the concentration of MIF for RA patients was 85. 7+/-35.2 ng/ml (mean+/-SD) (n=25). In comparison, the concentrations for osteoarthritis patients and normal volunteers were 19.5+/-5.3 ng/ml (n=12) and 10.4+/-1.1 ng/ml (n=5), respectively. The expression of MIF mRNA and presence of MIF protein in the synovial tissues of RA were demonstrated by Northern blot and Western blot analyses, respectively. Immunohistochemical analysis revealed that positive staining was largely observed in the cytoplasm of infiltrating T lymphocytes, which might be the major source of MIF detected in the synovial fluids. The pathophysiological role of MIF in RA remains to be elucidated; however, the present results for the first time suggest the possibility that MIF is involved in the potentiation of inflammatory and immunological responses in rheumatoid joints.     

Interference of macrophage migration inhibitory factor expression in a mouse melanoma inhibits tumor establishment by up-regulating thrombospondin-1

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with proinflammatory, proangiogenic, and protumorigenic properties. The molecular mechanisms underlying the role of MIF in tumorigenesis and angiogenesis are not well understood. To address these roles, an interfering MIF (iMIF) RNA was stably introduced into the B16-F10 mouse melanoma cell line, reducing MIF mRNA expression 1.6-fold and MIF protein expression 2.8-fold relative to control cells. When iMIF cells were subcutaneously injected into C57BL/6 mice, tumor establishment was significantly delayed and there was a marked absence of intratumoral vasculature in iMIF tumors relative to controls. A comparative gene expression analysis of iMIF and control melanoma cell lines revealed that thrombospondin-1 (TSP-1) mRNA expression was up-regulated 88-fold in the iMIF cells by real-time PCR. A 2-fold increase in TSP-1 protein levels was observed in iMIF cell culture supernatants. These results strongly suggest that the delayed tumor establishment and reduced vasculature in iMIF melanomas are linked to the up-regulation of the antiangiogenic TSP-1. They further define a novel function of MIF as a regulator of TSP-1 in a mouse melanoma model.     

Involvement of macrophage migration inhibitory factor (MIF) in experimental uric acid nephropathy

BACKGROUND: Deposition of uric acid in the kidney can lead to progressive tubulointerstitial injury with granuloma formation. We hypothesized that uric acid crystal deposition may induce granuloma formation by stimulating local expression of macrophage migration inhibitory factor (MIF), which is a known mediator of delayed type hypersensitivity (DTH). MATERIALS AND METHODS: A model of acute uric acid nephropathy was induced in rats by the administration of oxonic acid (an inhibitor of uricase), together with uric acid supplements. MIF expression and local cellular response were examined by in situ hybridization and immunohistochemistry. RESULTS: Kidney tissue examined at 35 days posttreatment showed widespread tubulointerstitial damage with intratubular uric acid crystal deposition and granuloma formation. Tubules within the areas of granuloma showed a six-fold increase in MIF mRNA, compared with uninvolved areas by in situ hybridization. Moreover, the areas of increased MIF mRNA expression correlated with sites of dense accumulation of macrophages and T cells, and these cells were activated when assessed by the expression of interleukin-2R (IL-2R) and (MHC) class II. Interestingly, cytoplasmic staining for MIF protein in the uric acid (UA) crystal-associated granulomatous lesions was reduced, indicating a rapid MIF secretion by damaged tubules and macrophages secondary to uric acid crystal stimulation. This was confirmed by the demonstration of a marked increase in urinary MIF protein by Western blot analysis. Control rats fed either a normal diet or only oxonic acid had no discernible evidence of renal disease by routine light microscopy and minimal tubular expression of MIF mRNA and protein. CONCLUSIONS: These data suggest that intrarenal granulomas in urate nephropathy may be the consequence of a crystal induced DTH reaction mediated by MIF.     

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.