Antisense Macrophage Migration Inhibitory Factor Research Articles

Molecular function of macrophage migration inhibitory factor and a novel therapy for inflammatory bowel disease

Macrophage migration inhibitory factor (MIF) is a unique protein that participates in inflammation, immune responses, and cell growth. An array of in vitro and in vivo experiments has demonstrated that MIF is profoundly involved in the pathogenesis of acute and chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Blockade of MIF bioactivities by either neutralizing anti-MIF antibodies or antagonists prevents inflammatory cytokine cascade, which strongly suggests that an anti-MIF therapeutic strategy is feasible for treatment of IBD. Recently, we developed a new therapeutic approach for IBD by administration of antisense MIF oligonucleotides in conjugation with schizophyllan (SPG), a member of the glucan family. SPG specifically binds Dectin-1 expressed in antigen-presenting cells (APCs), and the antisense MIF/SPG complex is incorporated into the cells. In in vivo experiments of colitis models in mice, we found that intraperitoneal administration of the complex ameliorated the clinical signs of colitis and improved the histological scores. This novel therapy designed to knock down the MIF production in APCs is expected to be clinically applicable for the treatment of IBD.

A New Therapeutic Approach Using a Schizophyllan-based Drug Delivery System for Inflammatory Bowel Disease

Antisense technologies for the targeted inhibition of gene expression could provide an effective strategy for the suppression of inflammation. However, the effective use of antisense oligonucleotides (ODN) has been limited because of several problems. Therefore, a delivery system for antisense ODNs that enhances antisense stability, while maintaining the specificity of antisense for its target RNA or DNA is needed. We have developed a delivery system for antisense ODN using schizophyllan (SPG), a polysaccharide that belongs to the β-(1-3) glucan family. This system has several advantages enabling the effective suppression of targeted RNA or DNA: the SPG complex is stable in vivo and does not dissolve in the presence of deoxyribonuclease, and the SPG complex is effectively taken up into macrophages by phagocytosis through Dectin-1. Macrophage-migration inhibitory factor (MIF), which is mainly produced by macrophages has been shown to have a pathogenetic role in inflammatory bowel disease (IBD). We developed a technique to create an SPG complex that highly conformed to the antisense MIF. The administration of antisense MIF/SPG complex effectively suppressed MIF production and significantly ameliorated intestinal inflammation. Our result demonstrated a possible new therapeutic approach, i.e., the administration of antisense MIF/SPG complex, for the treatment of IBD.

Inhibition of tumor growth and metastasis in vitro and in vivo by targeting macrophage migration inhibitory factor in human neuroblastoma

Macrophage migration inhibitory factor (MIF) has been defined as a novel oncogene. Our previous results have shown that MIF may contribute to the progression of neuroblastoma by (a) inducing N-Myc expression and (b) upregulating the expression of angiogenic factors. The aim of this study was to test whether tumor growth could be inhibited by reduction of endogenous MIF expression in neuroblastoma and clarify the molecular mechanisms underlying MIF reduction on the control of neuroblastoma growth. We established human neuroblastoma cell lines stably expressing antisense MIF (AS-MIF) cDNA. These stable transfectants were characterized by cell proliferation, gene expression profile, tumorigenicity and metastasis in vitro and in vivo. Decreased MIF expression was observed after transfection with AS-MIF in neuroblastoma cells and downregulation of MIF expression significantly correlated with decreased expression of N-Myc, Ras, c-Met and TrkB at protein level. Affymetrix microarray analysis revealed that expression of IL-8 and c-met was inhibited and neuroblastoma-favorable genes such as EPHB6 and BLU were upregulated in MIF reduced cells. Neuroblastoma cell growth exhibited a nearly 80% reduction in AS-MIF transfectants in vitro. Furthermore, mice in which tumors formed after subcutaneous injection of AS-MIF transfectants showed a 90% reduction in tumor growth compared to control. Metastasis in mice was also suppressed dramatically. Our data demonstrate that targeting MIF expression is a promising therapeutic strategy in human neuroblastoma therapy, and also identifies the MIF target genes for further study.

Macrophage migration inhibitory factor promotes innate immune responses by suppressing glucocorticoid‐induced expression of mitogen‐activated protein kinase phosphatase‐1

The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) acts as a physiological counter-regulator of the immuno-suppressive effects of glucocorticoids. However, the mechanisms whereby MIF exerts its counter-balancing effect remain largely unknown. Here we report that MAPK phosphatase 1 (MKP-1), an archetypal member of dual specificity phosphatase that inactivates MAPK activity in response to pro-inflammatory stimuli, is a critical target of MIF-glucocorticoid crosstalk. Recombinant MIF counter-regulated in a dose-dependent fashion dexamethasone inhibition of TNF and IL-8 production by RAW 264.7 macrophages and U-937 promonocytes stimulated with lipopolysaccharides (LPS) or with LPS plus phorbol 12-myristate 13-acetate. Stimulation of RAW 264.7 macrophages with dexamethasone or dexamethasone plus LPS led to a robust up-regulation of MKP-1 mRNA and protein expressions that were counter-regulated by addition of recombinant MIF. Antisense MIF macrophages expressing reduced levels of endogenous MIF produced higher amount of MKP-1 and lower amount of TNF after exposure to dexamethasone and dexamethasone plus LPS, indicating that endogenous MIF acts in an autocrine fashion to override glucocorticoid-induced MKP-1 expression and inhibition of cytokine production. Taken together, these data identify MKP-1 as a molecular target of MIF-glucocorticoid crosstalk and provide a molecular basis for the control of macrophage responses by a pair of physiological regulators of innate immunity.

Macrophage migration inhibitory factor (MIF) regulates host responses to endotoxin through modulation of Toll-like receptor 4 (TLR4).

The cytokine macrophage migration inhibitory factor (MIF) has emerged recently as an important mediator of inflammation and innate immunity. MIF is rapidly released by macrophages after stimulation with microbial products and pro-inflammatory cytokines and, in turn, stimulates the production of pro-inflammatory mediators by immune cells. Immunoneutralization of MIF or deletion of the Mif gene was shown to protect animals from lethal endotoxemia, staphylococcal toxic shock and septic shock in experimental models of bacterial peritonitis. To investigate the function of MIF in innate immunity, we studied the response of macrophages expressing reduced levels of MIF to microbial products. These cells were generated by transduction of an antisense MIF adenovirus or by stable transfection with an antisense MIF plasmid or were obtained from MIF-knockout mice. MIF-deficient macrophages were shown to be hyporesponsive to stimulation with LPS and Gram-negative bacteria. The defect was associated with a down-regulation of Toll-like receptor 4 (TLR4), the signal transducing molecule of the LPS receptor complex. Immunoneutralization of extracellular MIF decreased TLR4 expression and responses of macrophages to LPS, indicating that MIF may exert autocrine effects. These findings identify an important role for MIF in innate immunity and provide a rationale for the development of anti-MIF strategy for the treatment of patients with Gram-negative septic shock.

Macrophage migration inhibitory factor (MIF) regulates host responses to endotoxin through modulation of Toll-like receptor 4 (TLR4)

The cytokine macrophage migration inhibitory factor (MIF) has emerged recently as an important mediator of inflammation and innate immunity. MIF is rapidly released by macrophages after stimulation with microbial products and pro-inflammatory cytokines and, in turn, stimulates the production of pro-inflammatory mediators by immune cells. Immunoneutralization of MIF or deletion of the Mif gene was shown to protect animals from lethal endotoxemia, staphylococcal toxic shock and septic shock in experimental models of bacterial peritonitis. To investigate the function of MIF in innate immunity, we studied the response of macrophages expressing reduced levels of MIF to microbial products. These cells were generated by transduction of an antisense MIF adenovirus or by stable transfection with an antisense MIF plasmid or were obtained from MIFknockout mice. MIF-deficient macrophages were shown to be hyporesponsive to stimulation with LPS and Gram-negative bacteria. The defect was associated with a down-regulation of Toll-like receptor 4 (TLR4), the signal transducing molecule of the LPS receptor complex. Immunoneutralization of extracellular MIF decreased TLR4 expression and responses of macrophages to LPS, indicating that MIF may exert autocrine effects. These findings identify an important role for MIF in innate immunity and provide a rationale for the development of anti-MIF strategy for the treatment of patients with Gram-negative septic shock.

Recombinant Adenovirus Vector Bearing Antisense Macrophage Migration Inhibitory Factor cDNA Prevents Acute Lipopolysaccharide-Induced Liver Failure in Mice

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in delayed hypersensitivity and cellular immunity. MIF also acts as a proinflammatory cytokine and counterregulates the anti-inflammatory effects of glucocorticoids. Exogenous gene transfer mediated by adenovirus is useful to study a particular molecular function as well as to develop gene therapy strategies. A recombinant adenovirus containing sense and antisense murine MIF (mMIF) cDNA inserts was constructed using a cosmid-terminal protein complex method. The sense mMIF adenovirus (AxCA-mMIFS) efficiently induced mMIF in COS-7 cells that endogenously lack mMIF in a dose-dependent manner. In contrast, the antisense mMIF adenovirus (AxCA-mMIFAS) inhibited the expression of mMIF in NIH3T3 cells in a dose-dependent manner. To assess the pathophysiologic role of MIF in acute liver failure, we induced acute onset of liver damage in mice (male Jcl:ICR) by a combined treatment of Bacille Calmette-Guerin (BCG) and lipopolysaccharide (LPS). mMIF level in the liver of mice infected with AxCA-mMIFAS showed a significant reduction in MIF production in response to BCG-LPS compared with mice treated without viral infection and with AxCA-mMIFS. In addition, the immunohistochemical staining demonstrated that F4/80 antigen on macrophage was enhanced in liver infected with AxCA-mMIFS but reduced in liver infected with AxCA-mMIFAS. The staining intensity is correlated with the mMIF antigen level in liver tissue. The survival rate of mice infected with AxCA-mMIFAS was significantly higher than that of mice treated with PBS and infected with AxCA-LacZ in BCG-LPS. These results suggest that inhibition of MIF production, using recombinant adenovirus bearing the antisense MIF gene, reduced the mortality rate in BCG-LPS–induced liver failure in mice. This finding might aid in the further development of gene therapy targeting MIF.

Suppression of tumor growth through introduction of an antisense plasmid of macrophage migration inhibitory factor.

The potential role in cell growth of Macrophage migration inhibitory factor (MIF) has been studied, however, the mechanism of its anti-tumor effect is poorly understood. Antisense-MIF plasmids were directly injected into colon 26 tumors embedded in the back of mice. Furthermore, the role of MIF in the cell cycle was assessed with regard to retinoblastoma (Rb) protein and transcription factor E2F. Plasmids containing sense- and antisense-MIF genes were transfected into human colon cancer KM12SM cells in vitro. To examine the Rb protein-E2F pathway, plasmids containing each specific cis-acting enhancer for Rb protein and E2F with luciferase reporter genes, pRB-luc and pE2F-luc, respectively, were used. Antisense MIF treatment significantly reduced the tumor size. In vitro cell proliferation was significantly suppressed by the antisense treatment as examined by BrdU uptake. Transcriptions of Rb protein were 8.4x10(3) (RLU), 9.5x10(3) and 24.3x10(3) in the antisense MIF, PBK, and the sense MIF, respectively. As for E2F, transcription activities were 3.8x10(3), 3.6x10(3) and 7.7x10(3), respectively. These results indicate the possibility that MIF may promote tumor growth, in which the activation-inactivation mechanism of the Rb protein-E2F pathway could be profoundly involved.

Induction of cell arrest by transfection of macrophage migration inhibitory factor antisense plasmid

Macrophage migration inhibitory factor (MIF) is known to be a proinflammatory cytokine, glucocorticoid-induced immunomodulator. MIF is abundantly expressed in various cancer cells, and is considered to contribute to cell growth and differentiation. MIF inactivates the functions of wild-type p53. Consequently, if MIF expression in cancer cells can be suppressed, the functions of p53 and p21 will be restored and an anti-tumor effect can be expected due to the inducement of cell cycle arrest. In the p21 promoter there are three DNA binding sites of STAT-1, and p21 is induced without p53. In the present study, an investigation was made of associations between p53-p21 and STAT-1-p21 signal transmission by the introduction of antisense MIF plasmid and cell cycle. Two groups, namely a transfected antisense MIF plasmid group (antisense MIF group) and a transfected PBK group (PBK group, as a control), in a DLD-1 cell line were created and used in these experiments. The cell cycle, apoptosis, inhibition of growth by subcutaneous tumor, p21 promoter activity, p21 protein, p53 cis enhancer activity and STAT-1 protein were observed. In the antisense MIF group, a shift from the S phase to the G1 phase and an inhibition of growth was noted. Scarcely any apoptotic cells were noted in either group. In terms of p21 promoter, p53 cis enhancer and STAT-1 activity, an increase in activity was found in the anti-sense MIF group. In cancer cells with MIF expression, cell arrest via p21 began due to the inhibition of MIF expression, and increases in p53 transcription activity and in STAT-1 intervention at this time were confirmed.

Antisense macrophage migration inhibitory factor (MIF) prevents anti-IgM mediated growth arrest and apoptosis of a murine B cell line by regulating cell cycle progression.

Macrophage migration inhibitory factor (MIF) is involved in the generation of cell-mediated immune responses. Recently it has been reported that MIF also plays a role in cell proliferation and differentiation. In the present study, using a B-cell line, WEHI-231, and its stable MIF-antisense transfectant, WaM2, as a representative transfectant, we investigated the mechanism underlying regulation of the cell growth by MIF. WaM2 cells produced less MIF than vector control or parental WEHI-231 cells. Reduced and increased proportions were seen in G1 and S-phase cells, respectively, in WaM2 as compared with WEHI-231. Growth arrest and apoptosis after stimulation via surface Ig (sIg) were less prominent in WaM2 cells than those in WEHI-231. However, the addition of recombinant rat MIF did not reverse the inhibition of the growth arrest and apoptosis induced in WaM2 by cross-linking sIg. Almost the same amount of p27kip1 expression was detected in WaM2 cells as those in WEHI-231 and vector control cells. Cross-linking of sIg elevated the p27kip1 level equally in these cells irrespective of the MIF-antisense expression. Taken together, it seems that MIF plays a role in inducing apoptosis in B cells upon IgM cross-linking by regulating the cell cycle via a novel intracellular pathway.

Involvement of Macrophage Migration Inhibitory Factor (MIF) in the Mechanism of Tumor Cell Growth

Macrophage migration inhibitory factor (MIF) was recently rediscovered as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. MIF is constitutively expressed in various cells and enhances production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, and interferon gamma. Recently, it was reported that MIF mRNA was overexpressed in prostatic tumors, which suggests that MIF is a protein involved in tumor cell growth beyond inflammatory and immune responses. We examined the expression of MIF in the murine colon carcinoma cell line colon 26 by Western and Northern blot analyses and immunohistochemistry. Next, we investigated the effects of transforming growth factor (TGF) beta, basic fibroblast growth factor (b-FGF), and platelet-derived growth factor (PDGF) on the expression of MIF mRNA. Furthermore, we examined whether MIF is involved in tumor cell proliferation, using an MIF anti-sense plasmid transfection technique. We demonstrated that MIF protein and its mRNA were highly expressed in colon 26 cells, using Western and Northern blot analyses, respectively. By immunohistochemical analysis, we found that MIF was localized largely in the cytoplasm of the tumor cells. In response to TGF-beta, b-FGF, and PDGF, MIF mRNA expression was significantly up-regulated. Following this, we transfected the cells with an anti-sense MIF plasmid, which revealed that this treatment induced significant suppression of cell proliferation. Although MIF plays multifunctional roles in a broad spectrum of pathophysiological states, little has been done to investigate the role of this protein in association with tumor growth. The current results suggest the possibility that MIF induces tumor cell growth in concert with other growth factors, which encouraged us to investigate a novel approach for tumor therapy using an anti-MIF antibody and an MIF anti-sense plasmid transfection technique.

MIF Expression in the Rat Brain: Implications for Neuronal Function

The mediator known historically as macrophage migration inhibitory factor (MIF) has been identified recently as being released into the circulation by the anterior pituitary gland as a consequence of stress or during a systemic inflammatory response. Macrophages and T cells also secrete MIF, both in response to proinflammatory factors or upon stimulation with glucocorticoids. Once released, MIF "overrides" or counterregulates the immunosuppressive effects of steroids on cytokine production and immune cellular activation. To further investigate the biology of MIF and its role in the neuroendocrine system, we have studied the regional and cellular expression of MIF in brain tissue obtained from normal rats and rats administered LPS intracisternally. Rat brain sections were analyzed by immunohistochemistry utilizing an affinity-purified, anti-MIF antibody raised to recombinant MIF, and by in situ hybridization using a digoxigenin-labeled, antisense MIF cRNA probe. The kinetics of MIF mRNA expression in brain were compared with that of IL-1, IL-6, and TNF-alpha by RT-PCR of total brain RNA. The cerebrospinal fluid content of MIF and TNF-alpha proteins was analyzed by Western blotting and ELISA. A strong baseline expression pattern for MIF was observed in neurons of the cortex, hypothalamus, hippocampus, cerebellum, and pons. By in situ hybridization, MIF mRNA was found predominantly in cell bodies whereas MIF protein was detected mostly within the terminal fields associated with neurons. There was a marked pattern of MIF immunoreactivity within the mossy fibers of the dentate gyrus and dendrites of the hippocampal CA3 field. These structures have been shown previously to be involved in glucocorticoid-induced tissue damage within the hippocampus, suggesting an association between MIF and targets of glucocorticoid action. The intracisternal injection of LPS increased MIF mRNA and protein expression in brain and MIF immunoreactivity was due in part to infiltrating monocytes/macrophages. MIF protein also was found to be rapidly released into the cerebrospinal fluid. This response corresponded with that of LPS-induced cytokine release and MIF mRNA expression increased in a distribution that colocalized in large part with that of TNF-alpha, IL-1 beta, and IL-6. The significant levels of baseline and inducible MIF expression in the brain and its regional association with glucocorticoid action underscore the importance of this mediator as a physiological regulator of the inflammatory stress response and further define its role within the neuroendocrine system.