Recombinant Human Macrophage Migration Inhibitory Factor Research Articles

The Novel Role of MIF in the Secretion of IL-25, IL-31, and IL-33 from PBMC of Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune inflammatory joint disease with complex pathogenesis associated with cytokine dysregulation. Macrophage migration inhibitory factor (MIF) plays a role in systemic inflammation and joint destruction in RA and could be associated with the secretion of other immune-modulatory cytokines such as IL-25, IL-31, and IL-33. For the above, our main aim was to evaluate the IL-25, IL-31, and IL-33 secretion from recombinant human MIF (rhMIF)-stimulated peripheral blood mononuclear cells (PBMC) of RA patients. The rhMIF and lipopolysaccharide (LPS) plus rhMIF stimuli promote the secretion of IL-25, IL-31, and IL-33 (p < 0.05) from PBMC of RA patients. The study groups, the different stimuli, and the interaction between both showed a statistically significant effect on the secretion of IL-25 (p < 0.05) and IL-31 (p < 0.01). The study of the effect of the RA patient treatments and their interaction with the effect of stimuli did not show an interaction between them. In conclusion, our study generates new evidence for the role of MIF in the secretion of IL-25, IL-31, and IL-33 and its immunomodulatory effect on RA.

The role of macrophage migration inhibitory factor in promoting benign prostatic hyperplasia epithelial cell growth by modulating COX-2 and P53 signaling.

ABSTRACTInflammation and proinflammatory cytokines have been implicated in the progression of benign prostatic hyperplasia (BPH). Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine. Our previous study found that MIF is highly expressed in BPH epithelium. It has been reported that there is a correlation between MIF and clinical BPH progression. However, whether MIF has an effect on BPH epithelial cells is not clear. The aim of this study was to explore whether MIF has a role in BPH. Our results showed that immunohistochemistry (IHC) showed that MIF is highly expressed in the epithelium and that MIF and PCNA expression levels are higher in BPH samples than in control. CCK8 and flow cytometry assays showed that recombinant human MIF (rMIF) promoted the proliferation of BPH-1 and PWR-1E cells, while ISO-1 partially reversed this effect on proliferation. JC-1 assays showed that rMIF inhibited the apoptosis of BPH-1 and PWR-1E cells, and ISO-1 could partially reverse this inhibition. Moreover, western blotting indicated that rMIF downregulated P53 and upregulated COX-2. Furthermore, MIF-induced proliferation could be inhibited by celecoxib in the CCK8 and flow cytometry assay. MIF-inhibited apoptosis could be partially reversed by celecoxib in the JC-1 assay. Western blotting showed that celecoxib could partially reverse MIF-induced COX-2 upregulation and P53 downregulation. Together, MIF is highly expressed in BPH epithelium. In vitro, MIF promoted BPH epithelial cell growth by regulating COX-2 and P53 signaling. Targeting MIF may provide a new option for the improved treatment of BPH in the future.

Macrophage migration inhibitory factor modulates cytokine expression in the trophoblast cell line HTR-8/SVneo.

Extravillous trophoblasts are specific placental cells that invade the uterine stroma and spiral arteries modifying and adjusting them to pregnancy. Many pregnancy pathologies are associated with impairment of this process, including preeclampsia and intrauterine growth restriction, among others. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is abundant at the fetomaternal interface. Previous results from our group showed that MIF participates in trophoblast invasion and modulates the expression of molecules known to mediate stromal and endovascular trophoblast invasion. In this study we investigated the possibility that MIF could act as a regulator of cytokines known to modulate trophoblast invasion using the normal extravillous trophoblast-derived cell line HTR-8/SVneo. Expression of trophoblast MIF was attenuated by MIF mRNA-specific small interfering RNAs. Cytokine expression was assessed at the mRNA and protein levels using real-time quantitative polymerase chain reaction and flow cytometry respectively. Knockdown of MIF led to a significant decrease in mRNA for IL-1β (IL1B) and IL-8 (CXCL8) and interleukin (IL)-8 protein. The addition of recombinant human MIF to cell culture medium increased IL-6 after 24h treatment and IL-6 and IL-8 after 72h treatment. Cell viability was not affected by MIF silencing or rhMIF treatment. The results of this study imply that at least some of the effects of MIF on trophoblast invasion could be mediated through autocrine or paracrine modulation of trophoblast cytokine production.

Macrophage migration inhibitory factor enhances influenza-associated mortality in mice.

Influenza-associated mortality continues to occur annually despite available antiviral therapies. New therapies that improve host immunity could reduce influenza virus disease burden. Targeting macrophage migration inhibitory factor (MIF) has improved the outcomes of certain inflammatory diseases, but its role in influenza viral infection is unclear. Here, we showed that, during influenza viral infection, Mif-deficient mice have less inflammation, viral load, and mortality compared with WT control mice; conversely, Tg mice, overexpressing Mif in alveolar epithelial cells, had higher inflammation, viral load, and mortality. Antibody-mediated blockade of MIF in WT mice during influenza viral infection improved their survival. Mif-deficient murine lungs showed reduced levels of parkin, a mitophagy protein that negatively regulates antiviral signaling, prior to infection and augmented antiviral type I/III IFN levels in the airspaces after infection as compared with WT lungs. Additionally, in vitro assays with human lung epithelial cells showed that treatment with recombinant human MIF increased the percentage of influenza virus-infected cells. In conclusion, our study reveals that MIF impairs antiviral host immunity and increases inflammation during influenza infection and suggests that targeting MIF could be therapeutically beneficial during influenza viral infection.

Promoting migration and apoptosis of glioma cells by macrophage migration inhibitory factor

Objective: To investigate the effect of macrophage migration inhibitory factor (MIF) on the biology of glioma U87MG and U251 cells. Methods: Silencing MIF gene expression in U87MG cells by RNA interference was monitored by Western blot. MIF low expressing U251 cells were treated at different concentrations of recombinant human MIF (rhMIF) and scratching test and flow cytometry were used to detect cell migration and apoptosis. The protein expression of bcl-2, bax, AKT, p-AKT was detected by Western blot. Results: The ability of migration and anti-apoptosis of U87MG cells silenced by siRNA decreased significantly, and the expression levels of p-AKT and anti-apoptotic protein bcl-2 also decreased; in contrast, the expression level of apoptosis protein bax increased. With increase of rhMIF treatment concentration, the expression levels of MIF protein, p-AKT and bcl-2 in U251 cells were gradually enhanced, whereas the level of apoptosis protein bax was inhibited. Conclusion: MIF promotes cell migration and inhibits apoptosis of both U87MG and U251 cells, likely through the regulation of PI3K/AKT signaling pathway.

Functional MIF promoter haplotypes modulate Th17-related cytokine expression in peripheral blood mononuclear cells from control subjects and rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by elevated levels of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-17, and macrophage migration inhibitory factor (MIF). MIF induces IL-17 secretion and MIF promoter polymorphisms influence the expression of selected downstream mediators. The aim of this study was to investigate the relationship between known functional MIF haplotypes and Th17-related cytokine secretion profile in peripheral blood mononuclear cells (PBMC) from control subjects (CS) and RA patients stimulated with lipopolysaccharide (LPS) and recombinant human MIF (rhMIF). The −794 CATT5-8 and −173G > C polymorphisms of the MIF gene were determined by conventional PCR and PCR-RFLP, respectively. The most frequent haplotypes of the MIF polymorphism and PBMC were identified from three subjects homozygous for each haplotype and in both study groups, the PBMC were obtained and stimulated with LPS or rhMIF. The secretion of cytokines related to the Th17 profile was determined by a multiplex immunoassay (MAGPIX). LPS stimulation induced the secretion of cytokines related to the Th17 profile in PBMC from CS and RA patients, whereas, rhMIF only stimulated this response in PBMC from RA patients. PBMC from CS carriers of the MIF 7C haplotype showed more IL-17A, IL-17F, IL-22, and IL-23 secretion than non-7C carriers after LPS stimulation. In the case of rhMIF stimulation, the PBMC from CS carriers of the 7C haplotype secreted more IL-17A and IL-23 than non-7C carriers. In conclusion, genetic variants of the MIF promoter modulate the secretion of cytokines related to the Th17 profile in PBMC from CS inducing a differential response in comparison to PBMC from RA patients.

Macrophage migration inhibitory factor promotes lung fibrosis via reactive oxygen species-mediated up-regulation of aerobic glycolysis

To explore the role of macrophage migration inhibitory factor (MIF) in lung fibrosis and the possible molecular pathways involved. Twenty male adult mice were randomized into control group and pulmonary fibrosis model group to receive intratracheal instillation of normal saline and bleomycin, respectively. Thirty days after the instillation, the level of MIF in the lung tissue of the mice was measured. Human embryonic lung fibroblasts (HLFs) were stimulated with recombinant human MIF (rMIF) and the changes in reactive oxygen species (ROS) levels, aerobic glycolysis and collagen production were measured; the effects of ROS inhibitor and glycolysis inhibitor on collagen productions were tested in rMIFstimulated HLF cells. Compared with the control mice, the mice with bleomycin-induced lung fibrosis exhibited significantly increased levels of MIF in the lung tissue and bronchoalveolar lavage fluid (BALF). ROS levels, aerobic glycolysis and collagen production were all increased in HLFs in response to rMIF stimulation; the enhancement of aerobic glycolysis and collagen production induced by rMIF and hydrogen peroxide were obviously suppressed by ROS inhibitor; the application of glycolysis inhibitor obviously inhibited rMIF-and hydrogen peroxide-induced increase of collagen production in HLFs. rMIF participates in the development of pulmonary fibrosis in mice probably by up-regulating aerobic glycolysis via ROS to promote collagen production in fibroblasts.

Macrophage migration inhibitory factor enhances autophagy by regulating ROCK1 activity and contributes to the escape of dendritic cell surveillance in glioblastoma.

Macrophage migration inhibitory factor(MIF) is highly expressed in glioblastoma, promoting malignant progression and suppresses immune surveillance. However, the mechanism underlying its biological roles in human glioblastoma and the capability of MIF to escape dendritic cell(DC) surveillance remain poorly understood. In the present study, we found that recombinant human MIF(rhMIF) activated the RhoA‑ROCK1 pathway and simultaneously upregulated F‑actin fibre formation. Additionally, we showed that rhMIF increased autophagy in glioblastoma cells, and knockdown of endogenic MIF suppressed autophagy. In glioma specimens, MIF expression was significantly correlated with LC3B levels. Moreover, we confirmed that the activity of Rho‑associated coiled‑coil containing kinase(ROCK)1 played a crucial role in MIF‑induced autophagy. Y26736, a ROCK1 inhibitor, blocked the MIF‑mediated increase in migration and colony formation in glioblastoma multiforme(GBM) cells. Furthermore, exogenous rhMIF suppressed the migration of both immature DCs(iDCs) and mature DCs(mDCs). Addition of rhMIF during the maturation process of iDCs impaired the expression of co‑stimulatory markers. Taken together, our results identified ROCK1 as a critical mediator of MIF‑induced autophagy and the immunosuppressive effect of MIF on DC surveillance in glioblastoma.

Critical role of macrophage migration inhibitory factor in sepsis via interfere with MCP-1/CCL2 signaling pathway

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine produced by the multiple cell types, modulates the expression of several inflammatory molecules. Since MIF is a critical mediator of septic shock by modulation of innate immune responses and many studies demonstrated the role of MIF in sepsis pathogenesis and signaling pathways; however, the mechanisms underlying these changes remain unclear. MIF also promotes the migration and recruitment of immune cells inducing the expression of chemokines (monocyte chemoattractant protein (MCP)-1, adhesion molecules as intercellular adhesion molecule (I-CAM)-1 and vascular cell adhesion molecule (V-CAM)-1. Male MIF+/+ and MIF−/− mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. Mif(-/-) mice had enhanced susceptibility to bacterial infections and impaired tumor necrosis factor (TNF) compared with MIF+/+. Further, Mif(-/-) mice showed upregulation of proinflammatory cytokine and eleveated the levels of MCP-1. Interesting treatment with recombinant human MIF (rhMIF) before CLP protected the animals from sepsis. Together, these data suggest that potential of targeting or exploiting MIF for therapeutic strategies in the management of sepsis.

PP70 - Assessing inhibition of macrophage migration inhibitory factor by isothiocyanates

Isothiocyanates are a class of phytochemicals that occur in cruciferous vegetables, and have been shown to exhibit anti-inflammatory and anti-cancer activity in both cell and animal models. Macrophage migration inhibitory factor (MIF) was discovered as a major target of isothiocyanates. MIF is a highly conserved pro-inflammatory cytokine that has been associated with several human diseases including sepsis, cardiovascular disease and cancer. MIF has an unusually reactive N-terminal proline that bestows the protein with tautomerase activity. Isothiocyanates covalently modify this proline and inhibit MIF activity. In this study we have used a library of novel isothiocyanates to investigate their ability to inhibit the tautomerase activity of MIF, both with recombinant human MIF and in cell culture. IC 50 values as low as 0.25 µM were observed in cells. In cellular models of liver fibrosis, we have shown that isothiocyanates also inhibit the biological activity of MIF. There was no correlation between MIF inhibition and isothiocyanate cytotoxicity, suggesting independent pathways. Finally, we have shown that the reactive N-terminal proline makes MIF susceptible to other biological electrophiles, including epicatechins. This provides a novel mechanism to explain the anti-inflammatory activities of these dietary phytochemicals.

Virtual Screening of Some Active Human Macrophage Migration Inhibitory Factor Antagonists

Macrophage migration inhibitory factor (MIF) is an autocrine- and paracrine-acting cytokine that is involved in several inflammatory, autoimmune, infectious, and oncogenic diseases. Clinical data have shown that inhibition of MIF, especially its tautomerase activity, with small compounds has been beneficial in some disease models. A virtual screening (VS) experiment is conducted for searching some active compounds to inhibit the tautomerase activity of MIF from the ZINC database. By using an x-ray–determined structure OXIM-11 as the query and an in-house developed two-dimensional scaffold comparing method designated as Sfilter, we have screened out some 1500 compounds for ranking by our previously published docking method ADDock. After further ranking by ADDock on 119 compounds screened, we have decided to choose 17 of them for measuring their inhibitory activity IC50 against the MIF tautomerase experimentally. The IC50’s are measured using both human monocytic THP-1 cell lysate and purified recombinant human MIF protein. We have found that the IC50’s measured for three searched compounds (namely, ZINC02693801, ZINC00141102, and ZINC12368346) are better than that determined for ISO-1, a known MIF tautomerase inhibitor and standard used throughout our VS experiment. Moreover, the scaffolds of most of our active compounds searched are also quite different from those searched and published by others previously.

Macrophage Migration Inhibitory Factor Inhibits the Migration of Cartilage End Plate-Derived Stem Cells by Reacting CD74

Introduction Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that regulates inflammatory reactions and the pathophysiology of many inflammatory diseases. Intervertebral disc (IVD) degeneration is characterized by an inflammatory reaction, but the potential role of MIF in IVD degeneration has not yet been determined. Recent studies have shown that MIF and its receptor, CD74, are involved in regulating the migration of human mesenchymal stem cells (MSCs). Thus, MIF might impair the ability of mesenchymal stem cells (MSCs) to home to injured tissues. Our previous studies indicated that cartilage endplate-derived stem cells (CESCs) as a type of MSCs exist in human degenerate IVDs. Here, we investigate the role of MIF in regulating the migration of CESCs. Material and Method CESCs were isolated and identified. Results We have shown that MIF was distributed in human degenerate IVD tissues and was subject to regulation by the proinflammatory cytokine tumor necrosis factor-α. Furthermore, in-vitro cell migration assays revealed that nucleus pulposus (NP) cells inhibited the migration of CESCs in a number-dependent manner, and enzyme-linked immunosorbent assays revealed that the amount of MIF in conditioned medium was significantly increased as a function of increasing cell number. In addition, recombinant human MIF inhibited the migration of CESCs in a dose-dependent manner. CESCs migration was restored when an antagonist of MIF, (S, R)-3(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), was added. Finally, a CD74 activating antibody (CD74Ab) was used to examine the effect of CD74 on CESCs motility and inhibited the migration of CESCs in a dose-dependent manner. Conclusion We have identified and characterized a novel regulatory mechanism governing cell migration during IVD degeneration. The results will benefit understanding of another possible mechanism for IVD degeneration, and might provide a new method to repair degenerate IVD by enhancing CESCs migration to degenerated NP tissues to exert their regenerative effects. Disclosure of Interest None declared

Human single-chain variable fragment antibody inhibits macrophage migration inhibitory factor tautomerase activity

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, secreted from a variety of immune cells, that regulates innate and adaptive immune responses. Elevation of MIF levels in plasma correlates with the severity of inflammatory diseases in humans. Inhibition of MIF or its tautomerase activity ameliorates disease severity by reducing inflammatory responses. In this study, the human single-chain variable fragment (HuScFv) antibody specific to MIF was selected from the human antibody phage display library by using purified recombinant full-length human MIF (rMIF) as the target antigen. Monoclonal HuScFv was produced from phage-transformed bacteria and tested for their binding activities to rMIF by indirect enzyme-linked immunosorbent assay as well as to native MIF by western blot analysis and immunofluorescence assay. The HuScFv with highest binding signal to rMIF also inhibited the tautomerase activities of both rMIF and native MIF in human monoblastic leukemia (U937) cells in a dose-dependent manner. Mimotope searching and molecular docking concordantly demonstrated that the HuScFv interacted with Lys32 and Ile64 in the MIF tautomerase active site. To the best of our knowledge, this is the first study to focus on MIF-specific fully-human antibody fragment with a tautomerase-inhibitory effect that has potential to be developed as anti-inflammatory biomolecules for human use.

Overexpression of macrophage migration inhibitory factor in adenoid cystic carcinoma: correlation with enhanced metastatic potential

Adenoid cystic carcinoma (ACC) is a malignant tumor frequently arising in salivary glands with poor long-term prognosis due to high rates of local recurrences and distant metastases. Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine and has recently emerged as a pro-tumorigenic factor in various cancers. This study is designed to investigate the expression status and functional significance of MIF in ACC. Immunohistochemical staining was performed to evaluate the expression levels of MIF, HIF-1α, MMP-9, p53, and p-JNK in ACC tissues. In vitro, ACC-2 cells were exposed to recombinant human MIF (rMIF) or ISO-1 (an inhibitor of MIF) at different concentrations and times, followed by the detection of cell growth, viability, migration, and invasion, as well as the expression levels of several cellular signals. The immunohistochemical results demonstrated the overexpression of MIF in ACC tissues as well as its association with the distant metastasis. Further analyses showed a significant correlation between the staining of MIF and p-JNK. Moreover, the in vitro studies revealed that the treatment for ACC cells with ISO-1 significantly attenuated cell migratory and invasive capacity, as opposed to the limited promotive effects of rMIF. More importantly, MIF inhibition could cause the activation of JNK, correlating with the immunohistochemical findings on ACC tissues. The results suggest that MIF is likely to be an important player in the pathogenesis of ACC and may promote cancer metastasis, which possibly involves JNK inactivation. Further investigation of MIF-mediated molecular events may provide novel insights into the treatment for ACC.

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

IntroductionGlucocorticoid (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.MethodsSixty-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.ResultsMIF 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.ConclusionsOur 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.

Macrophage migration inhibitory factor induces ICAM-1and thrombomobulin expression in vitro

Macrophage migration inhibitory factor (MIF) is an important cytokine in the modulation of inflammatory and immune responses, but its role in coagulation remains to be elucidated. In this study, we investigated the potential role of MIF in coagulation through its influence on two factors, thrombomodulin (TM) and intercellular adhesion molecule-1 (ICAM-1). Recombinant human MIF was added to human microvascular endothelial cell line (HMEC-1) to investigate its influence on the expression of TM and ICAM-1. The results showed that both TM and ICAM-1 were induced with MIF addition in a dose-dependent and time-dependent manner. The expression of ICAM-1 and TM was increased as MIF doses were increased, with the highest expression seen at 12 hr after 400 ng/ml of MIF treatment. Besides, anti-MIF antibody treatment reduced the TM expression in HMEC-1 cells. In conclusion, our data support a role of MIF as an important factor in the regulation of coagulation.

P53 Mediates Cigarette Smoke–Induced Apoptosis of Pulmonary Endothelial Cells

Exposure to cigarette smoke (CS) is the most common cause of emphysema, a debilitating pulmonary disease histopathologically characterized by the irreversible destruction of lung architecture. Mounting evidence links enhanced endothelial apoptosis causally to the development of emphysema. However, the molecular determinants of human endothelial cell apoptosis and survival in response to CS are not fully defined. Such determinants could represent clinically relevant targets for intervention. We show here that CS extract (CSE) triggers the death of human pulmonary macrovascular endothelial cells (HPAECs) through a caspase 9-dependent apoptotic pathway. Exposure to CSE results in the increased expression of p53 in HPAECs. Using the p53 inhibitor, pifithrin-α (PFT-α), and RNA interference (RNAi) directed at p53, we demonstrate that p53 function and expression are required for CSE-mediated apoptosis. The expression of macrophage migration inhibitory factor (MIF), an antiapoptotic cytokine produced by HPAECs, also increases in response to CSE exposure. The addition of recombinant human MIF prevents cell death from exposure to CSE. Further, the suppression of MIF or its receptor/binding partner, Jun activation domain-binding protein 1 (Jab-1), with RNAi enhances the sensitivity of human pulmonary endothelial cells to CSE via a p53-dependent (PFT-α-inhibitable) pathway. Finally, we demonstrate that MIF is a negative regulator of p53 expression in response to CSE, placing MIF upstream of p53 as an antagonist of CSE-induced apoptosis. We conclude that MIF can protect human vascular endothelium from the toxic effects of CSE via the antagonism of p53-mediated apoptosis.

Macrophage migration inhibitory factor regulatesproliferation of gastric cancer cellsvia the PI3K/Akt pathway

To investigate the effects of macrophage migration inhibitory factor (MIF) on proliferation of human gastric cancer MGC-803 cells and expression of cyclin D1 and p27(Kip1) in them, and further determine whether the effects are related to the PI3K/Akt signal transduction pathway. Gastric cancer MGC-803 cells were cultured and then treated with 50 microg/L recombinant human MIF (rhMIF) with and without a PI3K inhibitor, LY294002 (25 micromol/L). MTT assay was used to detect the proliferation of MGC-803 cells. Cell cycle was detected by flow cytometry. Expression of cyclin D1 and p27(Kip1) mRNA was by reverse transcription-polymerase chain reaction. Protein expression of phosphorylated Akt (p-Akt), Akt, cyclin D1 and p27(Kip1) was examined by immunocytochemistry and Western blotting. rhMIF significantly stimulated the proliferation of MGC-803 cells and cell cycle progression from G1 phase to S phase in a concentration- and time-dependent manner. After the MGC-803 cells were treated with rhMIF for 24 h, the expression of cyclin D1 was significantly up-regulated compared with the cells not treated with rhMIF at both mRNA and protein levels (0.97 +/- 0.02 vs 0.74 +/- 0.01, P = 0.002; 0.98 +/- 0.05 vs 0.69 +/- 0.04, P = 0.003). The p27(Kip1) was down-regulated but only statistically significant at the protein level. rhMIF significantly increased the expression of p-Akt, which reached the peak at 30 min, but did not affect the expression of Akt. However, LY294002 inhibited all the effects of rhMIF. Macrophage MIF increases the proliferation of gastric cancer cells, induces the expression of cyclin D1 at the transcriptional level and inhibits the expression of p27(Kip1) at the post-transcriptional level via the PI3K/Akt pathway.

Macrophage Migration Inhibitory Factor Governs Endothelial Cell Sensitivity to LPS-Induced Apoptosis

Human endothelial cells (EC) are typically resistant to the apoptotic effects of stimuli associated with lung disease. The determinants of this resistance remain incompletely understood. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by human pulmonary artery EC (HPAEC). Its expression increases in response to various death-inducing stimuli, including lipopolysaccharide (LPS). We show here that silencing MIF expression by RNA interference (MIF siRNA) dramatically reduces MIF mRNA expression and the LPS-induced increase in MIF protein levels, thereby sensitizing HPAECs to LPS-induced cell death. Addition of recombinant human MIF (rhMIF) protein prevents the death-sensitizing effect of MIF siRNA. A common mediator of apoptosis resistance in ECs is the death effector domain (DED)-containing protein, FLIP (FLICE-like inhibitory protein). We show that LPS induces a transcription-independent increase in the short isoform of FLIP (FLIP(s)). This increase is blocked by MIF siRNA but restored with the addition of recombinant MIF protein (rHMIF). While FLIP(s) siRNA also sensitizes HPAECs to LPS-induced death, the addition of rhMIF does not affect this sensitization, placing MIF upstream of FLIP(s) in preventing HPAEC death. These studies demonstrate that MIF is an endogenous pro-survival factor in HPAECs and identify a novel mechanism for its role in apoptosis resistance through the regulation of FLIP(s). These results show that MIF can protect vascular endothelial cells from inflammation-associated cell damage.

Structural and Functional Characterization of a Secreted Hookworm Macrophage Migration Inhibitory Factor (MIF) That Interacts with the Human MIF Receptor CD74

Hookworms, parasitic nematodes that infect nearly one billion people worldwide, are a major cause of anemia and malnutrition. We hypothesize that hookworms actively manipulate the host immune response through the production of specific molecules designed to facilitate infection by larval stages and adult worm survival within the intestine. A full-length cDNA encoding a secreted orthologue of the human cytokine, Macrophage Migration Inhibitory Factor (MIF) has been cloned from the hookworm Ancylostoma ceylanicum. Elucidation of the three-dimensional crystal structure of recombinant AceMIF (rAceMIF) revealed an overall structural homology with significant differences in the tautomerase sites of the human and hookworm proteins. The relative bioactivities of human and hookworm MIF proteins were compared using in vitro assays of tautomerase activity, macrophage migration, and binding to MIF receptor CD74. The activity of rAceMIF was not inhibited by the ligand ISO-1, which was previously determined to be an inhibitor of the catalytic site of human MIF. These data define unique immunological, structural, and functional characteristics of AceMIF, thereby establishing the potential for selectively inhibiting the hookworm cytokine as a means of reducing parasite survival and disease pathogenesis.