Rediscovering MIF: New Tricks for an Old Cytokine.

Produced by many cell types, macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with critical and supporting roles in many disease states and conditions. Its disease associations, myriad functions, receptors, and downstream signaling have been the subject of considerable research, yet many questions remain. Moreover, the relevance of MIF's partially functionally redundant family member, D-dopachrome tautomerase (D-DT), also remains to be further characterized. Here, we discuss recent discoveries demonstrating direct roles of MIF in supporting NLR Family Pyrin Domain-Containing 3 (NRLP3) inflammasome activation, as well as acting as a molecular chaperone for intracellular proteins. These findings may offer new clues to understanding MIF's multiple functions, and assist the development of putative MIF-targeting therapeutics for a variety of pathologies.

Prostaglandin D2 synthase/prostaglandin D2/TWIST2 signaling inhibits breast cancer proliferation.

Though the past few years have witnessed exciting achievements in targeted and immunotherapeutic treatments of all breast cancer subtypes, yet the decline in breast cancer mortality has been slowed, urging the need for further expanding options of high-quality treatments. Prostaglandin D2 synthase (PTGDS)/prostaglandin D2 (PGD2) play important roles in a variety of cancer types and show tissue-specificity, however, there are limited relevant reports in breast cancer. Therefore, the aims of the present study were to investigate the effects of PTGDS/PGD2 in breast cancer by large-scale bioinformatic analysis and in vitro experiments conducted on human breast cancer cell lines. Results of our study indicated that patients with high levels of PTGDS expression showed a reduced potential of tumor proliferation. PGD2 treatment significantly inhibited the proliferation and migration of breast cancer cells, which was mediated by the reduced expression of TWIST2. Overexpression of TWIST2 reversed the inhibitory effects of PGD2 on breast cancer cell proliferation. These results provided the novel evidence that PTGDS may play a significant role in modulating breast cancer growth, with implications for its potential use in treating breast cancer.

15-Deoxy-Δ12,14-Prostaglandin J2 Reinforces the Anti-Inflammatory Capacity of Endothelial Cells With a Genetically Determined NO Deficit.

RATIONALE: Fluid shear stress (FSS) maintains NOS-3 (endothelial NO synthase) expression. Homozygosity for the C variant of the T-786C single-nucleotide polymorphism of the NOS3 gene, which solely exists in humans, renders the gene less sensitive to FSS, resulting in a reduced endothelial cell (EC) capacity to generate NO. Decreased bioavailability of NO in the arterial vessel wall facilitates atherosclerosis. Consequently, individuals homozygous for the C variant have an increased risk for coronary heart disease (CHD). OBJECTIVE: At least 2 compensatory mechanisms seem to minimize the deleterious effects of this single-nucleotide polymorphism in affected individuals, one of which is characterized herein. METHODS AND RESULTS: Human genotyped umbilical vein ECs and THP-1 monocytes were used to investigate the role of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in vitro. Its concentration in plasma samples from genotyped patients with CHD and age-matched CHD-free controls was determined using quantitative ultraperformance LC-MS/MS. Exposure of human ECs to FSS effectively reduced monocyte transmigration particularly through monolayers of CC-genotype ECs. Primarily in CC-genotype ECs, FSS elicited a marked rise in COX (cyclooxygenase)-2 and L-PGDS (lipocalin-type prostaglandin D synthase) expression, which appeared to be NO sensitive, and provoked a significant release of 15d-PGJ2 over baseline. Exogenous 15d-PGJ2 significantly reduced monocyte transmigration and exerted a pronounced anti-inflammatory effect on the transmigrated monocytes by downregulating, for example, transcription of the IL (interleukin)-1ß gene (IL1B). Reporter gene analyses verified that this effect is due to binding of Nrf2 (nuclear factor [erythroid-derived 2]-like 2) to 2 AREs (antioxidant response elements) in the proximal IL1B promoter. In patients with CHD, 15d-PGJ2 plasma levels were significantly upregulated compared with age-matched CHD-free controls, suggesting that this powerful anti-inflammatory prostanoid is part of an endogenous defence mechanism to counteract CHD. CONCLUSIONS: Despite a reduced capacity to form NO, CC-genotype ECs maintain a robust anti-inflammatory phenotype through an enhanced FSS-dependent release of 15d-PGJ2.

Prostaglandin D2 synthase modulates macrophage activity and accumulation in injured peripheral nerves.

We have previously reported that prostaglandin D2 Synthase (L-PGDS) participates in peripheral nervous system (PNS) myelination during development. We now describe the role of L-PGDS in the resolution of PNS injury, similarly to other members of the prostaglandin synthase family, which are important for Wallerian degeneration (WD) and axonal regeneration. Our analyses show that L-PGDS expression is modulated after injury in both sciatic nerves and dorsal root ganglia neurons, indicating that it might play a role in the WD process. Accordingly, our data reveals that L-PGDS regulates macrophages phagocytic activity through a non-cell autonomous mechanism, allowing myelin debris clearance and favoring axonal regeneration and remyelination. In addition, L-PGDS also appear to control macrophages accumulation in injured nerves, possibly by regulating the blood-nerve barrier permeability and SOX2 expression levels in Schwann cells. Collectively, our results suggest that L-PGDS has multiple functions during nerve regeneration and remyelination. Based on the results of this study, we posit that L-PGDS acts as an anti-inflammatory agent in the late phases of WD, and cooperates in the resolution of the inflammatory response. Thus, pharmacological activation of the L-PGDS pathway might prove beneficial in resolving peripheral nerve injury.

Perivascular clusters of dendritic cells provide critical survival signals to B cells in bone marrow niches.

Beyond its established function in hematopoiesis, the bone marrow hosts mature lymphocytes and acts as a secondary lymphoid organ in the initiation of T cell and B cell responses. Here we report the characterization of bone marrow-resident dendritic cells (bmDCs). Multiphoton imaging showed that bmDCs were organized into perivascular clusters that enveloped blood vessels and were seeded with mature B lymphocytes and T lymphocytes. Conditional ablation of bmDCs in these bone marrow immune niches led to the specific loss of mature B cells, a phenotype that could be reversed by overexpression of the antiapoptotic factor Bcl-2 in B cells. The presence of bmDCs promoted the survival of recirculating B cells in the bone marrow through the production of macrophage migration-inhibitory factor. Thus, bmDCs are critical for the maintenance of recirculating B cells in the bone marrow.

Inhibition of microRNA-451 is associated with increased expression of Macrophage Migration Inhibitory Factor and mitgation of the cardio-pulmonary phenotype in a murine model of Bronchopulmonary Dysplasia.

BACKGROUND: Macrophage migration inhibitory factor (MIF) has been implicated as a protective factor in the development of bronchopulmonary dysplasia (BPD) and is known to be regulated by MicroRNA-451 (miR-451). The aim of this study was to evaluate the role of miR-451 and the MIF signaling pathway in in vitro and in vivo models of BPD. METHODS: Studies were conducted in mouse lung endothelial cells (MLECs) exposed to hyperoxia and in a newborn mouse model of hyperoxia-induced BPD. Lung and cardiac morphometry as well as vascular markers were evaluated. RESULTS: Increased expression of miR-451 was noted in MLECs exposed to hyperoxia and in lungs of BPD mice. Administration of a miR-451 inhibitor to MLECs exposed to hyperoxia was associated with increased expression of MIF and decreased expression of angiopoietin (Ang) 2. Treatment with the miR-451 inhibitor was associated with improved lung morphometry indices, significant reduction in right ventricular hypertrophy, decreased mean arterial wall thickness and improvement in vascular density in BPD mice. Western blot analysis demonstrated preservation of MIF expression in BPD animals treated with a miR-451 inhibitor and increased expression of vascular endothelial growth factor-A (VEGF-A), Ang1, Ang2 and the Ang receptor, Tie2. CONCLUSION: We demonstrated that inhibition of miR-451 is associated with mitigation of the cardio-pulmonary phenotype, preservation of MIF expression and increased expression of several vascular growth factors.

Association of CD14 and macrophage migration inhibitory factor gene polymorphisms with inflammatory microRNAs expression levels in ankylosing spondylitis and polyarthralgia.

This study aimed to investigate the genetic basis of ankylosing spondylitis (AS) and polyarthralgia (PA) conditions among Indian subjects through genotyping two immune regulatory genes CD14 (-159C>T) and MIF (-173G>C) and find their association with the expression levels of three circulating inflammatory miRNAs. This investigation may provide early genetic cause of these two forms of arthritis and more optimal biological targets to predict early therapeutic outcomes. A total of 140 patients (AS: 70 and PA: 70) and 156 controls were recruited from Indian population. CD14 and MIF genotyping was performed using ARMS-PCR. Expression level of three inflammatory miRNAs (miRNA-146a, miRNA-155 and miRNA-181) was quantified using RT-qPCR. C/T genotype of CD14 gene was found to cause 2.06-fold risk of developing AS (CI 1.06-5.98, p = .04) as compared to others and G/C genotype in MIF also shown significant variation between AS and control subjects. In PA subjects, CD14 genotypes (C/T) was found to be associated with disease susceptibility and G/C genotype of MIF gene polymorphism showed 4.71-fold risk of developing PA (CI 2.58-8.62, p = .0001). The study also revealed significant upregulation of miRNA-155 expression in AS subjects (p = .0001) with more than 1.3-fold difference between AS and PA as compared to the control subjects. miRNA-155 had strong association with AS patients with CD14 genotypes (p < .05) than PA and control subjects. This study provides better understanding of the mechanisms and disease susceptibility for MIF and CD14 genetic variants and inflammatory miRNAs networks involved in AS and PA.

Effect of NF-kB signaling pathway on the expression of MIF, TNF-α, IL-6 in the regulation of intervertebral disc degeneration.

OBJECTIVE: Τo investigate the effect of NF-kB signaling pathway on the expression of MIF, TNF-α, and IL-6 in the regulation of disc degeneration. METHODS: The disc tissue was taken from 56 patients with cervical spondylosis. According to the preoperative MRI and intraoperative disc herniation, the patients were divided into two groups: degeneration group and herniation group. The control group was 34 patients with cervical trauma with no history of cervical spondylosis. According to the preoperative JOA scores of cervical spondylosis, patients were divided into three groups: mild, moderate and severe. ELISA was used to detect the expression of MIF, IL-6, and TNF-α in the cervical intervertebral disc. NF-kB mRNA expression in the intervertebral disc was detected by qRT-PCR. RESULTS: The expression levels of NF-kB mRNA, MIF, IL-6 and TNF-α in the control group were significantly higher than those in the degeneration group and the herniation group (p⟨0.05). There was a positive correlation between the expression of NF-kB mRNA, MIF, IL-6, TNF- and cervical intervertebral disc degeneration. The expression of MIF, IL-6, and TNF-α in the mild, moderate, and severe group was negatively correlated with the JOA score. CONCLUSIONS: The expressions of NF-kB, MIF, IL-6, and TNF-α in intervertebral disc tissue in patients with disc herniation were increased and related to the degree of disc herniation. It may play an important role in the pathophysiological process of disc herniation.

Lack of CUL4B leads to increased abundance of GFAP-positive cells that is mediated by PTGDS in mouse brain.

Astrocytes are the most abundant cell type in the mammalian brain and are important for the functions of the central nervous system. Glial fibrillary acidic protein (GFAP) is regarded as a hallmark of mature astrocytes, though some GFPA-positive cells may act as neural stem cells. Missense heterozygous mutations in GFAP cause Alexander disease that manifests leukodystrophy and intellectual disability. Here, we show that CUL4B, a scaffold protein that assembles E3 ubiquitin ligase, represses the expression of GFAP in neural progenitor cells (NPCs) during brain development. Lack of Cul4b in NPCs in cultures led to increased generation of astrocytes, marked by GFAP and S100ß. The GFAP+ cells were also found to be more abundant in the brains of nervous system-specific Cul4b knockout mice in vivo. Moreover, we demonstrated that the increased generation of GFAP+ cells from Cul4b-null NPCs was mediated by an upregulation of prostaglandin D2 synthase PTGDS. We showed that the increased GFAP expression can be attenuated by pharmacological inhibition of the PTGDS enzymatic activity or by shRNA-mediated knockdown of Ptgds. Importantly, exogenously added PTGDS could promote the generation of GFAP+ cells from wild-type NPCs. We further observed that Ptgds is targeted and repressed by the CUL4B/PRC2 complex. Together, our results demonstrate CUL4B as a negative regulator of GFAP expression during neural development.

The significant effect of chronic intermittent hypoxia on prostaglandin D2 biosynthesis in rat brain.

Obstructive sleep apnea (OSA) is a common disorder characterized by chronic intermittent hypoxia (CIH). Excessive daytime sleepiness (EDS) is one of severe complications frequently associated with OSA. Lipocalin-type prostaglandin synthase (L-PGDS) is potentially responsible for the production of prostaglandin D2 (PGD2) which is an endogenous sleep inducer. To date, whether the content of PGD2 and PGDS is related to intermittent hypoxia has never been reported. The aim of this study was to compare the content of PGD2 and L-PGDS in rats' brains with and without intermittent hypoxia. Adult male Wistar rats (n = 48; 8-10 weeks) were averagely divided into two groups. One was control group, and the other group was exposed to IH (12 h/day for 6 weeks). In each group there are four time-points including 0, 2, 4 and 6 weeks, and six rats were killed and studied at each time-point. At the end of 0, 2, 4 and 6 weeks, the concentrations of PGD2 in brains were measured by LC-MS/MS. In addition, the expressions of L-PGDS protein and mRNA in brains were investigated by western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed the concentrations of PGD2 in CIH rat brains were higher than those in control groups from the second week. At the end of 6 weeks, the concentrations of PGD2 in CIH and control groups were 11.1 and 5.9 ng/g, respectively. The levels of L-PGDS protein and mRNA followed the same trend during the whole 6 weeks. The results will provide a new idea to explore that patients with OSA are always accompanied by excessive daytime sleepiness.

L-PGDS Mediates Vagus Nerve Stimulation-Induced Neuroprotection in a Rat Model of Ischemic Stroke by Suppressing the Apoptotic Response.

The role of lipocalin prostaglandin D2 synthase (L-PGDS) in brain ischemia has not been fully clarified to date. Vagus nerve stimulation (VNS) protects against cerebral ischemia/reperfusion (I/R) injury, but the mechanisms involved need further exploration. This study investigated the role of L-PGDS in cerebral I/R and whether this process was involved in the mechanism of VNS-mediated neuroprotection. Male Sprague-Dawley rats were pretreated with a lentiviral vector (LV) through intracerebroventricular injection, followed by middle cerebral artery occlusion (MCAO) and VNS treatment. The expression of L-PGDS in the peri-infarct cortex was examined. The localization of L-PGDS was determined using double immunofluorescence staining. Neurologic scores, infarct volume and neuronal apoptosis were evaluated at 24 h after reperfusion. The expression of apoptosis-related molecules was measured by western blot analysis. The expression of L-PGDS in the peri-infarct cortex increased at 12 h, reached a peak at 24 h after reperfusion, and lasted up to 3 days. VNS treatment further enhanced the expression of L-PGDS following ischemic stroke. L-PGDS was mainly expressed in neurons in the peri-infarct cortex. I/R rats treated with VNS showed better neurological deficit scores, reduced infarct volume, and decreased neuronal apoptosis as indicated by the decreased levels of Bax and cleaved caspase-3 as well as increased levels of Bcl-2. Strikingly, the beneficial effects of VNS were weakened after L-PGDS down-regulation. In general, our results suggest that L-PGDS is a potential mediator of VNS-induced neuroprotection against I/R injury.

Altered expression of eNOS, prostacyclin synthase, prostaglandin G/H synthase, and thromboxane synthase in porcine aortic endothelial cells after exposure to human serum-relevance to xenotransplantation.

Under normal conditions, the activity of platelets is stringently and precisely balanced between activation and quiescent state. This guarantees rapid hemostasis and avoids uncontrolled thrombosis. However, excessive platelet activation and resulting thrombotic microangiopathy are frequently observed in pig-to-primate xenotransplantation models. Endothelium-derived inhibitory mechanisms play an important role in regulation of platelet activation. These mainly include nitric oxide (NO), prostacyclin PGI2 , and adenosine, which are synthesized by endothelial NO synthases (eNOS), prostacyclin synthase, and CD39/CD73, respectively. We investigated whether endothelium-derived regulatory mechanisms are affected in porcine aortic endothelial cells (PAECs) after exposure to human serum. In the present study, exposure of PAECs or porcine iliac arteries to human serum suppressed gene expression of eNOS and prostacyclin synthase, while induced gene expression of prostaglandin G/H synthase and thromboxane synthase. Simultaneously, exposure to human serum reduced NO and PGI2 production in PAEC culture supernatants. Thus, human serum altered the balance of endothelium-derived inhibitory mechanisms in PAECs, which may indicate a regulatory mechanism of excessive platelet activation in pig-to-primate xenotransplantation.

Combined Knockdown of D-dopachrome Tautomerase and Migration Inhibitory Factor Inhibits the Proliferation, Migration, and Invasion in Human Cervical Cancer.

OBJECTIVE: D-dopachrome tautomerase (D-DT) is a homologue of macrophage migration inhibitory factor (MIF) with similar functions. However, the possible biological roles of D-DT in cervical cancer remain unknown so far. METHODS: D-dopachrome tautomerase was assessed by immunohistochemistry in 83 cervical cancer and 31 normal cervix tissues. The stable knockdown of D-DT and MIF by lentivirus-delivered short hairpin RNA was established, and tumor growth was examined in vitro and in vivo. The effects of D-DT and MIF on the migration and invasion were further detected by wound healing assay and transwell assay. Western blot was used to explore the mechanism of D-DT and MIF in cervical cancer pathogenesis. RESULTS: We found that D-DT was significantly high in cervical cancer, which correlated with lymph node metastasis. The knockdown of D-DT and MIF, individually and additively, inhibited the proliferation, migration, and invasion in HeLa and SiHa cells and restrained the growth of xenograft tumor. The ablation of D-DT and MIF rescued the expression of E-cadherin and inhibited the expression of PCNA, cyclin D1, gankyrin, Sam68, and vimentin, as well as phospho-Akt and phospho-glycogen synthase kinase 3-ß. CONCLUSIONS: The inhibition of D-DT and MIF in combination may represent a potential therapeutic strategy for cervical cancer.

Metabolomic analysis revealed the role of DNA methylation in the balance of arachidonic acid metabolism and endothelial activation.

Arachidonic acid (AA) metabolism plays an important role in vascular homeostasis. We reported that DNA hypomethylation of EPHX2 induced a pro-inflammatory response in vascular endothelial cells (ECs). However, the change in the whole AA metabolism by DNA methylation is still unknown. Using a metabolomic approach, we investigated the effect of DNA methylation on the balance of AA metabolism and the underlying mechanism. ECs were treated with a DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-AZA), and AA metabolic profiles were analyzed. Levels of prostaglandin D2 (PGD2) and thromboxane B2 (TXB2), metabolites in the cyclooxygenase (COX) pathway, were significantly increased by 5-AZA treatment in ECs resulting from the induction of PGD2 synthase (PTGDS) and thromboxane A synthase 1 (TBXAS1) expression by DNA hypomethylation. This phenomenon was also observed in liver and kidney cell lines, indicating a universal mechanism. Pathophysiologically, homocysteine, known to cause DNA demethylation, induced a similar pattern of the change of AA metabolism. Furthermore, 5-AZA activated ECs, as evidenced by the upregulation of adhesion molecules. Indomethacin, a COX inhibitor, reversed the effects of 5-AZA on the levels of PGD2 and TXB2, EC activation and monocyte adhesion. In vivo, the plasma levels of PGD2 and TXB2 and the expression of In vivo PTGDS and TBXAS1 as well as adhesion molecules were increased in the aorta of the mice injected with 5-AZA. In conclusion, using a metabolomic approach, our study uncovered that DNA demethylation increased AA metabolites PGD2 and TXB2 by upregulating the expression of the corresponding enzymes, which might contribute to the DNA hypomethylation-induced endothelial activation.

Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2.

Melanocyte differentiation antigens, such as gp100, tyrosinase, and Melan-A and their corresponding antibodies HMB45, T311, and A103, are major diagnostic tools in surgical pathology. Little is known about tyrosinase-related protein 2 (TRP-2, or dopachrome tautomerase/DCT) another melanocyte differentiation antigen, which is an enzymatic component of melanogenesis. We identified a commercial reagent to TRP-2, monoclonal antibody (mAb) C-9 and undertook a comprehensive analysis to assess its specificity and usefulness for surgical pathology. Subsequently, we analyzed panels of normal tissues and tumors. We show that TRP-2 is regularly expressed in melanocytes of the normal skin. In cutaneous nevi, TRP-2 is present in junctional as well as in dermal nevocytes. In malignant tumors, C-9 reactivity is restricted to melanocytic and related lesions and present in 84% and 58% of primary and metastatic melanomas, respectively. Ten primary melanomas of the anorectal mucosa were all positive. Like the other melanocyte differentiation antigens, TRP-2 was absent in 6 desmoplastic melanomas. Also, only 2 of 9 angiomyolipomas were TRP-2 positive. We conclude that mAb C-9 is a valuable reagent for the analysis of TRP-2 expression in archival surgical pathology material. The expression pattern of TRP-2 in melanocytic and related lesions appears to parallel other melanocyte differentiation antigens, although the overall incidence is lower than other antigens, such as Melan-A or gp100.

Paracrine in vivo inhibitory effects of adipose tissue-derived mesenchymal stromal cells in the early stages of the acute inflammatory response.

BACKGROUND AIMS: Excessive or unresolved inflammation leads to tissue lesions. Adipose tissue-derived mesenchymal stromal cells (AMSCs) have shown protective effects that may be dependent on the modulation of inflammation by secreted factors. METHODS: We used the zymosan-induced mouse air pouch model at two time points (4 h and 18 h) to evaluate the in vivo effects of AMSCs and their conditioned medium (CM) on key steps of the early inflammatory response. We assessed the effects of AMSCs and CM on leukocyte migration and myeloperoxidase activity. The levels of chemokines, cytokines and eicosanoids in exudates were measured by use of enzyme-linked immunoassay or radio-immunoassay. In addition, the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 (mPGES-1) was studied by use of Western blotting and the phosphorylation of p65 nuclear factor-κB (NF-κB) by immunofluorescence. RESULTS: All inflammatory parameters were significantly reduced by CM and AMSCs to a similar extent at 4 h after zymosan injection with lower effects at 18 h. The observed inhibition of leukocyte migration was associated with reduced levels of chemokines and leukotriene B4. Interleukin-1ß, interleukin-6, tumor necrosis factor-α and tumor necrosis factor-stimulated gene 6 levels were significantly decreased. The downregulation of mPGES-1 was associated with inhibition of prostaglandin E2 production. Our results suggest that these anti-inflammatory effects are related, in part, to the inhibition of NF-κB activation. CONCLUSIONS: AMSCs dampen the early process of inflammation in the zymosan-induced mouse air pouch model through paracrine mechanisms. These results support the potential utility of these cells as a source of novel treatment approaches for inflammatory pathologies.

Macrophage migration inhibitory factor - a therapeutic target in gallbladder cancer.

BACKGROUND: Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer. METHODS: Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA. RESULTS: The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells. CONCLUSIONS: Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.

Improved survival of mesenchymal stem cells by macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a critical inflammatory cytokine that was recently associated with progenitor cell survival and potently inhibits apoptosis. We examined the protective effect of MIF on hypoxia/serum deprivation (SD)-induced apoptosis of mesenchymal stem cells (MSCs), as well as the possible mechanisms. MSCs were obtained from rat bone marrow and cultured in vitro. Apoptosis was induced by culturing MSCs under hypoxia/SD conditions for up to 24 h and assessed by flow cytometry. Expression levels of c-Met, Akt, and FOXO3a were detected by Western blotting. CD74 expression was detected by qRT-PCR, Western blot, and immunofluorescence. Oxidative stress under hypoxia/SD was examined by detection of reactive oxygen species (ROS) and activity of superoxide dismutase (SOD) and malondialdehyde (MDA). Hypoxia/SD-induced apoptosis was significantly attenuated by recombinant rat MIF in a concentration-dependent manner. MIF induced CD74-asssociated c-Met activation, which was blocked by knocking down CD74 expression using siRNA. MIF also induced Akt and associated FOXO3a phosphorylation, and this effect was abolished by knocking down either CD74 or Akt. In addition, MIF decreased oxidative stress in MSCs, as shown by decreased ROS and MDA, and increased the activity of SOD. Knockdown of CD74, Akt, or FOXO3a largely attenuated the anti-apoptotic effect of MIF and its ability to protect against oxidative stress. MIF protected MSCs from hypoxia/SD-induced apoptosis by interacting with CD74 to stimulate c-Met, leading to downstream PI3K/Akt-FOXO3a signaling and decreased oxidative stress.

Gremlin-1 is an inhibitor of macrophage migration inhibitory factor and attenuates atherosclerotic plaque growth in ApoE-/- Mice.

Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE(-/-) mice and is secreted from activated monocytes and during macrophage development in vitro. Gremlin-1 reduces macrophage formation by inhibiting macrophage migration inhibitory factor (MIF), a cytokine critically involved in atherosclerotic plaque progression and vulnerability. Gremlin-1 binds with high affinity to MIF (KD = 54 nm), as evidenced by surface plasmon resonance analysis and co-immunoprecipitation, and reduces MIF-induced release of TNF-α from macrophages. Treatment of ApoE(-/-) mice with a dimeric recombinant fusion protein, mGremlin1-Fc, but not with equimolar control Fc or inactivated mGremlin1-Fc, reduced TNF-α expression, the content of monocytes/macrophages of atherosclerotic lesions, and attenuated atheroprogression. The present data disclose that Gremlin-1 is an endogenous antagonist of MIF and define a role for Gremlin-1/MIF interaction in atherosclerosis.

CD74: a new prognostic factor for patients with malignant pleural mesothelioma.

BACKGROUND: The pro-inflammatory cytokine migration inhibitory factor (MIF) and its receptor CD74 have been proposed as possible therapeutic targets in several cancers. We studied the expression of MIF and CD74 together with calretinin in specimens of malignant pleural mesothelioma (MPM), correlating their expression levels with clinico-pathologic parameters, in particular overall survival (OS). METHODS: Migration inhibitory factor, CD74, and calretinin immunoreactivity were investigated in a tissue microarray of 352 patients diagnosed with MPM. Protein expression intensities were semiquantitatively scored in the tumour cells and in the peritumoral stroma. Markers were matched with OS, age, gender, and histological subtype. RESULTS: Clinical data from 135 patients were available. Tumour cell expressions of MIF and CD74 were observed in 95% and 98% of MPM specimens, respectively, with a homogenous distribution between the different histotypes. CD74 (P<0.001) but not MIF overexpression (P=0.231) emerged as an independent prognostic factor for prolonged OS. High expression of tumour cell calretinin correlated with the epithelioid histotype and was also predictive of longer OS (P<0.001). When compared with previously characterised putative epithelial-to-mesenchymal transition markers, CD74 correlated positively with tumoral PTEN and podoplanin expressions, but was inversely related with periostin expression. CONCLUSIONS: High expression of CD74 is an independent prognostic factor for prolonged OS in mesothelioma patients.