Macrophage migration inhibitory factor promotes renal injury induced by ischemic reperfusion

Macrophage migration inhibitory factor (MIF) is pleiotropic cytokine that has multiple effects in many inflammatory and immune diseases. This study reveals a potential role of MIF in acute kidney injury (AKI) in patients and in kidney ischemic reperfusion injury (IRI) mouse model in MIF wild‐type (WT) and MIF knockout (KO) mice. Clinically, plasma and urinary MIF levels were largely elevated at the onset of AKI, declined to normal levels when AKI was resolved and correlated tightly with serum creatinine independent of disease causes. Experimentally, MIF levels in plasma and urine were rapidly elevated after IRI‐AKI and associated with the elevation of serum creatinine and the severity of tubular necrosis, which were suppressed in MIF KO mice. It was possible that MIF may mediate AKI via CD74/TLR4‐NF‐κB signalling as mice lacking MIF were protected from AKI by largely suppressing CD74/TLR‐4‐NF‐κB associated renal inflammation, including the expression of MCP‐1, TNF‐α, IL‐1β, IL‐6, iNOS, CXCL15(IL‐8 in human) and infiltration of macrophages, neutrophil, and T cells. In conclusion, our study suggests that MIF may be pathogenic in AKI and levels of plasma and urinary MIF may correlate with the progression and regression of AKI.     

Macrophage migration inhibitory factor (MIF) promotes rat airway muscle cell proliferation and migration mediated by ERK1/2 and FAK signaling

Macrophage migration inhibitory factor (MIF) is an inflammatory mediator that contributes to asthmatic airway remodeling; however, little is known regarding the effects of MIF on airway smooth muscle cells (ASMCs). In the present study, we found that an enhanced expression of MIF promoted ASMC proliferation, increased the population of cells in the S/G2 phase, downregulated P21 expression, and upregulated cyclin D1, cyclin D3, and Cdk6 expression. In addition, the apoptosis of ASMCs was significantly decreased in response to MIF overexpression, compared with the negative control. Moreover, MIF facilitated the migration of ASMCs by upregulating the expression of matrix metalloproteinase (MMP)‐2. Finally, we showed that MIF increased the phosphorylation of extracellular regulated protein kinases (ERK) 1/2 and focal adhesion kinase (FAK), which are associated with proliferation and migration. In conclusion, this study demonstrated that MIF overexpression promotes the proliferation and migration of ASMCs by upregulating the activity of the ERK1/2 and FAK signaling pathways in these cells, further indicating that inhibition of MIF may prove to be an effective strategy for treating asthma patients with airway remodeling.     

Macrophage migration inhibitory factor-deficient mice are resistant to ovariectomy-induced bone loss

A link between macrophage migration inhibitory factor (MIF) and estrogen has recently emerged. We examined the involvement of MIF in osteoporotic changes in bone after ovariectomy (OVX), and revealed that MIF-deficient mice (MIF-KO) were completely protected from this phenomenon. The increase in osteoclast number per bone surface and serum IL-1β levels, which were observed in wild-type mice after OVX, did not occur in MIF KO. Our data suggest that MIF plays an important role in the pathogenesis of postmenopausal osteoporosis, and could be a novel target for the treatment of this disease.     

Macrophage migration inhibitory factor (MIF): a key player in protozoan infections

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine produced by the pituitary gland and multiple cell types, including macrophages (Mø), dendritic cells (DC) and T-cells. Upon releases MIF modulates the expression of several inflammatory molecules, such as TNF-α, nitric oxide and cyclooxygenase 2 (COX-2). These important MIF characteristics have prompted investigators to study its role in parasite infections. Several reports have demonstrated that MIF plays either a protective or deleterious role in the immune response to different pathogens. Here, we review the role of MIF in the host defense response to some important protozoan infections.     

Blockage of macrophage migration inhibitory factor (MIF) suppressed uric acid-induced vascular inflammation,smooth muscle cell de-differentiation,and remodeling

Hyperuricemia contributes to vascular injury and dysfunction, yet the potential mechanisms are not well understood. Uric acid (UA) has been found to stimulate macrophage migration inhibitory factor (MIF) up-regulation in renal tubules from rats subjected to UA-induced nephropathy. Given that MIF is able to induce vascular smooth muscle cell (VSMC) de-differentiation (from contractile state to a secretory state), we thus hypothesized that UA-induced vascular injury is via up-regulating of MIF in VSMCs, which enhancing vascular inflammation and VSMC transition. Within a mouse model of UA injection (500?mg/kg, twice/day, 14 days), we measured circulating and vascular MIF levels under UA stimulation at 6?h, day 1, and 14. We tested the efficacy of MIF inhibitor (10?mg/kg, twice/day, 14 days) on UA-induced vascular inflammation and remodeling. High plasma level of UA induced vascular MIF release into the plasma at acute phase. In the chronic phase, the protein level of MIF is up-regulated in the vessels. MIF inhibitor suppressed vascular inflammatory responses, repressed VSMC de-differentiation, and attenuated vascular remodeling and dysfunction following UA stimulation. Knockdown of MIF in cultured VSMCs repressed UA-induced de-differentiation. Our results provided a novel mechanism for MIF-mediated vascular injury in response to UA stimulation, and suggested that anti-MIF interventions may be of therapeutic value in hyperuricemic patients.     

Macrophage migration inhibitory factor (MIF) is rendered enzymatically inactive by myeloperoxidase-derived oxidants but retains its immunomodulatory function

Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF.     

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.     

A functional heteromeric MIF receptor formed by CD74 and CXCR4

MIF is a chemokine-like inflammatory mediator that triggers leukocyte recruitment by binding to CXCR2 and CXCR4. MIF also interacts with CD74/invariant chain, a single-pass membrane-receptor. We identified complexes between CD74 and CXCR2 with a role in leukocyte recruitment. It is unknown whether CD74 also binds to CXCR4. We demonstrate that CD74/CXCR4 complexes formed when CD74 was expressed with CXCR4 in HEK293 cells. Expression of CD74-variants lacking an ER-retention signal showed CD74/CXCR4 complexes at the cell surface. Importantly, endogenous CD74/CXCR4 complexes were isolated by co-immunoprecipitation from monocytes. Finally, MIF-stimulated CD74-dependent AKT activation was blocked by anti-CXCR4 and anti-CD74 antibodies and AMD3100, whereas CXCL12-stimulated AKT activation was not reduced by anti-CD74. Thus, CD74 forms functional complexes with CXCR4 that mediate MIF-specific signaling.

Structured summary

MINT-7234512, MINT-7234528: CD74 (uniprotkb:P04233) physically interacts (MI:0915) with CXCR4 (uniprotkb:P61073) by anti tag coimmunoprecipitation (MI:0007)MINT-7234542: CD74 (uniprotkb:P04233) and CXCR4 (uniprotkb:P61073) physically interact (MI:0915) by anti bait coimmunoprecipitation (MI:0006)MINT-7234499: CXCR4 (uniprotkb:P61073) and CD74 (uniprotkb:P04233) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Macrophage migration inhibitory factor regulates joint capsule fibrosis by promoting TGF-β1 production in fibroblasts

Joint capsule fibrosis caused by excessive inflammation results in post-traumatic joint contracture (PTJC). Transforming growth factor (TGF)-β1 plays a key role in PTJC by regulating fibroblast functions, however, cytokine-induced TGF-β1 expression in specific cell types remains poorly characterized. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in inflammation- and fibrosis-associated pathophysiology. In this study, we investigated whether MIF can facilitate TGF-β1 production from fibroblasts and regulate joint capsule fibrosis following PTJC. Our data demonstrated that MIF and TGF-β1 significantly increased in fibroblasts of injured rat posterior joint capsules. Treatment the lesion sites with MIF inhibitor 4-Iodo-6-phenylpyrimidine (4-IPP) reduced TGF-β1 production and relieved joint capsule inflammation and fibrosis. In vitro, MIF facilitated TGF-β1 expression in primary joint capsule fibroblasts by activating mitogen-activated protein kinase (MAPK) (P38, ERK) signaling through coupling with membrane surface receptor CD74, which in turn affected fibroblast functions and promoted MIF production. Our results reveal a novel function of trauma-induced MIF in the occurrence and development of joint capsule fibrosis. Further investigation of the underlying mechanism may provide potential therapeutic targets for PTJC.     

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.     

胰腺癌组织中巨噬细胞移动抑制因子的表达及其意义

目的研究巨噬细胞移动抑制因子（MIF）在胰腺癌发生发展中的作用,与肿瘤标志物CEA、CA199的关系。方法应用免疫组化方法检测31例胰腺癌组织、癌旁组织以及14例正常胰腺组织中MIF表达水平,分析MIF表达与各项临床病理特点及血清CEA、CA199水平的关系。结果 MIF在胰腺癌组织中的表达为87.1%,高于癌旁组织的54.8%和正常胰腺组织的7.4%（P〈0.01）;癌旁组织的MIF表达高于正常组织（P〈0.01）。MIF的表达与肿瘤分化程度及远处转移有关（P〈0.05）,MIF表达阳性患者的血清CA199水平高于MIF表达阴性患者,而血清CEA水平两组间无显著统计学意义。结论 MIF对胰腺癌的发生发展起重要作用,可能促进正常腺体组织向胰腺癌发生和发展。MIF可作为胰腺癌的一种血清标志物,联合CA199的检测可更好的发现胰腺癌。     

Early release of macrophage migration inhibitory factor after liver ischemia and reperfusion injury in rats

Macrophage migration inhibitory factor (MIF) is an important mediator of ischemia/reperfusion (I/R) injury in heart, brain and intestine. We previously demonstrated that MIF was released during warm/cold ischemia in vitro. However, the role of MIF in liver I/R injury remains unclear. We aimed to test the hypothesis that MIF acts as an early proinflammatory cytokine and could mediate the inflammatory injury in liver I/R. Rats (n = 6 per group) were subjected to 90 min warm ischemia followed by 0.5 h, 6 h and 24 h reperfusion, respectively to liver transplantation (LTx) after 6 h of cold ischemia followed by 24 h of reperfusion. The expression of MIF, its receptor (cluster of differentiation 74 (CD74)) and the downstream inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)) were analyzed. Peritoneal macrophages were cultured for 6 h alone or in the presence of effluent from cold-preserved livers or effluent depleted of MIF. Warm I/R increased hepatic MIF-mRNA and protein expression. MIF-protein was released into peripheral circulation in vivo with a maximum at 0.5 h after reperfusion. Induction of MIF-expression was associated with the expression of proinflammatory cytokines and its receptor in both models. MIF released by isolated cold preserved livers, induced TNF-α and IL-1β production by cultured peritoneal macrophages. Intrahepatic upregulation of MIF, release into systemic circulation and the associated upregulation of the proinflammatory mediators suggest a role of MIF in mediating the inflammatory response to I/R injury. Blocking experiments will help to elucidate its role as potential molecular target for preventing hepatic I/R injury.     

Macrophage migration inhibitory factor diminishes muscle glucose transport induced by insulin and AICAR in a muscle type-dependent manner

Skeletal muscle is a primary organ that uses blood glucose. Insulin- and 5′AMP-activated protein kinase (AMPK)-regulated intracellular signaling pathways are known as major mechanisms that regulate muscle glucose transport. It has been reported that macrophage migration inhibitory factor (MIF) is secreted from adipose tissue and heart, and affects these two pathways. In this study, we examined whether MIF is a myokine that is secreted from skeletal muscles and affects muscle glucose transport induced by these two pathways. We found that MIF is expressed in several different types of skeletal muscle. Its secretion was also confirmed in C2C12 myotubes, a skeletal muscle cell line. Next, the extensor digitorum longus (EDL) and soleus muscles were isolated from mice and treated with recombinant MIF in an in vitro muscle incubation system. MIF itself did not have any effect on glucose transport in both types of muscles. However, glucose transport induced by a submaximal dose of insulin was diminished by co-incubation with MIF in the soleus muscle. MIF also diminished glucose transport induced by a maximal dose of 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), an AMPK activator, in the EDL muscle. These results suggest that MIF is a negative regulator of insulin- and AICAR-induced glucose transport in skeletal muscle. Since MIF secretion from C2C12 myotubes to the culture medium decreased during contraction evoked by electrical stimulations, MIF may be involved in the mechanisms underlying exercise-induced sensitization of glucose transport in skeletal muscle.     

Specific reduction of insulin disulfides by macrophage migration inhibitory factor (MIF) with glutathione and dihydrolipoamide: potential role in cellular redox processes

The molecular mechanism of action of MIF, a cytokine that plays a critical role in the host immune and inflammatory response, has not yet been identified. We recently demonstrated that MIF is an enzyme that exhibits oxidoreductase activity by a cysteine thiol-mediated mechanism. Here we further investigated this function by examining the reduction of insulin disulfides by wild-type human MIF (wtMIF) using various substrates, namely glutathione (GSH), dihydrolipoamide, l-cysteine, β-mercaptoethanol and dithiothreitol. The activity of wtMIF was compared to that of the relevant cysteine mutants of MIF and to two carboxy-truncated mutants. Only GSH and dihydrolipoamide were found to serve as reductants, whereas the other substrates were not utilized by MIF. Reduction of insulin disulfides by MIF was closely dependent on the presence of the Cys⁵⁷-Ala-Leu-Cys⁶⁰ (CALC) motif-forming cysteines C57 and C60, whereas C81 was not involved (activities: 51±13%, 14±5%, and 70±12% of wtMIF, respectively, and 20±3% for the double mutant C57S/C60S). Confirming the notion that the activity of MIF was dependent on the CALC motif in the central region of the MIF sequence, the C-terminal deletion mutants MIF(1–105) and MIF(1–110) were found to be fully active. The favored use of GSH and dihydrolipoamide indicated that MIF may be involved in the regulation of cellular redox processes and was supported further by the finding that MIF expression by the cell lines COS-1 and RAW 264.7 was significantly induced upon treatment with the oxidant hydrogen peroxide.     

Hypoxia‐induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non‐cognate ligand of the CXC‐family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine‐like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC‐derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia‐induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non‐classical mechanism and originated from pre‐formed MIF stores. Early hypoxia‐triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia‐inducible factor (HIF)‐1α‐induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF‐1α‐induced de novo synthesis of MIF. To functionally investigate the role of hypoxia‐inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL⁺ KDR⁺ CD31⁺ EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose‐dependent bell‐shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia‐conditioned HUVECs, an effect that was completely abrogated by anti‐MIF‐ or anti‐CXCR4‐antibodies. Thus, hypoxia‐induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia‐induced myocardial damage.     

A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli

Mouse embryonic stem cells (mESCs) rely on a cytokine named leukemia inhibitory factor (LIF) to maintain their undifferentiated state and pluripotency. However, the progress of mESC research is restricted and limited to highly funded laboratories due to the cost of commercial LIF. Here we presented the homemade hLIF which is biologically active. The hLIF cDNA was cloned into two different vectors in order to produce N-terminal His₆-tag and Trx-His₆-tag hLIF fusion proteins in Origami(DE3) Escherichia coli. The His₆-hLIF fusion protein was not as soluble as the Trx-His₆-hLIF fusion protein. One-step immobilized metal affinity chromatography (IMAC) was done to recover high purity (>95% pure) His₆-hLIF and Trx-His₆-hLIF fusion proteins with the yields of 100 and 200 mg/l of cell culture, respectively. The hLIF fusion proteins were identified by Western blot and verified by mass spectrometry (LC/MS/MS). The hLIF fusion proteins specifically promote the proliferation of TF-1 cells in a dose-dependent manner. They also demonstrate the potency to retain the morphology of undifferentiated mESCs, in that they were positive for mESC markers (Oct-4, Sox-2, Nanog, SSEA-1 and alkaline phosphatase activity). These results demonstrated that the N-terminal fusion tags of the His₆-hLIF and Trx-His₆-hLIF fusion proteins do not interfere with their biological activity. This expression and purification approach to produce recombinant hLIF is a simple, reliable, cost effective and user-friendly method.