anti-MIF Antibody Research Articles

MIF is essential to the establishment of house dust mite-induced airway inflammation and tissue remodeling in mice.

Macrophage migration inhibitory factor (MIF) is present in high amounts in the bronchoalveolar lavage fluid (BALF) and serum of asthmatic patients contributing to the pathogenesis of experimental asthma induced by ovalbumin (OVA) in mice. Whether MIF contributes to the physiopathology on a more complex and relevant asthma model has not been characterized. Mif-deficient (Mif-/- ) or wild-type (WT) mice treated with anti-MIF antibody were challenged multiple times using house dust mite (HDM) extract by intranasal route. HDM-challenged Mif-/- mice presented decreased airway hyperresponsiveness, lung infiltration of eosinophils, mucus hypersecretion and subepithelial fibrosis compared to HDM-challenged WT mice. Amounts IL-4, IL-5 and IL-13 were decreased in the lungs of Mif-/- mice upon HDM challenges, but the increase of CCL11 was preserved, compared to HDM-challenged WT mice. We also observed increased numbers of group 2 innate lymphoid cells and Th2 cells in the BALF and mediastinal lymph nodes (mLN) induced challenged by HDM of WT mice, but not in HDM-challenged Mif-/- mice. Anti-MIF treatment abrogated the airways infiltration of eosinophils, mucus hypersecretion and subepithelial fibrosis in the lungs of HDM-challenged mice. In conclusion, MIF ablation prevents the pathologic hallmarks of asthma in HDM-challenged mice, reinforcing the promising target of MIF for asthma therapy. This article is protected by copyright. All rights reserved.

MIF but not MIF-2 recruits inflammatory macrophages in an experimental polymicrobial sepsis model.

Excessive inflammation drives the progression from sepsis to septic shock. Macrophage migration inhibitory factor (MIF) is of interest because MIF promoter polymorphisms predict mortality in different infections, and anti-MIF antibody improves survival in experimental models when administered 8 hours after infectious insult. The recent description of a second MIF superfamily member, D-dopachrome tautomerase (D-DT/MIF-2), prompted closer investigation of MIF-dependent responses. We subjected Mif-/- and Mif-2-/- mice to polymicrobial sepsis and observed a survival benefit with Mif but not Mif-2 deficiency. Survival was associated with reduced numbers of small peritoneal macrophages (SPMs) that, in contrast to large peritoneal macrophages (LPMs), were recruited into the peritoneal cavity. LPMs produced higher quantities of MIF than SPMs, but SPMs expressed higher levels of inflammatory cytokines and the MIF receptors CD74 and CXCR2. Adoptive transfer of WT SPMs into Mif-/- hosts reduced the protective effect of Mif deficiency in polymicrobial sepsis. Notably, MIF-2 lacks the pseudo-(E)LR motif present in MIF that mediates CXCR2 engagement and SPM migration, supporting a specific role for MIF in the recruitment and accumulation of inflammatory SPMs.

MP28-13 ACTIVATION OF INTRAVESICAL MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) RECEPTORS MEDIATES BLADDER PAIN

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I (MP28)1 Apr 2020MP28-13 ACTIVATION OF INTRAVESICAL MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) RECEPTORS MEDIATES BLADDER PAIN Shaojing Ye, Fei Ma, Katherine Meyer-Siegler, Lin Leng, Richard Bucala, and Pedro Vera* Shaojing YeShaojing Ye More articles by this author , Fei MaFei Ma More articles by this author , Katherine Meyer-SieglerKatherine Meyer-Siegler More articles by this author , Lin LengLin Leng More articles by this author , Richard BucalaRichard Bucala More articles by this author , and Pedro Vera*Pedro Vera* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000867.013AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Activation of intravesical protease activated receptor-4 (PAR4) releases urothelial MIF to mediate release of urothelial high mobility group box1 (HMGB1) and mediate bladder pain. MIF receptors (CD74, CXCR2 OR CXCR4) are all localized within the urothelium and their role in bladder pain remains to be determined. We hypothesized that MIF mediates bladder pain by interacting with urothelial MIF receptors. Therefore, we tested whether intravesical antagonism of MIF reduces or prevents bladder pain. METHODS: PAR4 (100 µM; 1hr) was intravesically infused in female C57BL/6 mice under isoflurane anesthesia. Bladder pain (50% mechanical threshold on lower abdomen by von Frey (VF) stimulation) was measured before (baseline) and 24 hr post-PAR4 infusion. Scrambled PAR4 peptide (Scramb) was used as control. Intravesical pre-treatment (10 min prior to PAR4) with PBS, isotype control antibodies, neutralizing anti-MIF or anti-CD74 monoclonal antibodies, AMD-3100 (CXCR4 antagonist) and SB225002 (CXCR2 antagonist) tested the effect of specific MIF receptor antagonism on PAR4-induced bladder pain. Voided volume and frequency were also measured at 24 hr. At the end of experiments, bladders were collected for histology. ANOVA with Tukey post-hoc tests was used. RESULTS: Intravesical scrambled peptide did not produce bladder pain while intravesical PAR4 induced bladder pain. Pre-treatment with neutralizing anti-MIF or anti-CD74 antibodies, or CXCR4 antagonist prevented PAR4-induced bladder pain (Figure 1). Pretreatment with CXCR2 antagonist was not effective. No significant effects were noted on micturition volume while anti-CD74 pretreatment increased frequency slightly (Table 1). CONCLUSIONS: Intravesical MIF receptors (CD74 and CXCR4, but not CXCR2) mediate bladder pain. Future experiments will examine how intravesical antagonism of MIF or its receptors contributes to downstream mechanisms (e.g. release of urothelial HMGB1) that mediate bladder pain. Source of Funding: DK121695 (PLV); AR049610 (RB) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e425-e425 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shaojing Ye More articles by this author Fei Ma More articles by this author Katherine Meyer-Siegler More articles by this author Lin Leng More articles by this author Richard Bucala More articles by this author Pedro Vera* More articles by this author Expand All Advertisement PDF downloadLoading ...

Selective Recruitment of Lethal Pro-inflammatory Macrophages in Sepsis by MIF but not D-DT (MIF-2)

Abstract Sepsis is a leading cause of mortality with an estimated global incidence of 30 million cases annually. Current paradigms emphasize the role of an overwhelming or dysregulated inflammatory response in sepsis lethality, but to date interventions aimed at particular inflammatory pathways have been unsuccessful. Macrophage migration inhibitory factor (MIF) is of interest because MIF promoter polymorphisms predict mortality in different infections and anti-MIF antibody improves survival in experimental models when administered eight hours after infectious insult. The recent description of a second MIF superfamily member, D-dopachrome tautomerase (D-DT/MIF-2), prompts closer investigation of MIF-dependent responses. This study provides new insight into the pathophysiology of polymicrobial sepsis by revealing a direct role for MIF, but not its close structural homolog D-DT/MIF-2, in activating and recruiting a lethal macrophage subset. We subjected Mif −/− and Mif-2−/− mice to polymicrobial sepsis and observed a survival benefit with Mif but not Mif-2 deficiency. Survival was associated with lower levels of small peritoneal macrophages (SPMs) that, in contrast to large peritoneal macrophages (LPMs), are recruited to site of peritoneal inflammation during sepsis. LPMs produce higher quantities of MIF than SPMs, but SPMs express higher levels of inflammatory cytokines and MIF receptors CD74 and CXCR2. Adoptive transfer of SPMs into Mif −/− hosts abrogated the protective effect of Mif deficiency. Notably, MIF-2 lacks the pseudo-(E)LR motif present in MIF that mediates CXCR2 engagement and SPM migration, supporting MIF’s selective role in the recruitment and accumulation of lethal inflammatory SPMs.

Role of Macrophage Migration Inhibitory Factor in Granulomatosis With Polyangiitis.

To examine the association between macrophage migration inhibitory factor (MIF) promoter polymorphisms and granulomatosis with polyangiitis (GPA) in human subjects, and to assess the role of MIF in a murine model of granulomatous vasculitis. The human study involved 1,077 patients with GPA and healthy controls whose serum was genotyped by capillary electrophoresis for the MIF -794 CATT5-8 promoter microsatellite (rs5844572). MIF promoter, CATT-length-dependent gene expression in response to β-glucan was assessed by gene reporter assays. In mouse studies, granulomatous disease was induced by injection of Candida albicans β-glucan into wild-type (WT) or Mif-knockout (Mif-KO) C57BL/6 mice and C57BL/6 mice transgenically overexpressing Mif in lung epithelium (Mif lung-Tg2.1). Mice were treated with a neutralizing anti-MIF antibody and analyzed for the density of pulmonary granulomas, expression of inflammatory chemokines, and frequency of mortality. The percentage of human subjects carrying >5 CATT repeats in each MIF allele (high genotypic MIF expressers) was 60.2% among patients with GPA and 53.9% among healthy controls (adjusted P = 0.049). In response to granulomatous stimulation, human MIF gene expression increased proportionally with CATT length. Mif lung-Tg2.1 mice exhibited more pulmonary granulomas than WT mice, which in turn showed more granulomas than Mif-KO mice. A significantly higher percentage of Mif lung-Tg2.1 mice, compared to Mif-KO or WT mice, died when injected with Candida albicans β-glucan, and treatment of these mice with an anti-MIF monoclonal antibody protected against a lethal outcome. Levels of MIF-dependent neutrophil/macrophage chemokines were elevated in the bronchoalveolar lavage fluid or plasma of Mif lung-Tg2.1 mice. Patients with GPA have an increased frequency of high MIF expression CATT alleles. Higher Mif expression increases the incidence of mortality and pulmonary granulomas in Mif lung-Tg2.1 mice, while anti-MIF treatment protects these mice against death. Blockade of MIF in high genotypic MIF expressers may therefore offer a selective pharmacologic therapy for GPA.

Macrophage migration inhibitory factor mediates metabolic dysfunction induced by atypical antipsychotic therapy.

Atypical antipsychotics are highly effective antischizophrenic medications but their clinical utility is limited by adverse metabolic sequelae. We investigated whether upregulation of macrophage migration inhibitory factor (MIF) underlies the insulin resistance that develops during treatment with the most commonly prescribed atypical antipsychotic, olanzapine. Olanzapine monotherapy increased BMI and circulating insulin, triglyceride, and MIF concentrations in drug-naive schizophrenic patients with normal MIF expression, but not in genotypic low MIF expressers. Olanzapine administration to mice increased their food intake and hypothalamic MIF expression, which led to activation of the appetite-related AMP-activated protein kinase and Agouti-related protein pathway. Olanzapine also upregulated MIF expression in adipose tissue, which reduced lipolysis and increased lipogenic pathways. Increased plasma lipid concentrations were associated with abnormal fat deposition in liver and skeletal muscle, which are important determinants of insulin resistance. Global MIF-gene deletion protected mice from olanzapine-induced insulin resistance, as did intracerebroventricular injection of neutralizing anti-MIF antibody, supporting the role of increased hypothalamic MIF expression in metabolic dysfunction. These findings uphold the potential pharmacogenomic value of MIF genotype determination and suggest that MIF may be a tractable target for reducing the metabolic side effects of atypical antipsychotic therapy.

Role of the Macrophage Migration Inhibitory Factor (MIF) in the survival of first trimester human placenta under induced stress conditions

Macrophage Migration Inhibitory Factor (MIF) is a multifunctional molecule highly secreted by human placenta mainly in the early phases of pregnancy. Studies in different cells show that MIF is a pro-survival factor by binding to its receptor CD74. By using the in vitro model of placental explants from first trimester pregnancy, we investigated the role of MIF in the survival of placental cells under induced stress conditions that promote apoptosis or mimic the hypoxia/re-oxygenation (H/R) injury that placenta could suffer in vivo. We demonstrated that recombinant MIF (rMIF) treatment was able to reduce caspase-3 activation when cultures were challenged with the apoptosis-inducer Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) while, in the cultures exposed to H/R, the treatment with rMIF did not show any effect. However, a significant increase in caspase-3 and caspase-8 activation was found when H/R-exposed cultures, were treated with anti-MIF or anti-CD74 antibody. We also observed that under H/R, a significant amount of endogenous MIF was released into the medium, which could account for the lack of effect of rMIF added to the cultures. Our results demonstrate for the first time that the MIF/CD74 axis contributes to maintain trophoblast homeostasis, by preventing abnormal apoptotic death.

Neutralization of macrophage migration inhibitory factor improves host survival after Clostridium difficile infection

Clostridium difficile is an important cause of nosocomial diarrhea in the western world. Toxins (A, B, and binary toxins) generated by C. difficile bacteria damage intestinal epithelial cells. Hallmarks of host response to C. difficile infection (CDI) include upregulation of inflammatory mediators and tissue infiltration by immune cells. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that is known to enhance the host immune response to infectious pathogens. Additionally, MIF can adversely impact host survival to numerous infections. The role of MIF in the pathogenesis of CDI remains poorly understood. Here, we show that patients with CDI had significantly higher circulating MIF compared to patients who had diarrhea but tested negative for C. difficile (non-CDI controls). Similarly, in a mouse model, C. difficile challenge significantly increased levels of plasma and tissue MIF. Antibody-mediated depletion of MIF decreased C. difficile-induced inflammatory responses, clinical disease, and mortality. Together, these results uncover a potential role for MIF in exacerbating CDI and suggest that use of anti-MIF antibodies may represent a therapeutic strategy to curb host inflammatory responses and improve disease outcomes in CDI.

Oxidized macrophage migration inhibitory factor is a potential new tissue marker and drug target in cancer.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, which was shown to be upregulated in cancers and to exhibit tumor promoting properties. Unlike other cytokines, MIF is ubiquitously present in the circulation and tissue of healthy subjects. We recently described a previously unrecognized, disease-related isoform of MIF, designated oxMIF, which is present in the circulation of patients with different inflammatory diseases. In this article, we report that oxMIF is also linked to different solid tumors as it is specifically expressed in tumor tissue from patients with colorectal, pancreatic, ovarian and lung cancer. Furthermore, oxMIF can be specifically targeted by a subset of phage display-derived fully human, monoclonal anti-MIF antibodies (mAbs) that were shown to neutralize pro-tumorigenic activities of MIF in vivo. We further demonstrate that anti-oxMIF mAbs sensitize human cancer cell lines (LNCaP, PC3, A2780 and A2780ADR) to the action of cytotoxic drugs (mitoxantrone, cisplatin and doxorubicin) in vitro and in an A2780 xenograft mouse model of ovarian cancer. We conclude that oxMIF is the disease related isoform of MIF in solid tumors and a potential new diagnostic marker and drug target in cancer.

Macrophage Migration Inhibitory Factor (MIF) Supports Homing of Osteoclast Precursors to Peripheral Osteolytic Lesions.

By binding to its chemokine receptor CXCR4 on osteoclast precursor cells (OCPs), it is well known that stromal cell-derived factor-1 (SDF-1) promotes the chemotactic recruitment of circulating OCPs to the homeostatic bone remodeling site. However, the engagement of circulating OCPs in pathogenic bone resorption remains to be elucidated. The present study investigated a possible chemoattractant role of macrophage migration inhibitory factor (MIF), another ligand for C-X-C chemokine receptor type 4 (CXCR4), in the recruitment of circulating OCPs to the bone lytic lesion. To accomplish this, we used Csf1r-eGFP-knock-in (KI) mice to establish an animal model of polymethylmethacrylate (PMMA) particle-induced calvarial osteolysis. In the circulating Csf1r-eGFP+ cells of healthy Csf1r-eGFP-KI mice, Csf1r+/CD11b+ cells showed a greater degree of RANKL-induced osteoclastogenesis compared to a subset of Csf1r+/RANK+ cells in vitro. Therefore, Csf1r-eGFP+/CD11b+ cells were targeted as functionally relevant OCPs in the present study. Although expression of the two cognate receptors for MIF, CXCR2 and CXCR4, was elevated on Csf1r+/CD11b+ cells, transmigration of OCPs toward recombinant MIF in vitro was facilitated by ligation with CXCR4, but not CXCR2. Meanwhile, the level of PMMA-induced bone resorption in calvaria was markedly greater in wild-type (WT) mice compared to that detected in MIF-knockout (KO) mice. Interestingly, in contrast to the elevated MIF, diminished SDF-1 was detected in a particle-induced bone lytic lesion of WT mice in conjunction with an increased number of infiltrating CXCR4+ OCPs. However, such diminished SDF-1 was not found in the PMMA-injected calvaria of MIF-KO mice. Furthermore, stimulation of osteoblasts with MIF in vitro suppressed their production of SDF-1, suggesting that MIF can downmodulate SDF-1 production in bone tissue. Systemically administered anti-MIF neutralizing monoclonal antibody (mAb) inhibited the homing of CXCR4+ OCPs, as well as bone resorption, in the PMMA-injected calvaria, while increasing locally produced SDF-1. Collectively, these data suggest that locally produced MIF in the inflammatory bone lytic site is engaged in the chemoattraction of circulating CXCR4+ OCPs. © 2016 American Society for Bone and Mineral Research.

Targeting MIF in Cancer: Therapeutic Strategies, Current Developments, and Future Opportunities.

Strong evidence has been presented linking chronic inflammation to the onset and pathogenesis of cancer. The multifunctional pro-inflammatory protein macrophage migration inhibitory factor (MIF) occupies a central role in the inflammatory pathway and has been implicated in the tumorigenesis, angiogenesis, and metastasis of many cancer phenotypes. This review highlights the current state of the art, which presents MIF, and the second member of the MIF structural superfamily, D-DT (MIF2), as significant mediators in the inflammatory-cancer axis. Although the mechanism by which MIF asserts its biological activity has yet to be fully understood, it has become clear in recent years that for certain phenotypes of cancer, MIF represents a valid therapeutic target. Current research efforts have focused on small molecule approaches that target MIF's unique tautomerase active site and neutralization of MIF with anti-MIF antibodies. These approaches have yielded promising results in a number of preclinical murine cancer models and have helped to increase our understanding of MIF biological activity. More recently, MIF's involvement in a number of key protein-protein interactions, such as with CD74 and HSP90, has been highlighted and provides a novel platform for the development of anti-MIF chemotherapeutic strategies in the future.

Positive correlations of age and parity with plasma concentration of macrophage migration inhibitory factor in Japanese black cows.

Plasma Macrophage migration inhibitory factor (MIF) concentration correlates positively with age, and negatively with self-rated health in women, and optimal MIF concentration may promote proper reproductive function. This study was conducted to evaluate the hypotheses that plasma MIF concentration changes with parturition or postpartum first ovulation, and that age in months and parity correlate with plasma MIF concentration in Japanese black cows. Western blotting utilizing an anti-MIF mouse monoclonal antibody of various tissues and plasma from females indicated that MIF expression was stronger in the anterior pituitary than in other tissues. We developed a competitive EIA utilizing the same anti-MIF mouse monoclonal antibody with sufficient sensitivity and reliable performance for measuring bovine plasma samples. We then measured MIF concentrations in bovine plasma collected from 4 weeks before parturition to 4 weeks after postpartum first ovulation. There was no significant difference in plasma MIF concentration pre- and post-parturition, or before and after the postpartum first ovulation. Plasma MIF concentrations were positively correlated (P < 0.01) with parity (r = 0.703), age in months on the day of parturition (r = 0.647), and age in months on the day of the postpartum first ovulation (r = 0.553) when we used almost all data, except for that from a third-parity cow with an abnormally high plasma MIF concentration. We therefore concluded that plasma MIF concentrations may increase with age in months and parity, but do not change either before and after parturition or before and after postpartum first ovulation in Japanese black cows.

MIF contributes to Trypanosoma brucei associated immunopathogenicity development.

African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6Chigh inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity.

The critical role of macrophage migration inhibitory factor in insulin activity

Macrophage migration inhibitory factor (MIF) is a molecule with plethora of functions such as regulation of immune response, hormone-like, enzymatic and chaperone-like activity. Further, MIF is a major participant in glucose homeostasis since it is an autocrine stimulator of insulin secretion. MIF absence in male knockout mice (MIF-KO) results in development of glucose intolerance, while sensitivity to insulin is fully preserved. Since our results confirm that beta cells from MIF-KO mice express, produce and secrete insulin similarly to beta cells of their wild type (WT) counterparts C57BL/6 mice, we hypothesize that MIF-KO-derived insulin is less active. Indeed, insulin from MIF-KO islets is unable to significantly induce glucose uptake into hepatocytes and to efficiently promote insulin-triggered Akt phosphorylation determined by immunoblot. However, MIF’s tautomerase function is not crucial for insulin biosynthesis since MIF inhibitors had no impact on WT insulin activity. Importantly, MIF recognition by anti-MIF antibody (ELISA) after in vitro co-incubation with purified insulin was significantly lower suggesting that insulin covers MIF immunodominant epitope. In addition, MIF binds insulin within beta cell as confirmed by co-immunoprecipitation. WT and MIF-KO-derived insulin exhibited different cleavage patterns suggesting different protein conformations. Finally, pre-incubation of recombinant MIF with insulin promotes formation of insulin hexamers. These results imply that MIF probably enables proper insulin folding what results in insulin full activity. This newly discovered feature of the cytokine MIF could be potentially important for commercially produced insulin, for increasing its stability and/or bioavailability.

Compartmentalized Protective and Detrimental Effects of Endogenous Macrophage Migration-Inhibitory Factor Mediated by CXCR2 in a Mouse Model of Myocardial Ischemia/Reperfusion

Here, we aimed to clarify the role of CXC chemokine receptor (CXCR) 2 in macrophage migration-inhibitory factor (MIF)-mediated effects after myocardial ischemia and reperfusion. As a pleiotropic chemokine-like cytokine, MIF has been identified to activate multiple receptors, including CD74 and CXCR2. In models of myocardial infarction, MIF exerts both proinflammatory effects and protective effects in cardiomyocytes. Similarly, CXCR2 displays opposing effects in resident versus circulating cells. Using bone marrow transplantation, we generated chimeric mice with Cxcr2(-/-) bone marrow-derived inflammatory cells and wild-type (wt) resident cells (wt/Cxcr2(-/-)), Cxcr2(-/-) cardiomyocytes and wt bone marrow-derived cells (Cxcr2(-/-)/wt), and wt controls reconstituted with wt bone marrow (wt/wt). All groups were treated with anti-MIF or isotype control antibody before they underwent myocardial ischemia and reperfusion. Blocking MIF increased infarction size and impaired cardiac function in wt/wt and wt/CXCR2(-/-) mice but ameliorated functional parameters in Cxcr2(-/-)/wt mice, as analyzed by echocardiography and Langendorff perfusion. Neutrophil infiltration and angiogenesis were unaltered by MIF blockade or Cxcr2 deficiency. Monocyte infiltration was blunted in wt/Cxcr2(-/-) mice and reduced by MIF blockade in wt/wt and Cxcr2(-/-)/wt mice. Furthermore, MIF blockade attenuated collagen content in all groups in a CXCR2-independent manner. The compartmentalized and opposing effects of MIF after myocardial ischemia and reperfusion are largely mediated by CXCR2. Although MIF confers protective effects by improving myocardial healing and function through CXCR2 in resident cells, thereby complementing paracrine effects through CD74/AMP-activated protein kinase, it exerts detrimental effects on CXCR2-bearing inflammatory cells by increasing monocyte infiltration and impairing heart function. These dichotomous findings should be considered when developing novel therapeutic strategies to treat myocardial infarction.

Human Anti-Macrophage Migration Inhibitory Factor Antibodies Inhibit Growth of Human Prostate Cancer Cells In Vitro and In Vivo

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biologic properties in immunology and oncology. Circulating MIF levels are elevated in several types of human cancer including prostate cancer. MIF is released presumably by both stromal and tumor cells and enhances malignant growth and metastasis by diverse mechanisms, such as stimulating tumor cell proliferation, suppressing apoptotic death, facilitating invasion of the extracellular matrix, and promoting angiogenesis. Recently described fully human anti-MIF antibodies were tested in vitro and in vivo for their ability to influence growth rate and invasion of the human PC3 prostate cancer cell line. In vitro, the selected candidate antibodies BaxG03, BaxB01, and BaxM159 reduced cell growth and viability by inhibiting MIF-induced phosphorylation of the central kinases p44/42 mitogen-activated protein kinase [extracellular signal-regulated kinase-1 and -2 (ERK1/2)] and protein kinase B (AKT). Incubation of cells in the presence of the antibodies also promoted activation of caspase-3/7. The antibodies furthermore inhibited MIF-promoted invasion and chemotaxis as transmigration through Matrigel along a MIF gradient was impaired. In vivo, pharmacokinetic parameters (half-life, volume of distribution, and bioavailability) of the antibodies were determined and a proof-of-concept was obtained in a PC3-xenograft mouse model. Treatment with human anti-MIF antibodies blunted xenograft tumor growth in a dose-dependent manner. We therefore conclude that the anti-MIF antibodies described neutralize some of the key tumor-promoting activities of MIF and thus limit tumor growth in vivo.

Abstract 1235: Human antibodies specific for a novel isoform of the macrophage migration inhibitory factor are potential therapeutics for prostate cancer.

Abstract Macrophage migration inhibitory factor (MIF) was found to be upregulated in many cancers and to act in para- and autocrine loops within the tumor microenvironment. Thereby, MIF contributes to the modulation of cytokine patterns and triggers anti-apoptotic and pro-proliferative cell signaling events. MIF was also described as a pro-angiogenic factor promoting neoangiogenesis and vascularization. In prostate cancer, MIF mRNA and protein levels were shown to be increased and to contribute to tumor development. We have discovered a novel, previously unrecognized conformational isoform of MIF (designated ‘active MIF’) that is produced in diseases and cannot be detected in healthy subjects. A set of phage display derived human monoclonal anti-MIF antibodies neutralizing MIF biological activity in vitro and in vivo specifically recognize active MIF. In vitro, these antibodies elicited both, a growth inhibitory effect and a pro-apoptotic response on PC3 and DU145 prostate cancer cells by interfering with ERK1/2 and Akt/PKB signaling pathways and by promoting activation of caspase-3. Furthermore, the antibodies inhibited tumor cell invasion of PC3 cells in a transwell assay in a dose-dependent manner. In vivo, treatment with human antibodies specific for active MIF dose-dependently inhibited the growth of the human prostate cancer cell line PC3 xenografted into MF1 nude mice. New ELISA methods have been established to allow the detection of active MIF as well as non-active MIF in plasma samples. In the plasma of PC3 xenografted nude mice, we observed a reduction of active MIF upon antibody treatment. In addition we were able to prove the occurrence of active MIF in the plasma of prostate cancer patients (median 2445 pg/mL, p &amp;lt; 0.002 versus controls), whereas active MIF was not detectable in plasma from healthy donors. These studies demonstrate that active MIF represents a new target in prostate cancer, as well as a promising marker for disease activity or disease progression. The data further suggest that our fully human anti-MIF antibodies have a good potential for clinical use in oncology. Citation Format: Michael Thiele, Michael Freissmuth, Filza Hussain, Patrice Douillard, Dirk Völkel, Hartmut Ehrlich, Friedrich Scheiflinger, Randolf Kerschbaumer. Human antibodies specific for a novel isoform of the macrophage migration inhibitory factor are potential therapeutics for prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2013-1235

Delayed development of chronic lymphocytic leukemia in the absence of macrophage migration inhibitory factor

AbstractSurvival of chronic lymphocytic leukemia (CLL) cells depends on stimuli provided by a suitable microenvironment. The factors and mechanisms providing this growth support for CLL cells are not fully understood. We found that plasma levels of macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, were elevated in CLL patients. Therefore, we characterized the functional role of MIF in a CLL mouse model. For this purpose, we crossed Eμ-TCL1 mice with MIF knockout (MIF−/−) mice. The resulting TCL1+/wtMIF−/− mice showed a delayed onset of leukemia, reduced splenomegaly and hepatomegaly, and a longer survival than TCL1+/wtMIFwt/wt controls. Immunohistochemical examination of the lymphoid organs showed that the numbers of macrophages were significantly reduced in the spleen and bone marrow of TCL1+/wtMIF−/− mice compared with TCL1+/wtMIFwt/wt controls. Mechanistic studies in vitro revealed that the absence of MIF rendered CLL cells more susceptible to apoptosis. Accordingly, incubation with an anti-MIF antibody reduced the survival of CLL cells on a macrophage feeder layer. In addition, the migratory activity of TCL1+/wtMIF−/− macrophages was decreased compared with TCL1+/wtMIFwt/wt macrophages. Taken together, our results provide evidence that MIF supports the development of CLL by enhancing the interaction of CLL cells with macrophages.

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.

Neutralization of Macrophage Migration Inhibitory Factor (MIF) by Fully Human Antibodies Correlates with Their Specificity for the β-Sheet Structure of MIF

The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50-68 or 86-102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a β-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this β-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.