Macrophage Migration Inhibitory Factor Antagonism Research Articles

MIF-Modulated Spinal Proteins Associated with Persistent Bladder Pain: A Proteomics Study.

Bladder pain is a prominent symptom in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). We studied spinal mechanisms of bladder pain in mice using a model where repeated activation of intravesical Protease Activated Receptor-4 (PAR4) results in persistent bladder hyperalgesia (BHA) with little or no bladder inflammation. Persistent BHA is mediated by spinal macrophage migration inhibitory factor (MIF), and is associated with changes in lumbosacral proteomics. We investigated the contribution of individual spinal MIF receptors to persistent bladder pain as well as the spinal proteomics changes associated with relief of persistent BHA by spinal MIF antagonism. Female mice with persistent BHA received either intrathecal (i.t.) MIF monoclonal antibodies (mAb) or mouse IgG1 (isotype control antibody). MIF antagonism temporarily reversed persistent BHA (peak effect: 2 h), while control IgG1 had no effect. Moreover, i.t. antagonism of the MIF receptors CD74 and C-X-C chemokine receptor type 4 (CXCR4) partially reversed persistent BHA. For proteomics experiments, four separate groups of mice received either repeated intravesical scrambled peptide and sham i.t. injection (control, no pain group) or repeated intravesical PAR4 and: sham i.t.; isotype IgG1 i.t. (15 μg); or MIF mAb (15 μg). L6-S1 spinal segments were excised 2 h post-injection and examined for proteomics changes using LC-MS/MS. Unbiased proteomics analysis identified and relatively quantified 6739 proteins. We selected proteins that showed significant changes compared to control (no pain group) after intravesical PAR4 (sham or IgG i.t. treatment) and showed no significant change after i.t. MIF antagonism. Six proteins decreased during persistent BHA (V-set transmembrane domain-containing protein 2-like confirmed by immunohistochemistry), while two proteins increased. Spinal MIF antagonism reversed protein changes. Therefore, spinal MIF and MIF receptors mediate persistent BHA and changes in specific spinal proteins. These novel MIF-modulated spinal proteins represent possible new targets to disrupt spinal mechanisms that mediate persistent bladder pain.

Bladder Oxidative Stress and HMGB1 Release Contribute to PAR4-Mediated Bladder Pain in Mice.

Activation of intravesical PAR4 receptors leads to bladder hyperalgesia (BHA) through release of urothelial macrophage migration inhibitory factor (MIF) and urothelial high mobility group box-1 (HMGB1). MIF deficiency and/or MIF antagonism at the bladder block BHA in mice yet the mechanisms are not clear. Since oxidative stress and ERK phosphorylation are involved in MIF signaling we hypothesized that oxidative stress and/or ERK signaling, activated by MIF release, promote intravesical HMGB1 release to induce BHA. We induced BHA by intravesical PAR4 infusion in female C57BL/6 mice. Mechanical sensitivity was evaluated by measuring abdominal von Frey (VF) 50% thresholds before (baseline) and 24 h post-infusion. Intravesical pre-treatment (10 min infusion prior to PAR4) with N-acetylcysteine amide (NACA; reactive-oxygen species scavenger; 3 mg in 50 μl), FR180204 (selective ERK1/2 inhibitor; 200 μg in 50 μl), ethyl pyruvate (EP; HMGB1 release inhibitor; 600 μg in 50 μl), or diluent controls (50 μl) tested the effects of pre-treatment on PAR4-induced BHA. Intravesical fluid was collected after each treatment and HMGB1 concentration was measured using ELISA. Awake micturition parameters (volume and frequency) were assessed at the end of the experiments. Bladders were collected and examined for histological signs of edema and inflammation. Pre-treatment with PBS followed by PAR4 induced BHA in mice but PBS followed by scrambled peptide did not. Pre-treatment with NACA or EP partially blocked PAR4-induced BHA while FR180204 had no effect. A significant correlation between intravesical HMGB1 levels and 50% VF thresholds was observed. All PAR4 treated groups had increased levels of HMGB1 in the intravesical fluid compared to PBS-Scrambled group although not statistically significant. No significant effects were noted on awake micturition volume, micturition frequency or histological evidence of bladder edema or inflammation. Our results show that intravesical antagonism of bladder reactive-oxygen species accumulation was effective in reducing PAR4-induced bladder pain. The correlation between intravesical levels of HMGB1 and bladder pain indicates that released HMGB1 is pivotal to bladder pain. Thus, modulating events in the MIF signaling cascade triggered by PAR4 activation (including bladder oxidative stress and HMGB1 release) warrant further investigation as possible therapeutic strategies.

Targeting Macrophage Migration Inhibitory Factor in Acute Pancreatitis and Pancreatic Cancer.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine implicated in the pathogenesis of inflammation and cancer. It is produced by various cells and circulating MIF has been identified as a biomarker for a range of diseases. Extracellular MIF mainly binds to the cluster of differentiation 74 (CD74)/CD44 to activate downstream signaling pathways. These in turn activate immune responses, enhance inflammation and can promote cancer cell proliferation and invasion. Extracellular MIF also binds to the C-X-C chemokine receptors cooperating with or without CD74 to activate chemokine response. Intracellular MIF is involved in Toll-like receptor and inflammasome-mediated inflammatory response. Pharmacological inhibition of MIF has been shown to hold great promise in treating inflammatory diseases and cancer, including small molecule MIF inhibitors targeting the tautomerase active site of MIF and antibodies that neutralize MIF. In the current review, we discuss the role of MIF signaling pathways in inflammation and cancer and summarize the recent advances of the role of MIF in experimental and clinical exocrine pancreatic diseases. We expect to provide insights into clinical translation of MIF antagonism as a strategy for treating acute pancreatitis and pancreatic cancer.

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 ...

The small molecule macrophage migration inhibitory factor antagonist MIF098, inhibits pulmonary hypertension associated with murine SLE

Pulmonary arterial hypertension (PAH) is a severe complication of systemic lupus erythematosus (SLE), with unclear etiopathogenesis. We evaluated the role of macrophage migration inhibitory factor (MIF), which has been implicated in idiopathic pulmonary hypertension (PH), in SLE-associated PAH. Circulating MIF was measured in SLE patients, SLE-PAH patients, and healthy donors. In situ pulmonary artery MIF protein expression was determined in spontaneous SLE mice (MRL/lpr) and hypoxia-induced C57BL/6J mice. Daily MIF098 was administered to C57BL/6J mice, and these mice were maintained in a hypoxic chamber for 4 weeks. The right ventricular systolic pressure (RVSP) and pathological characteristics of the pulmonary artery (PA), such as hyperproliferation, muscularization, and fibrosis were then measured in each group of mice. Data were also obtained in vitro using pulmonary smooth muscle cells (PASMC) challenged with platelet-derived growth factor (PDGF)-BB or 1% O2 hypoxia. As a result, circulating MIF was elevated in SLE-PAH patients compared with SLE patients or healthy donors. Higher RVSP SLE mice produced more MIF protein than lower RVSP SLE mice in the pulmonary artery. MIF098 decreased RVSP and inhibited distal pulmonary artery hyperproliferation, muscularization, and collagen deposition in hypoxia challenged mice. In addition, MIF098 inhibited PASMC proliferation and migration by regulating mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK1/2) signal- and cell-cycle-related proteins. MIF098 also reduced collagen synthesis by inhibiting the TGFβ1/Smad2/Smad3 pathway in cell-based experiments. In conclusion, MIF may serve as a biomarker and a therapeutic target of SLE-associated PAH. Pharmacologic MIF antagonism may be an effective means to ameliorate SLE-PAH.

HIF-1α-regulated MIF activation and Nox2-dependent ROS generation promote Leishmania amazonensis killing by macrophages under hypoxia

Increasing attention is given to the finding that macrophages under hypoxia are capable of controlling infection by the intracellular protozoan parasite Leishmania amazonensis. The hypoxia-inducible factor (HIF)-1α has been shown to play an essential role in this enhanced innate immune response. Our study aimed to explore the HIF-1α-dependent mechanisms leading to reduced survival of the parasites residing in macrophages under low oxygen conditions. Hypoxia triggered (P < 0.01) NADPH oxidase 2 (Nox2) expression and reactive oxygen species (ROS) production in J774 macrophages upon 24-h infection with L. amazonensis. Furthermore, increased (P < 0.01) expression levels of HIF-1α and macrophage migration inhibitory factor (MIF) were detected in the infected cells grown at 3% oxygen tension. We found that either HIF-1α silencing, Nox2 inhibition or MIF antagonism caused a significant (P < 0.05) reversal of the improved leishmanicidal activity displayed by the hypoxic phagocytes. Taken together, our current results suggest that, under conditions of limited availability of oxygen, activation of the HIF-1α/MIF axis via Nox2/ROS induction promotes killing of L. amazonensis amastigotes by macrophages. Such protective mechanism might operate in L. amazonensis-infected tissues where low oxygen levels prevail.

MIF inhibition interferes with the inflammatory and T cell-stimulatory capacity of NOD macrophages and delays autoimmune diabetes onset

Macrophages contribute in the initiation and progression of insulitis during type 1 diabetes (T1D). However, the mechanisms governing their recruitment into the islets as well as the manner of retention and activation are incompletely understood. Here, we investigated a role for macrophage migration inhibitory factor (MIF) and its transmembrane receptor, CD74, in the progression of T1D. Our data indicated elevated MIF concentrations especially in long-standing T1D patients and mice. Additionally, NOD mice featured increased MIF gene expression and CD74+ leukocyte frequencies in the pancreas. We identified F4/80+ macrophages as the main immune cells in the pancreas expressing CD74 and showed that MIF antagonism of NOD macrophages prevented their activation-induced cytokine production. The physiological importance was highlighted by the fact that inhibition of MIF delayed the onset of autoimmune diabetes in two different diabetogenic T cell transfer models. Mechanistically, macrophages pre-conditioned with the MIF inhibitor featured a refractory capacity to trigger T cell activation by keeping them in a naïve state. This study underlines a possible role for MIF/CD74 signaling pathways in promoting macrophage-mediated inflammation in T1D. As therapies directed at the MIF/CD74 pathway are in clinical development, new opportunities may be proposed for arresting T1D progression.

MIF, MIF Alleles, and Prospects for Therapeutic Intervention in Autoimmunity

Macrophage migration inhibitory factor (MIF) is an innate cytokine whose main actions include counter-regulating the immunosuppressive action of glucocorticoids and inhibiting activation-induced apoptosis. MIF is encoded in a functionally polymorphic locus and human genetic studies have shown significant relationships between high-expression MIF alleles, host inflammatory responses, and improved clinical outcome from infections. A recently completed candidate gene association study in the autoimmune disease systemic lupus erythematosus (SLE) indicates that individuals with a high-expression MIF allele have reduced incidence of SLE. Among patients with established disease however, those with end-organ complications have increased frequency of high-expression MIF alleles. Plasma MIF levels and Toll-like receptor (TLR) stimulated MIF production also reflect the underlying MIF genotype. These data suggest that MIF exerts a dual influence on the immunopathogenesis of SLE: high-expression MIF alleles are associated with a reduced susceptibility to SLE, perhaps by enhancing clearance of autoimmunogenic pathogens; once SLE develops however, low-expression MIF alleles protect from ensuing inflammatory end-organ damage. These data thus provide an example of the potential evolutionary advantage of maintaining an autoimmunity susceptibility gene in the population in that high-expression MIF alleles may allow for a maximal anti-infective response despite risk of autoimmunity. These results also support the clinical feasibility of pharmacologic MIF antagonism as such therapies may be most effectively applied in those individuals who, on the basis of their genotype, manifest a MIF dependent form of autoimmunity.

Role of MIF gene polymorphisms in systemic lupus erythematosus and prospects for therapeutic intervention

The cytokine macrophage migration inhibitory factor (MIF) counter-regulates the immunosuppressive action of glucocorticoids and inhibits activation-induced apoptosis. MIF is encoded in a polymorphic locus with a variant allele frequency >5% and high-expression alleles are associated with clinical severity of rheumatoid arthritis, scleroderma, and asthma but improved outcome from pneumonia. We completed a candidate gene association study in SLE (3195 patients and controls) to examine the relationship between two promoter polymorphisms in MIF: a -794 CATT5-8 microsatellite repeat (rs5844572), where increased repeat number leads to greater MIF expression; and a -173 G/C SNP (rs755622) that is in linkage disequilibrium with CATT7. Patients with the high-expression CATT7 allele had lower SLE incidence: OR = 0.63, P = 0.001 in Caucasians and OR = 0.46, P = 0.012 in African-Americans. Among patients with established SLE, those with serositis, nephritis, and cerebritis had reduced frequencies of low-expression MIF genotypes (CATT5) when compared with patients without end-organ involvement (P = 0.005 for serositis, P = 0.023 for nephritis, and P = 0.04 for cerebritis). Plasma MIF levels and TLR4, TLR7, and TLR9 stimulated MIF production reflected the underlying MIF genotype of the studied groups. These data suggest that MIF exerts a dual influence on the immunopathogenesis of SLE. High-expression MIF genotypes are associated with a reduced susceptibility to SLE, perhaps by contributing to enhanced clearance of infectious agents. Once SLE develops, however, low-expression MIF genotypes may protect from ensuing inflammatory end-organ damage [1]. We also examined the therapeutic impact of MIF antagonism in two mouse models of spontaneous SLE. In both the NZB/NZW F1 and the MRL/lpr mouse strains, anti-MIF or small molecule MIF receptor antagonism reduced functional and histological indices of glomerulonephritis, MIF-R+ leukocyte recruitment, and proinflammatory cytokine and chemokine expression [2]. A humanized anti-MIF developed from our studies has recently entered phase I clinical testing [3]. These data highlight the importance of MIF in the development of autoimmunity and support the potential clinical feasibility of pharmacologic MIF antagonism. We anticipate that MIF antagonists may be most effectively applied in those individuals who, on the basis of their genotype, manifest a MIF-dependent form of autoimmunity.

A small-molecule macrophage migration inhibitory factor antagonist protects against glomerulonephritis in lupus-prone NZB/NZW F1 and MRL/lpr mice.

Autoimmunity leads to the activation of innate effector pathways, proinflammatory cytokine production, and end-organ injury. Macrophage migration inhibitory factor (MIF) is an upstream activator of the innate response that mediates the recruitment and retention of monocytes via CD74 and associated chemokine receptors, and it has a role in the maintenance of B lymphocytes. High-expression MIF alleles also are associated with end-organ damage in different autoimmune diseases. We assessed the therapeutic efficacy of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an orally bioavailable MIF antagonist, in two distinct models of systemic lupus erythematosus: the NZB/NZW F1 and the MRL/lpr mouse strains. ISO-1, like anti-MIF, inhibited the interaction between MIF and its receptor, CD74, and in each model of disease, it reduced functional and histological indices of glomerulonephritis, CD74(+) and CXCR4(+) leukocyte recruitment, and proinflammatory cytokine and chemokine expression. Neither autoantibody production nor T and B cell activation were significantly affected, pointing to the specificity of MIF antagonism in reducing excessive proinflammatory responses. These data highlight the feasibility of targeting the MIF-MIF receptor interaction by small-molecule antagonism and support the therapeutic value of downregulating MIF-dependent pathways of tissue damage in systemic lupus erythematosus.

Autoimmune diseases: MIF as a therapeutic target

Importance of the field: Autoimmune inflammatory diseases occur commonly in developed countries. The treatment of these diseases is usually non-curative and is aimed at suppressing inflammatory end-organ damage. Macrophage migration inhibitory factor (MIF) is a multipotent cytokine that has been implicated in the pathogenesis of numerous autoimmune inflammatory disorders. The selective targeting of MIF with either anti-MIF antibody or specific MIF antagonists may offer new therapeutic avenues for these diseases.Areas covered in this review: Our aim is to discuss MIF-directed therapies as a novel therapeutic approach. The review covers literature from the past 10 years.What the reader will gain: MIF inhibition has been shown to be efficacious in many experimental and pre-clinical studies of autoimmune inflammatory diseases. The close regulatory relationship between MIF and glucocorticoids makes therapeutic antagonism of MIF a potential steroid-sparing therapy in patients with refractory autoimmune diseases.Take home message: We expect that MIF antagonism by either small-molecule- or antibody-based approaches will find wide application in the treatment of autoimmune inflammatory diseases. Such therapy also may be informed by the MIF genotype of affected patients.

The Macrophage Migration Inhibitory Factor-Glucocorticoid Dyad: Regulation of Inflammation and Immunity

The cytokine macrophage migration inhibitory factor (MIF) occupies a unique position in physiology by its ability to directly regulate the immunosuppressive actions of glucocorticoids. We review herein the interactions between MIF and glucocorticoids within the immune system and discuss the relevance of the MIF-glucocorticoid regulatory dyad in physiology and immunopathology. Therapeutic antagonism of MIF may be an effective approach for steroid-sparing therapies in patients with refractory autoimmune or inflammatory diseases.

The role of macrophage migration inhibitory factor in the inflammatory immune response and rheumatoid arthritis

Rheumatoid arthritis (RA) is a debilitating disease of unknown etiology. Although the pathogenesis of RA is multifactorial, the contribution of cytokines is undoubtedly pivotal in the progression of the inflammatory process. One cytokine gaining recognition for its importance in inflammation is macrophage migration inhibitory factor (MIF). Initially described as a biological activity, a broad range of functions of MIF has emerged including induction of proinflammatory mediators as well as demonstrated roles in both innate and adaptive immunity. In RA, increased MIF levels have been demonstrated in serum, synovial fluid and tissue with the latter correlating with disease activity. In vitro, MIF induces production of key proinflammatory genes operative in arthritis, including IL-1, TNF, IL-6, IL-8, COX-2, PLA2, and MMPs. In addition, MIF regulates proliferation and apoptosis via direct effects on the tumor suppressor protein p53 implicating a role for MIF in synovial hyperplasia. In vivo, MIF antagonism or MIF deficiency result in decreased disease severity in animal models of RA further confirming a role for MIF in joint inflammation. Interestingly, MIF is induced by glucocorticoids and MIF in turn antagonises glucocorticoid effects. This unique relationship presents antagonism of MIF as a potentially effective steroid-sparing therapy.

New therapeutic target in inflammatory disease: macrophage migration inhibitory factor

The cytokine macrophage migration inhibitory factor (MIF) participates in fundamental events in innate and adaptive immunity. The profile of activities of MIF in vivo and in vitro is strongly suggestive of a role for MIF in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (RA), and hence antagonism of MIF is suggested as a potential therapeutic strategy in inflammatory disease. The best developed case for therapeutic antagonism of MIF is in RA. In RA, MIF is abundantly expressed in serum and synovial tissue. MIF induces synovial expression of key pro-inflammatory genes, regulates the function of endothelial cells and leucocytes, and is implicated in the control of synoviocyte proliferation and apoptosis via direct effects on the expression of the tumour suppressor protein p53. In animal models of RA, anti-MIF antibodies or genetic MIF deficiency are associated with significant inhibition of disease. A similar case has been made, for example using MIF-deficient mice, in models of atheroma, colitis, multiple sclerosis and other inflammatory diseases. The relationship with p53 also means MIF may be important in the link between inflammatory disease and cancer, such as is seen in RA or colitis. MIF also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of MIF is in fact induced by glucocorticoids. Thus, MIF functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. This may be entrained by selective activation of mitogen-activated protein kinases rather than nuclear factor kappa B. Therapeutic MIF antagonism may therefore provide a specific means of 'steroid sparing'. Exploitation of antibody, soluble receptor or small molecule technologies may soon lead to the ability to test in the clinic the importance of MIF in human inflammatory diseases.

Macrophage migration inhibitory factor in rheumatoid arthritis

Rheumatoid arthritis is characterised by the interaction of multiple mediators, among the most important of which are cytokines. In recent years, extensive data demonstrates a pivotal role for one cytokine, macrophage migration inhibitory factor (MIF), in fundamental events in innate and adaptive immunity. MIF has now been demonstrated to be involved in the pathogenesis of many diseases, but in the case of RA the evidence for a role of MIF is very strong. MIF is abundantly expressed in the serum of RA patients, and in RA synovial tissue where it correlates with disease activity. MIF induces synoviocyte expression of key proinflammatory genes including TNF, IL-1, IL-6, IL-8, cPLA2, COX2 and MMPs. MIF also regulates the function of endothelial cells and B cells. Moreover, MIF is implicated in the control of synoviocyte proliferation and apoptosis via direct effects on the expression of the tumor suppressor protein p53. In multiple rat and mouse models of RA, anti-MIF antibodies or genetic MIF deficiency are associated with significant inhibition of disease. MIF -/- mice further demonstrate increases in synovial apoptosis. That the human Mif gene is encoded by different functional alleles in subjects with inflammatory disease also provides evidence for the role of MIF in RA. The mechanism of action of MIF is becoming better understood. MIF appears to interact with cell surface CD74, with consequent activation of MAP kinases but possibly not NFkappaB intracellular signal transduction. This apparent selectivity may be implicated in the ability of MIF to antagonise the effects of glucocorticoids. As MIF expression is induced by glucocorticoids, inhibition of its antagonistic effects may permit enhanced therapeutic effect of glucocorticoids, or "steroid sparing". To date there are no clinical trials of MIF antagonism in any disease, but exploitation of antibody, soluble receptor, or small molecule approaches enabled by the unique crystal structure of MIF, may soon lead to the ability to test in the clinic the importance of this cytokine in human RA.

Control of fibroblast-like synoviocyte proliferation by macrophage migration inhibitory factor.

The hyperplasia of fibroblast-like synoviocytes (FLS) is considered essential to the evolution of joint destruction in rheumatoid arthritis (RA), but the mechanisms underlying FLS proliferation remain poorly understood. Macrophage migration inhibitory factor (MIF) is a cytokine that has recently been shown to exert proinflammatory effects on RA FLS. This study sought to identify the mechanisms of activation of FLS by MIF, and to assess the effects of MIF on synovial cell proliferation. Human RA FLS were treated with recombinant MIF, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and/or anti-MIF monoclonal antibodies (mAb). Proliferation was measured with tritiated thymidine incorporation. Nuclear factor kappa B (NF-kappa B) and mitogen-activated protein (MAP) kinase activation were measured with immunohistochemistry and Western blotting, respectively. FLS proliferation was significantly increased by MIF. IL-1 beta and TNFalpha also induced proliferation, but these effects were prevented by neutralization with anti-MIF mAb. Activation of NF-kappa B was induced by IL-1 beta, but not by MIF. Anti-MIF mAb had no effect on IL-1 beta-induced NF-kappa B nuclear translocation. By contrast, MIF induced phosphorylation of extracellular signal-regulated kinase (ERK) MAP kinase. ERK antagonism, but not NF-kappa B antagonism, prevented the effect of MIF on FLS proliferation. These data suggest that MIF may regulate RA synovial hyperplasia by acting directly and via involvement in the effects of IL-1 beta and TNFalpha. In addition, the effects of MIF on FLS activation are independent of NF-kappa B, and dependent on ERK MAP kinase. These data suggest an important therapeutic potential for MIF antagonism in RA.

Essential role for migration inhibitory factor in IL-1 activation of mitogen-activated protein kinases

Macrophage migration inhibitory factor (MIF) is a pluripotential proinflammatory cytokine with a possible role in the pathogenesis of rheumatoid arthritis (RA). MIF is expressed in RA synovium, and directly activates RA synoviocyte gene expression and proliferation, as well as exerting anti-apoptotic effects via inhibition of p53. MIF activates ERK and p38 mitogen-activated protein (MAP) kinase, but evidence for direct activation of NF-κB by MIF is lacking. Anti-MIF monoclonal antibodies prevent IL-1 activation of fibroblast-like synoviocytes in vitro, implicating MIF in the activation of cells by IL-1. The mechanism of this action of MIF has not been adduced. We studied the activation of signal transduction pathways by IL-1 in cells deficient in MIF. Dermal fibroblasts were cultured from MIF-/- and WT mice, and were exposed to IL-1. MAP kinase and NF-κB activation were studied by Western blotting, electrophoretic mobility shift assay (EMSA), and reporter gene assays. IL-1 rapidly induced phosphorylation of p38, JNK, and ERK MAP kinases in WT cells. In contrast, MIF-/- cell p38, JNK, and ERK activation in response to IL-1 was reduced. Consistent with this observation, MIF-/-cells were hyporesponsive to IL-1-induced AP-1 DNA binding as measured by EMSA (P = 0.03). IL-1-induced activation of an AP-1 luciferase reporter gene system was also reduced in MIF-/- cells (P = 0.037). Confirming the functional significance of these results, MIF-/-cells were hyporesponsive to IL-1-induced proliferation. In contrast, MIF-/- cells were normally responsive to IL-1-induced MKK3 and MKK7 phosphorylation. No significant difference in NF-κB activation was detected between MIF-/- and WT cells, as measured by cellular IκB content, NF-κB EMSA, or NF-κB luciferase reporter gene assay. These data demonstrate that MIF is required for cellular MAP kinase responses to IL-1. This represents a novel mechanism of action of MIF in the support of the inflammatory response. The absence of an effect on MKK3/MKK7 or NF-κB suggests the effects may be mediated distally in the signal transduction cascade. Therapeutic MIF antagonism may limit the cellular effects of IL-1.

Regulation of synoviocyte phospholipase A2 and cyclooxygenase 2 by macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with known actions in macrophage and T cell activation. MIF also has the unique capacity to reverse the inhibitory effects of glucocorticoids on these cells. We have recently demonstrated MIF expression in human rheumatoid arthritis (RA) synovium and cultured fibroblast-like synoviocytes (FLS), as well as the ability of FLS-derived MIF to induce monocyte release of tumor necrosis factor alpha. We investigated the effects of MIF on aspects of RA FLS activation, including the induction of phospholipase A2 (PLA2) and cyclooxygenase (COX). PLA2 activity was measured by 3H-arachidonic acid released from treated FLS supernatants. COX activity was measured by prostaglandin E2 enzyme-linked immunosorbent assay. Cytosolic PLA2 (cPLA2) and COX-2 messenger RNA (mRNA) were determined using semiquantitative reverse transcriptase-polymerase chain reaction. Constitutive PLA2 activity was detected in RA FLS. Recombinant human MIF up-regulated PLA2 activity (P < 0.01) and cPLA2 mRNA expression, but had no effect on secretory PLA2. Recombinant human MIF up-regulated COX activity (P < 0.05) and COX-2 mRNA, but had no observable effect on COX-1. Interleukin-1beta (IL-1beta) significantly up-regulated PLA2 activity (P < 0.005) and cPLA2 mRNA expression while anti-MIF monoclonal antibody (mAb) significantly inhibited this IL-1beta-induced PLA2 activity (P < 0.02). Anti-MIF mAb significantly reduced IL-1beta-induced COX activity (P < 0.05) and COX-2 mRNA expression. MIF exerts a proinflammatory effect on key aspects of RA FLS activation. That anti-MIF mAb inhibited IL-1beta up-regulation of FLS indicates an additional cofactor role for MIF in IL-1beta-induced FLS activation. These data suggest that MIF antagonism has important therapeutic potential in RA.