MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment

The cytokine macrophage migration inhibitory factor (MIF) plays a critical role in inflammatory diseases and atherogenesis. We identify the chemokine receptors CXCR2 and CXCR4 as functional receptors for MIF. MIF triggered G(alphai)- and integrin-dependent arrest and chemotaxis of monocytes and T cells, rapid integrin activation and calcium influx through CXCR2 or CXCR4. MIF competed with cognate ligands for CXCR4 and CXCR2 binding, and directly bound to CXCR2. CXCR2 and CD74 formed a receptor complex, and monocyte arrest elicited by MIF in inflamed or atherosclerotic arteries involved both CXCR2 and CD74. In vivo, Mif deficiency impaired monocyte adhesion to the arterial wall in atherosclerosis-prone mice, and MIF-induced leukocyte recruitment required Il8rb (which encodes Cxcr2). Blockade of Mif but not of canonical ligands of Cxcr2 or Cxcr4 in mice with advanced atherosclerosis led to plaque regression and reduced monocyte and T-cell content in plaques. By activating both CXCR2 and CXCR4, MIF displays chemokine-like functions and acts as a major regulator of inflammatory cell recruitment and atherogenesis. Targeting MIF in individuals with manifest atherosclerosis can potentially be used to treat this condition.     

Cyclophosphamide-Induced Cystitis Increases Bladder CXCR4 Expression and CXCR4-Macrophage Migration Inhibitory Factor Association

Background

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro. We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis.

Methods and Findings

Twenty male rats received saline or cyclophosphamide (40 mg/kg; i.p.; every 3^rd day) to induce persistent cystitis. After eight days, urine was collected and bladders excised under anesthesia. Bladder CXCR4 and CXCR4-MIF co-localization were examined with immunhistochemistry. ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels. Bladder CXCR4 expression (real-time RTC-PCR) and protein levels (Western blotting) were examined. Co-immunoprecipitations studied MIF-CXCR4 associations.Urothelial basal and intermediate (but not superficial) cells in saline-treated rats contained CXCR4, co-localized with MIF. Cyclophosphamide treatment caused: 1) significant redistribution of CXCR4 immunostaining to all urothelial layers (especially apical surface of superficial cells) and increased bladder CXCR4 expression; 2) increased urine MIF with decreased bladder MIF; 3) increased bladder SDF-1; 4) increased CXCR4-MIF associations.

Conclusions

These data demonstrate CXCR4-MIF associations occur in vivo in rat bladder and increase in experimental cystitis. Thus, CXCR4 represents an alternative pathway for MIF-mediated signal transduction during bladder inflammation. In the bladder, MIF may compete with SDF-1 (cognate ligand) to activate signal transduction mediated by CXCR4.     

Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.     

MIF,CD74 and other partners in kidney disease: Tales of a promiscuous couple

Macrophage migration inhibitory factor (MIF) is increased in kidney and urine during kidney disease. MIF binds to and activates CD74 and chemokine receptors CXCR2 and CXCR4. CD74 is a protein trafficking regulator and a cell membrane receptor for MIF, D-dopachrome tautomerase (D-DT/MIF-2) and bacterial proteins. MIF signaling through CD74 requires CD44. CD74, CD44 and CXCR4 are upregulated in renal cells in diseased kidneys and MIF activation of CD74 in kidney cells promotes an inflammatory response. MIF or CXCR2 targeting protects from experimental kidney injury, CD44 deficiency modulates kidney injury and CXCR4 activation promotes glomerular injury. However, the contribution of MIF or MIF-2 to these actions of MIF receptors has not been explored. The safety and efficacy of strategies targeting MIF, CD74, CD44 and CXCR4 are under study in humans.     

Activation of the JNK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on CXCR4 and CD74

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family and controls essential processes such as inflammation, cell differentiation, and apoptosis. JNK signalling is triggered by extracellular signals such as cytokines and environmental stresses. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine with chemokine-like functions in leukocyte recruitment and atherosclerosis. MIF promotes MAPK signalling through ERK1/2, while it can either activate or inhibit JNK phosphorylation, depending on the cell type and underlying stimulation context. MIF activities are mediated by non-cognate interactions with the CXC chemokine receptors CXCR2 and CXCR4 or by ligation of CD74, which is the cell surface expressed form of the class II invariant chain. ERK1/2 signalling stimulated by MIF is dependent on CD74, but the receptor pathway involved in MIF activation of the JNK pathway is unknown. Here we comprehensively characterize the stimulatory effect of MIF on the canonical JNK/c-Jun/AP-1 pathway in fibroblasts and T cell lines and identify the upstream signalling components. Physiological concentrations of recombinant MIF triggered the phosphorylation of JNK and c-Jun and rapidly activated AP-1. In T cells, MIF-mediated activation of the JNK pathway led to upregulated gene expression of the inflammatory chemokine CXCL8. Activation of JNK signalling by MIF involved the upstream kinases PI3K and SRC and was found to be dependent on CXCR4 and CD74. Together, these data show that the CXCR4/CD74/SRC/PI3K axis mediates a rapid and transient activation of the JNK pathway as triggered by the inflammatory cytokine MIF in T cells and fibroblasts.     

LPS-mediated cell surface expression of CD74 promotes the proliferation of B cells in response to MIF

Macrophage migration inhibitory factor (MIF) is a chemokine-like inflammatory cytokine, which plays a pivotal role in the pathogenesis of inflammatory and cardiovascular diseases as well as cancer. We previously identified MIF as a novel B cell chemokine that promotes B cell migration through non-cognate interaction with the CXC chemokine receptor CXCR4 and CD74, the surface form of MHC class II invariant chain. In this study, we have analyzed the regulation of the MIF receptors under inflammatory conditions by investigating the impact of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) on CD74 and CXCR4 expression in B lymphocytes. We found that both LPS and TNF-α stimulation of primary B cells and the human B myeloma cell line RPMI-8226 enhanced protein expression as well as mRNA levels of CD74 in a time- and dose-dependent manner. By contrast, no effect on CXCR4 expression was observed. Selective inhibition of IκBα phosphorylation significantly attenuated LPS-induced expression of CD74, suggesting the contribution of NF-κB signaling pathways to the regulation of CD74 expression. Importantly, individual or simultaneous blockade of MIF or CD74 using specific neutralizing antibodies markedly affected B cell proliferation after LPS exposure. Taken together, our findings unveil a connection between the pro-proliferative activity of MIF/CD74 signaling in B cells and inflammation, offering novel target mechanisms in inflammatory cardiovascular or autoimmune pathogenesis.     

The effects of a hypothalamic peptide factor,MIF and its cyclic analog on tolerance to haloperidol in the rat

The effects of the hypothalamic peptide, ProLeuGlyNH₂ (MIF) and its analog, cyclo (LeuGly) (CLG) on the development of tolerance to haloperidol were investigated in male Sprague-Dawley rats. Chronic oral administration of haloperidol (1.5 mg/kg/day) for 21 days resulted in the development of tolerance to its pharmacological effects. A dose of haloperidol (3 mg/kg ip) exhibited an absence of cataleptic as well as hypothermic response in chronically haloperidol treated rats. Subcutaneous administration of MIF or CLG (2 mg/kg each) daily one hour prior to haloperidol injection blocked the haloperidol-induced tolerance as evidenced by the appearance of both the cataleptic and hypothermic responses. It is concluded that the hypothalamic peptide hormone, MIF may be important in regulating chronic effects of neuroleptic drugs.     

Comparative effects of Pro-Leu-Gly-NH2 and cyclo(Leu-Gly) administered orally on the development of tolerance to the analgesic effect of morphine in the rat

Comparative effects of Pro-Leu-Gly-NH2 (MIF) and cyclo(Leu-Gly) (CLG) administered orally at different stages of chronic morphine treatment on the development of tolerance to the analgesic effect of morphine in the rat were determined. Male Sprague-Dawley rats were implanted with either 6 placebo or morphine pellets during a 7-day period. Implantation of morphine pellets resulted in the development of a high degree of tolerance as evidenced by a decrease in the analgesic response to morphine. Administration of CLG (8 and 16 mg/kg/day) on day 5, 6 and 7 of implantation inhibited the development of tolerance to morphine but 4 and 32 mg/kg doses had no effect. Further, CLG (2 mg/kg/day for 7 days) inhibited the development of tolerance but higher doses (4 and 8 mg/kg) had no effect. MIF (26 and 52 mg/kg) administered orally on the last three days of the implantation schedule inhibited the development of tolerance to morphine. MIF (6.5 mg/kg/day for 7 days) inhibited the development of tolerance but the higher doses had no effect. Concurrent administration of MIF (6.5 mg/kg) and CLG (2 mg/kg) for seven days failed to inhibit the development of tolerance. A single dose of MIF or CLG administered a day before the assessment of tolerance did not affect the morphine tolerance. Thus, even after a significant degree of tolerance to morphine had developed, neuropeptides like MIF and CLG given orally, in appropriate doses, can inhibit development of tolerance to morphine and restore the analgesic effect of morphine.     

Activation of NFKB-JMJD3 signaling promotes bladder fibrosis via boosting bladder smooth muscle cell proliferation and collagen accumulation

Chronic cystitis is characterized by the hyperplasia and fibrosis of the bladder wall as well as attenuated compliance of the bladder. To further unravel its underlying molecular mechanism, the role of NFκB-JMJD3 signaling pathway in cystitis induced bladder fibrosis was investigated. Jmjd3 and Col1/3 expression was detected in a cystitis mouse model that was developed by intraperitoneal injection of cyclophosphamide (CYP). Human bladder smooth muscle cells (hBSMCs) were stimulated in vitro with lipopolysaccharide (LPS), and the cell proliferation and collagen accumulation were detected using EdU, CCK8, flow cytometry, qPCR, western blotting and immunofluorescence assays. Furthermore, the effects of NFκB and JMJD3 on cell proliferation and collagen accumulation were investigated using its selective antagonists, JSH23 and GSK-J4, respectively. CYP induced cystitis significantly increased Jmjd3, Col1 and Col3 expression in the bladder muscle cells. Furthermore, LPS stimulation markedly activated NFκB signaling and elevated JMJD3 expression in hBSMCs, and the activation of NFκB-JMJD3 signaling significantly promoted cell proliferation and collagen accumulation by upregulating CCND1 and COL1/3 expression, respectively. Our study reveals the critical role of NFκB-JMJD3 signaling in cystitis induced bladder reconstruction by regulating hBSMC proliferation and extracellular matrix (ECM) deposition, and these findings provide an avenue for effective treatment of patients with cystitis.     

Role for pAKT in rat urinary bladder with cyclophosphamide (CYP)-induced cystitis

AKT phosphorylation following peripheral nerve injury or inflammation may play a role in somatic pain processes and visceral inflammation. To examine such a role in micturition reflexes with bladder inflammation, we induced bladder inflammation in adult female Wistar rats (200-300 g) by injecting cyclophosphamide (CYP) intraperitoneally at acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every third day for 10 days) time points. Western blot analyses of whole urinary bladders showed significant increases (P ≤ 0.01) in phosphorylated (p) AKT at all time points; however, the magnitude of AKT phosphorylation varied with duration of CYP treatment. Immunohistochemical analyses of pAKT immunoreactivity (pAKT-IR) in cryostat bladder sections demonstrated duration-dependent, significant (P ≤ 0.01) increases in pAKT-IR in both the urothelium and detrusor smooth muscle of CYP-inflamed bladders. Additionally, a suburothelial population of pAKT-IR macrophages (CD68-, MAC2-, and F4/80-positive) was present in chronic CYP-treated bladders. The functional role of pAKT in micturition was evaluated using open, conscious cystometry with continuous instillation of saline in conjunction with administration of an inhibitor of AKT phosphorylation, deguelin (1.0 μg/10 μl), or vehicle (1% DMSO in saline) in control (no inflammation) and CYP (48 h)-treated rats. Bladder capacity, void volume, and intercontraction void interval increased significantly (P ≤ 0.05) following intravesical instillation of deguelin in CYP (48 h)-treated rats. These results demonstrate increased AKT phosphorylation in the urinary bladder with urinary bladder inflammation and that blockade of AKT phosphorylation in the urothelium improves overall bladder function.     

Intraluminal Blockade of Cell-Surface CD74 and Glucose Regulated Protein 78 Prevents Substance P-Induced Bladder Inflammatory Changes in the Rat

Background

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine constitutively expressed by urothelial cells. During inflammatory stimuli, MIF is released into the lumen complexed to other proteins and these complexes can bind to urothelial cell-surface receptors to activate signaling pathways. Since MIF is complexed to α1-inhibitor III (A1-I3; a member of the α2-macroglubulin family) and glucose regulated protein 78 (GRP78) is a receptor for A1-I3 the goals of this study were to determine if substance P elicits urothelial cell-surface expression of GRP78 and to assess the functional role of CD74 (receptor for MIF) or GRP78 in substance P-induced bladder inflammatory changes.

Methodology/Principal Findings

Anesthetized male Sprague-Dawley rats received either saline or substance P (s.c.), bladders were collected 1 hour after treatment and processed for histology or protein/mRNA. The expression of GRP78 at urothelial cell-surface was determined by performing in vivo biotinylation of urothelial cell-surface proteins. Finally, in order to determine the effects of receptor blockade on substance P-induced MIF release and inflammatory changes, rats received either intraluminal antibodies to CD74, GRP78, both, or non-specific IgG (as a control).GRP78 and MIF immunostaining was simultaneously visualized in umbrella cells only after substance P treatment. Immunoprecipitation studies showed GRP78-MIF complexes increased after substance P while in vivo biotinylation confirmed substance P-induced GRP78 cell-surface expression in urothelial cells. Intraluminal blockade of CD74 and/or GRP78 prevented substance P-induced changes, including bladder edema, intraluminal MIF release by urothelial cells and production of inflammatory cytokines by urothelial cells.

Conclusions/Significance

GRP78 is expressed on the surface of urothelial cells after substance P treatment where it can bind MIF complexes. Blocking CD74 (receptor for MIF) and/or GRP78 prevented substance P-induced inflammatory changes in bladder and urothelium, indicating that these urothelial receptors are effective targets for disrupting MIF-mediated bladder inflammation.     

Effect of silymarin on bladder overactivity in cyclophosphamide-induced cystitis rat model

The purpose of this study was to investigate the effects of silymarin, a phytotherapeutic agent, on bladder overactivity in a cyclophosphamide (CYP)-induced cystitis rat model. Female Wistar Albino rats received a single intraperitoneal injection of CYP (150 mg/kg) or saline and after 72 h, bladder function was evaluated by in vitro preparations of whole bladders and cystometry with continuous saline infusion under urethane anesthesia. Silymarin or a vehicle was orally given for 7 days in rats. CYP was injected on the 5th day of silymarin or vehicle treatment and then the animals were killed on the 8th day. CYP-treatment dramatically potentiated the basal spontaneous contractions of isolated whole bladders compared to control rats. In anesthetized rats, during continuous infusion cystometry, intercontraction interval (ICI) was significantly shorter, but bladder voiding pressure was not significantly changed in CYP-injected rats compared to control rats. In the CYP-injected group, silymarin treatment significantly decreased the amplitude, frequency (contractions/min) and area under the curve of spontaneous contractions, but failed to change carbachol-induced contraction in isolated whole bladder. Also, silymarin treatment significantly increased the ICI in comparison to the vehicle treatment. In the saline-injected group, no significant changes in the bladder function were observed between the silymarin and vehicle-treated groups. Histopathological examination showed that CYP-induced bladder inflammation tended to be lower in the silymarin+CYP-treated group. In conclusion, the oral administration of silymarin suppressed CYP-induced bladder overactivity. Silymarin may be considered as an attractive treatment for CYP-induced bladder overactivity.     

Role for CD74 and CXCR4 in clathrin-dependent endocytosis of the cytokine MIF

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that plays a role in innate and adaptive immunity. Depending on the cellular context and disease state, MIF signaling is mediated by its receptors CXCR2, CXCR4 and/or CD74. Although it is known that MIF is endocytosed, the exact mechanism has remained unknown. In exploring the mechanism of MIF endocytosis with biologically active Alexa(546)MIF, pathway-specific inhibitors (monodansylcadaverine, MDC; chlorpromazine, CPZ; dynasore; dominant-negative dynamin, bafilomycin, nocodazole) and receptor overexpression and blockade approaches, we identified a clathrin/dynamin-dependent endocytosis pathway as the main track for MIF internalization. MIF endocytosis was rapid and colocalization with both early and late endosomal vesicles in a microtubule- and acidification-dependent manner was observed. LDL endocytosis (which is clathrin-mediated) served as a control and was similarly inhibited by MDC or dynasore. When MIF endocytosis was compared to that of transferrin, acetylated LDL, and choleratoxin B (the latter internalized by a clathrin-independent pathway) by colocalization studies, the MIF internalization pathway clearly resembled that of LDL but also shared early trafficking with transferrin, whereas no colocalization with choleratoxin was noted. To identify the receptors involved in MIF endocytosis, we focused on CD74 and CXCR4 which form a heteromeric complex. Ectopic overexpression of CD74 in HEK293 and HeLa cells, which do not endogenously express CD74, led to a marked acceleration of MIF endocytosis while pharmacological blockade of CXCR4, which is endogenously expressed on these cells, with AMD3100 led to a 20% reduction of MIF endocytosis in HEK293-CD74 transfectants, whereas in untransfected cells, a blockade of 40% was observed. Of note, both CD74 and CXCR4 strongly colocalize with Alexa(546)MIF both on the plasma membrane and in endosomal compartments. Moreover, MIF-stimulated AKT signaling, which was previously shown to involve both CD74 and CXCR4, was reduced by endocytosis inhibitors, indicating that MIF signaling is at least in part due to endosomal signaling mechanisms. Thus, MIF uptake follows a rapid LDL-like, clathrin- and dynamin-dependent endocytosis pathway, which is dependent on the receptors CD74 and CXCR4 and leads to the initiation of endosomal signaling responses.     

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.     

COX-2 and prostanoid expression in micturition pathways after cyclophosphamide-induced cystitis in the rat

The purpose of this study was to determine the role of cyclooxygenase-2 (COX-2) and its metabolites in lower urinary tract function after induction of acute (4 h), intermediate (48 h), or chronic (10 day) cyclophosphamide (CYP)-induced cystitis. Bladders were harvested from euthanized female rats for analyses. Conscious cystometry was used to assess the effects of a COX-2-specific inhibitor, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl2(5H)-furanone (DFU, 5 mg/kg sc), a disubstituted furanone, in CYP-induced cystitis. COX-2 mRNA was increased in inflamed bladders after acute (12-fold) and chronic (9-fold) treatment. COX-2 protein expression in inflamed bladders paralleled COX-2 mRNA expression. Prostaglandin D2-methoxime expression in the bladder was significantly (P < or = 0.01) increased in acute (3-fold) and chronic (5.5-fold) cystitis. Prostaglandin E2 was significantly (P < or = 0.01) increased (2-fold) in the bladder with intermediate (1.7-fold) and chronic (2.6-fold) cystitis. COX-2-immunoreactive cell profiles were distributed throughout the inflamed bladder and coexpressed histamine immunoreactivity. Conscious cystometry in rats treated with CYP + DFU showed increased micturition intervals 4 and 48 h after CYP treatment and decreased intravesical pressures during filling and micturition compared with rats treated with CYP + vehicle. These studies suggest an involvement of urinary bladder COX-2 and its metabolites in altered micturition reflexes with CYP-induced cystitis.     

Modeling of both shared and distinct interactions between MIF and its homologue D-DT with their common receptor CD74

D-dopachrome tautomerase (D-DT) shares amino acid sequence similarity, structural architecture and biological activity with the cytokine MIF. Recent studies show that the two protein homologs also bind to the same cell surface receptor, CD74, to activate the ERK1/2 pathway that ultimately leads to pro-inflammatory and pro-survival gene expression. We recently showed that RTL1000 and DRa1-MOG-35-55, two biological drugs with potent anti-inflammatory properties that treat experimental autoimmune encephalomyelitis (EAE) in mice, bind to the cell surface receptor CD74 with high affinity and compete with MIF for binding to the same regions of CD74. Computational modeling of MIF and RTL1000 binding interactions with CD74 predicted the presence of three CD74 binding regions for each MIF homotrimer. Through a similar approach we have now expanded our work to study the D-DT (MIF-2) interaction with CD74 that is mainly defined by three elements scattered throughout the disordered regions of the interacting molecules. The model predicted: (a) a hydrophobic cradle between CD74 and D-DT consisting of N-terminal tyrosine residues of three CD74 monomers arranged in a planar alignment interacts with aromatic amino acid residues located in the disordered D-DT C-terminus; (b) a triad consisting of the E103 residue on one D-DT monomer in close contact with R179 and S181 on one chain of the CD74 trimer forms an intermolecular salt bridge; and (c) amino acid residues on the C-terminus random coil of CD74 chain C form a long interacting area of ∼500 Å² with a disordered region of D-DT chain B. These three binding elements were also present in MIF/CD74 binding interactions, with involvement of identical or highly similar amino acid residues in each MIF homotrimer that partner with the exact same residues in CD74. Topologically, however, the location of the three CD74 binding regions of the D-DT homotrimer differs substantially from that of the three MIF binding regions. This key difference in orientation appears to derive from a sequence insertion in D-DT that topologically limits binding to only one CD74 molecule per D-DT homotrimer, in contrast to predicted binding of up to three CD74 molecules per MIF homotrimer. These results have implications for the manner in which D-DT and MIF compete with each other for binding to the CD74 receptor and for the relative potency of DRa1-MOG-35-55 and RTL1000 for competitive inhibition of D-DT and MIF binding and activation through CD74.     

The N-terminus of MIF regulates the dynamic profile of residues involved in CD74 activation

Macrophage migration inhibitory factor (MIF) is an immunomodulatory protein with a pathogenic activity in various inflammatory disorders, autoimmune diseases, and cancer. The majority of MIF-triggered pathological conditions are associated with activation of the cell surface receptor CD74. In the absence of small molecule antagonists that directly target CD74, MIF variants and MIF-ligand complexes have served as modulators of CD74 activity. These molecules have been reported to have either antagonistic or agonistic effects against the receptor, although the mechanistic parameters that distinguish the two groups are largely unknown. Through molecular dynamics simulations and NMR experiments, we explored the relationship between MIF’s catalytically active N-terminus and the surface residues important for the activation of CD74. We found that the two sites are connected via backbone dynamics that are propagated to the CD74 activation surface of MIF, from the β2 and β4 strands. Our results also provide mechanistic evidence that explain the functional characteristics of MIF variants, serving as CD74 agonists or antagonists. Such findings are of high importance for understanding the MIF-induced activation of CD74 as well as for the development of highly potent CD74 therapeutics.     

Uroprotective Effect of Pentoxifylline in Cyclophosphamide‐Induced Hemorrhagic Cystitis in Rats

The role of phosphodiesterase inhibitor, pentoxifylline, in the prevention of cyclophosphamide‐induced hemorrhagic cystitis was evaluated in a rat model. Hemorrhagic cystitis was induced in rats by an intraperitoneal (i.p.) injection of a single dose of cyclophosphamide (150 mg/kg). Pentoxifylline (150 mg/kg/day/ip) was administered for 10 days followed by cyclophosphamide. Hemorrhagic cystitis was well characterized macroscopically, microscopically, and biochemically. Cyclophosphamide induced bladder injury including acute severe inflammation, vascular congestion, severe edema, hemorrhage, inflammatory cell infiltration in the lamina propria, and epithelial denudation; as well as it notably elevated serum inflammatory cytokines (tumor necrosis factor‐α, interleukin‐6, and interleukin‐1β), bladder content of malondialdehyde and total nitrate, accompanied with depletion of bladder antioxidant enzymes activities (glutathione peroxidase, superoxide dismutase, glutathione‐S‐transferase, and catalase). Prior administration of pentoxifylline improved all biochemical and histologic alterations induced by the cytotoxic drug cyclophosphamide. In conclusion, pentoxifylline has proven uroprotective efficacy in the cyclophosphamide‐induced hemorrhagic cystitis model, possibly through modulating the release of inflammatory cytokines and nitric oxide and restoring the oxidant/antioxidant balance. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:343‐350, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21494     

Optimization of N-benzyl-benzoxazol-2-ones as receptor antagonists of macrophage migration inhibitory factor (MIF)

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also exhibits keto–enol tautomerase activity, believed to be vestigial in mammals. Starting from a 1 μM hit from virtual screening, substituted benzoxazol-2-ones have been discovered as antagonists with IC₅₀ values as low as 7.5 nM in a tautomerase assay and 80 nM in a MIF–CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts.     

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)