A macrophage migration inhibitory factor (MIF), originally described as a product of activated lymphocytes, has been defined as a 12 kDa protein, expressed in a wide variety of tissues. Here MIF is identified as a phenylpyruvate tautomerase (EC 5.3.2.1) having p-hydroxyphenylpyruvate and phenylpyruvate as its natural substrates. The definition of MIF as an enzyme may yield insight into the mechanism of action of this proinflammatory and immunomodulating cytokine.

Macrophage migration inhibitory factor (MIF) is a pivotal regulator of the immune response. Neutralization or genetic deletion of MIF does not completely abrogate activation responses, however, and deletion of the MIF receptor, CD74, produces a more pronounced phenotype than MIF deficiency. We hypothesized that these observations may be explained by a second MIF-like ligand, and we considered a probable candidate to be the protein encoded by the homologous, D-dopachrome tautomerase (D-DT) gene. We show that recombinant D-DT protein binds CD74 with high affinity, leading to activation of ERK1/2 MAP kinase and downstream proinflammatory pathways. Circulating D-DT levels correlate with disease severity in sepsis or malignancy, and the specific immunoneutralization of D-DT protects mice from lethal endotoxemia by reducing the expression of downstream effector cytokines. These data indicate that D-DT is a MIF-like cytokine with an overlapping spectrum of activities that are important for our understanding of MIF-dependent physiology and pathology.

In recent years, studies have advocated neuropeptide systems as modulators for the behavioral states found in mood disorders such as depression and anxiety disorders. Neuropeptides have been tested in traditional animal models and screening procedures that have been validated by known antidepressants and anxiolytics. However, it has become clear that although these tests are very useful, neuropeptides have distinct behavioral effects and dose-dependent characteristics, and therefore, use of these tests with neuropeptides must be done with an understanding of their unique characteristics. This review will focus on the behavioral actions of neuropeptides and their synthetic analogs, particularly in studies utilizing various preclinical tests of depression and anxiety. Specifically, the following neuropeptide systems will be reviewed: corticotropin-releasing factor (CRF), urocortin (Ucn), teneurin C-terminal associated peptide (TCAP), neuropeptide Y (NPY), arginine vasopressin (AVP), oxytocin, the Tyr-MIF-1 family, cholecystokinin (CCK), galanin, and substance P. These neuropeptide systems each have a unique role in the regulation of stress-like behavior, and therefore provide intriguing therapeutic targets for mood disorder treatment.

The absence of MHC class I Ags on the corneal endothelium, which lines the anterior chamber of the eye, makes this cell layer potentially vulnerable to lysis by NK cells. However, aqueous humor (AH), which bathes the corneal endothelium, contains a 12-kDa protein which inhibits the NK-mediated lysis of corneal endothelial cells. An amino acid sequence analysis of AH revealed that this factor shared >90% homology with macrophage migration inhibitory factor (MIF). The NK inhibitory effect of AH was neutralized with anti-human MIF Ab. Moreover, mouse rMIF produced a similar inhibition of NK cell activity. However, neither rMIF nor AH inhibited the CTL-mediated Lysis of allogeneic cells. rMIF prevented the release of perforin granules by NK cells but not CTLs. Although MIF displays proinflammatory properties, these results indicate that it can also inhibit at least one immune effector element, NK cells, and thereby contribute to immune privilege in the eye.

OBJECTIVE

To measure levels of various inflammatory cytokines in the aqueous humor of patients with primary open-angle glaucoma (POAG), exfoliation glaucoma (EXG), and senile cataract.

METHODS

Aqueous humor samples were obtained from 64 eyes of 64 Japanese subjects (POAG, 20 eyes; EXG, 23 eyes; and cataract, 21 control eyes). The levels of eight cytokines including interleukin (IL)1-β, IL-6, IL-8, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, serum amyloid A (SAA), migration inhibitory factor (MIF), and vascular endothelial growth factor (VEGF)-A were estimated using the multiplex bead immunoassay technique.

RESULTS

Compared with the cataract group, the levels of TGF-β1, IL-8, and SAA were significantly higher in aqueous humor samples from the POAG (5.0-fold, 2.3-fold, and 11.9-fold, respectively) and EXG (12.5-fold, 4.0-fold, and 18.3-fold, respectively) groups. Except for a significant decrease in the IL-6 level in the POAG (0.23-fold) group, no other cytokine levels differed in the POAG and EXG groups compared with the cataract group. The levels of TGF-β1, IL-8, and SAA were positively correlated with each other (ρ = 0.723-0.786; P < 0.0001), the intraocular pressure (IOP) (ρ = 0.392-0.662; P < 0.0001-0.0019), and the number of glaucoma medications (ρ = 0.478-0.659; P < 0.0001-0.0001).

CONCLUSIONS

Cytokine networks including TGF-β1, IL-8, and SAA in aqueous humor may have critical roles in IOP elevations in patients with open-angle glaucoma.

Although chemokine and growth factor receptors are attractive and popular targets for cancer therapeutic intervention, structure-based targeting of the ligands themselves is generally not considered practical. New evidence indicates that a notable exception to this is macrophage migration inhibitory factor (MIF). MIF, an autocrine- and paracrine-acting cytokine/growth factor, plays a pivotal role in both the initiation and maintenance of neoplastic diseases. MIF possesses a nonphysiologic enzymatic activity that is evolutionarily well-conserved. Although small molecule antagonists of MIFs enzymatic active site have been reported to inhibit biological activities of MIF, universally high IC(50)s have limited their clinical appeal. Using a computational virtual screening strategy, we have identified a unique small molecule inhibitor that serves as a suicide substrate for MIF, resulting in the covalent modification of the catalytically active NH(2)-terminal proline. Our studies further reveal that this compound, 4-iodo-6-phenylpyrimidine (4-IPP), is approximately 5x to 10x times more potent in blocking MIF-dependent catalysis and lung adenocarcinoma cell migration and anchorage-independent growth than the prototypical MIF inhibitor, ISO-1. Finally, using an in silico combinatorial optimization strategy, we have identified four unique congeners of 4-IPP that exhibit MIF inhibitory activity at concentrations 10x to 20x lower than that of parental 4-IPP.

OBJECTIVE

Angiogenesis is the formation of new capillaries from pre-existing vessels, whereas vasculogenesis is de-novo capillary formation from endothelial precursor cells (EPCs). Current understanding of the role of angiogenesis and vasculogenesis in rheumatoid arthritis (RA) and possibilities of therapeutic intervention should be summarized.

RESULTS

There have been many recent studies on the role of the hypoxia and hypoxia-inducible factor (HIF)-vascular endothelial growth factor (VEGF)-angiopoietin axis in angiogenesis associated with RA. The role of additional growth factors, chemokines, cytokines, matrix components and adhesion molecules has been further characterized. Macrophage migration inhibitory factor (MIF) may link inflammation, angiogenesis and atherosclerosis. Junctional adhesion molecules (JAMs) and focal adhesion kinases (FAKs) have recently been implicated in inflammatory angiogenesis. Novel information regarding the role of serum amyloid A (SAA) and sphingosine kinase has become available. Most of these angiogenic factors have recently been targeted using various techniques and arthritis models. Whereas angiogenesis is abundant in RA, there is defective EPC function and vasculogenesis leading to atherosclerosis and vascular disease in arthritis. Treatment with EPCs already under investigation in vascular diseases may also be attempted in RA.

CONCLUSIONS

Targeting angiogenesis and restoration of vasculogenesis may be beneficial for the therapy and outcome of RA.

OBJECTIVE

We investigated the effects of macrophage migration inhibitory factor (MIF) antibodies in experimental colitis-induced dextran sulfate sodium (DSS) and trinitrobenzenesulfonic acid (TNBS) and examined whether plasma levels of MIF were elevated in patients with inflammatory bowel disease (IBD).

METHODS

BALB/c or C57BL/6 mice were fed 4% DSS in their drinking water for up to 7 days with and without administration of an anti-MIF antibody every 2 days. The severity of inflammation in the cecum and colon was assessed by clinical signs and histologic scoring. Tissue levels of MIF, tumor necrosis factor (TNF)-alpha, interferon gamma (IFN-gamma), interleukin (IL)-4, and matrix metalloproteinase (MMP)-13 messenger RNA (mRNA) were measured. The effects of anti-MIF antibody on chronic colitis induced by TNBS was assessed in BALB/c mice. Plasma MIF concentrations were assayed in patients with Crohn's disease, ulcerative colitis, and healthy controls.

RESULTS

During DSS-induced colitis, colonic MIF mRNA expression was increased. Clinical signs and histopathologic features were significantly improved in animals given anti-MIF antibody. DSS-induced up-regulation of colonic TNF-alpha and IFN-gamma were significantly suppressed in animals given the anti-MIF antibody. Colonic IL-4 was decreased during DSS but restored to baseline by the anti-MIF antibody. The anti-MIF antibody prevented MMP-13 up-regulation by DSS and ameliorated TNBS colitis. Plasma MIF was elevated in patients with Crohn's disease or ulcerative colitis compared with healthy controls.

CONCLUSIONS

We conclude that anti-MIF antibodies reduce the severity of experimental colitis and limit the up-regulation of Th1-type cytokines. Anti-MIF antibodies are of potential therapeutic use in IBD.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into lineages of mesenchymal tissues that are currently under investigation for a variety of therapeutic applications. The purpose of this study was to compare cytokine gene expression in MSCs from human placenta, cord blood (CB) and bone marrow (BM). The cytokine expression profiles of MSCs from BM, CB and placenta (amnion, decidua) were compared by proteome profiler array analysis. The cytokines that were expressed differently, in each type of MSC, were analyzed by real-time PCR. We evaluated 36 cytokines. Most types of MSCs had a common expression pattern including MIF (GIF, DER6), IL-8 (CXCL8), Serpin E1 (PAI-1), GROalpha(CXCL1), and IL-6. MCP-1, however, was expressed in both the MSCs from the BM and the amnion. sICAM-1 was expressed in both the amnion and decidua MSCs. SDF-1 was expressed only in the BM MSCs. Real-time PCR demonstrated the expression of the cytokines in each of the MSCs. The MSCs from bone marrow, placenta (amnion and decidua) and cord blood expressed the cytokines differently. These results suggest that cytokine induction and signal transduction are different in MSCs from different tissues.

Falciparum malaria is a complex disease with no simple explanation, affecting organs where the parasite is rare as well as those organs where it is more common. We continue to argue that it can best be understood in terms of excessive stimulation of normally useful pathways mediated by inflammatory cytokines, the prototype being tumor necrosis factor (TNF). These pathways involve downstream mediators, such as nitric oxide (NO) that the host normally uses to control parasites, but which, when uncontrolled, have bioenergetic failure of patient tissues as their predictable end point. Falciparum malaria is no different from many other infectious diseases that are clinically confused with it. The sequestration of parasitized red blood cells, prominent in some tissues but absent in others with equal functional loss, exacerbates, but does not change, these overriding principles. Recent opportunities to stain a wide range of tissues from African pediatric cases of falciparum malaria and sepsis for the inducible NO synthase (iNOS) and migration inhibitory factor (MIF) have strengthened these arguments considerably. The recent demonstration of bioenergetic failure in tissue removed from sepsis patients being able to predict a fatal outcome fulfils a prediction of these principles, and it is plausible that this will be demonstrable in severe falciparum malaria. Understanding the disease caused by falciparum malaria at a molecular level requires an appreciation of the universality of poly(ADP-ribose) polymerase-1 (PARP-1) and Na(+)/K(+)-ATPase and the protean effects of activation by inflammation of the former that include inactivation of the latter.

The objective of the study was to determine whether plasma migration inhibitor factor (MIF) concentration and mononuclear cell (MNC) mRNA are elevated in obesity and whether treatment with metformin reduces plasma MIF concentration. Forty obese subjects [body mass index (BMI), 37.5 +/- 4.9 kg/m(2)] and 40 nonobese healthy subjects (BMI, 22.6 +/- 3.4 kg/m(2)) had their plasma MIF, glucose, insulin, free fatty acids (FFAs) and C-reactive protein (CRP) concentrations measured. Sixteen obese patients and 16 nonobese healthy subjects had RNA prepared from MNCs. Eight obese subjects with normal glucose concentration were treated with metformin 1 g (Glucophage XR; 1000 mg twice daily) twice daily for 6 wk. Eight obese subjects were used as controls. Plasma concentration of glucose, insulin, FFAs, and MIF was measured by appropriate assays. mRNA for MIF was measured by real-time PCR. Forty obese subjects had a fasting concentration of MIF of 2.8 +/- 2.0 ng/ml, whereas 40 nonobese subjects had a fasting MIF concentration of 1.2 +/- 0.6 ng/ml (P < 0.001). Plasma MIF concentrations were significantly related to BMI (r = 0.52; P < 0.001). mRNA for MIF was correlated to plasma FFAs (r = 0.40; P < 0.05) and plasma CRP (r = 0.42; P < 0.05) concentrations. Eight obese subjects had their fasting blood samples taken before and after taking a slow-release preparation of metformin at 1, 2, 4, and 6 wk. The mean plasma concentration fell from 2.3 +/- 1.4 to 1.6 +/- 1.2 ng/ml at 6 wk (P < 0.05). Obese subjects not on treatment with metformin showed no change. During the period of treatment with metformin, the body weight did not change and the plasma concentration of glucose, insulin, and FFAs did not alter. We conclude that: 1) plasma MIF concentrations and MIF mRNA expression in the MNCs are elevated in the obese, consistent with a proinflammatory state in obesity; 2) these increases in MIF are related to BMI, FFA concentrations, and CRP; 3) metformin suppresses plasma MIF concentrations in the obese, suggestive of an antiinflammatory effect of this drug; and 4) this action of metformin may contribute to a potential antiatherogenic effect, which may have implications for the reduced cardiovascular mortality observed with metformin therapy in type 2 diabetes mellitus.

The goal of this study was to examine the role of E(2) in regulating innate immune protection by human uterine epithelial cells (UECs). Recognizing that UECs produce cytokines and chemokines to recruit and activate immune cells as well as viral and bacterial antimicrobials, we sought to examine the effect of E(2) on constitutive and Toll-like receptor (TLR) agonist (lipopolysaccharide (LPS) and poly (I:C))-induced immune responses. The secretion by polarized UECs in culture of interleukin (IL)-6, macrophage inhibitory factor (MIF), and secretory leukocyte protease inhibitor (SLPI) was examined as well as the mRNA expression of human beta-defensin-2 (HBD2), tumor necrosis factor (TNF)-alpha, IL-8, and nuclear factor (NF)-kB. When incubated with E(2) for 24-48 h, we found that E(2) stimulated UEC secretion of SLPI (fourfold) and mRNA expression of HBD2 (fivefold). Moreover, when antibacterial activity in UEC secretions was measured using Staphylococcus aureus, E(2) increased the secretion of soluble factor(s) with antibacterial activity. In contrast, E(2) had no effect on constitutive secretion of proinflammatory cytokines and chemokines by UECs but completely inhibited LPS- and poly (I:C)-induced secretion of MIF, IL-6, and IL-8. Estradiol also reversed the stimulatory effects of IL-1beta on mRNA expression of TNF-alpha, IL-8, and NF-kB by 85, 95, and 70%, respectively. As SLPI is known to inhibit NF-kB expression, these findings suggest that E(2) inhibition of proinflammatory cytokines may be mediated through SLPI regulation of NF-kB. Overall, these findings indicate that the production of cytokines, chemokines, and antimicrobials by UECs are differentially regulated by E(2). Further, it suggests that with E(2) regulation, epithelial cells that line the uterine cavity have evolved immunologically to be sensitive to viral and bacterial infections as well as the constraints of procreation.

OBJECTIVE

To address the functional and prognostic relevance of the -173 single-nucleotide G-to-C polymorphism of the macrophage migration inhibitory factor (MIF) gene in patients with systemic-onset juvenile idiopathic arthritis (systemic-onset JIA) by evaluating its association with serum and synovial fluid levels of MIF, with glucocorticoid requirement, and with the outcome of the disease.

METHODS

A total of 136 patients with systemic-onset JIA were studied, including 98 patients from the British Paediatric Rheumatology Study Group's National Repository for JIA and 38 patients who were followed up at the IRCCS Policlinico San Matteo (Pavia, Italy) and the IRCCS G. Gaslini (Genoa, Italy). The MIF-173 polymorphism was genotyped using SnaPshot ddNTP primer extension and capillary electrophoresis. MIF levels were measured by enzyme-linked immunosorbent assay. The evaluation of the association of the MIF-173 polymorphism with outcome was performed only in Italian patients who were followed up for >5 years, by analyzing retrospectively 1) the number of joints with active arthritis and the number of joints with limited range of motion; 2) the score, at the last visit, on the Italian version of the Childhood Health Assessment Questionnaire (C-HAQ); and 3) data concerning the treatment regimens during the disease course.

RESULTS

Systemic-onset JIA patients carrying a MIF-173*C allele had serum and synovial fluid levels of MIF significantly higher than those in patients with the GG genotype. The duration of glucocorticoid treatment on a daily regimen was significantly longer in patients carrying a MIF-173*C allele than in MIF-173 GG homozygous patients. Moreover, the duration of clinical response to intraarticular injection of triamcinolone hexacetonide was significantly shorter in patients carrying a MIF-173*C allele. At the last visit, the numbers of joints with active arthritis, the C-HAQ scores, and the numbers of joints with limited range of motion were significantly higher in patients carrying the MIF-173*C allele.

CONCLUSIONS

Our study shows the functional relevance of the MIF-173 polymorphism and suggests that the MIF-173*C allele is a predictor of poor outcome in systemic-onset JIA.

We have recently identified the archaic cytokine macrophage migration inhibitory factor (MIF) as a non-canonical ligand of the CXC chemokine receptors CXCR2 and CXCR4 in inflammatory and atherogenic cell recruitment. Because its affinity for CXCR2 was particularly high, we hypothesized that MIF may feature structural motives shared by canonical CXCR2 ligands, namely the conserved N-terminal Glu-Leu-Arg (ELR) motif. Sequence alignment and structural modeling indeed revealed a pseudo-(E)LR motif (Asp-44-X-Arg-11) constituted by non-adjacent residues in neighboring loops but with identical parallel spacing as in the authentic ELR motif. Structure-function analysis demonstrated that mutation of residues R11, D44, or both preserve proper folding and the intrinsic catalytic property of MIF but severely compromises its binding to CXCR2 and abrogates MIF/CXCR2-mediated functions in chemotaxis and arrest of monocytes on endothelium under flow conditions. R11A-MIF and the R11A/D44A-MIF double-mutant exhibited a pronounced defect in triggering leukocyte recruitment to early atherosclerotic endothelium in carotid arteries perfused ex vivo and upon application in a peritonitis model. The function of D44A-MIF in peritoneal leukocyte recruitment was preserved as a result of compensatory use of CXCR4. In conjunction, our data identify a pseudo-(E)LR motif as the structural determinant for MIF's activity as a non-canonical CXCR2 ligand, epitomizing the structural resemblance of chemokine-like ligands with chemokines and enabling selective targeting of pro-inflammatory MIF/CXCR2 interactions.

Macrophage migration inhibitory factor (MIF) is a secreted protein that activates macrophages, neutrophils and T cells, and is implicated in sepsis, adult respiratory distress syndrome and rheumatoid arthritis. The mechanism of MIF function, however, is unknown. The three-dimensional structure of MIF is unlike that of any other cytokine, but bears striking resemblance to three microbial enzymes, two of which possess an N-terminal proline that serves as a catalytic base. Human MIF also possesses an N-terminal proline (Pro-1) that is invariant among all known homologues. Multiple sequence alignment of these MIF homologues reveals additional invariant residues that span the entire polypeptide but are in close proximity to the N-terminal proline in the folded protein. We find that p-hydroxyphenylpyruvate, a catalytic substrate of MIF, binds to the N-terminal region and interacts with Pro-1. Mutation of Pro-1 to a glycine substantially reduces the catalytic and cytokine activity of MIF. We suggest that the underlying biological activity of MIF may be based on an enzymatic reaction. The identification of the active site should facilitate the development of structure-based inhibitors.

OBJECTIVE

To determine if polymorphisms of the macrophage migration inhibitory factor (MIF) gene are associated with systemic-onset juvenile idiopathic arthritis (JIA).

METHODS

Denaturing high-performance liquid chromatography was used to screen for the MIF gene in 32 healthy Caucasian subjects. One hundred seventeen UK Caucasian patients with systemic-onset JIA and 172 unrelated healthy UK Caucasian controls were genotyped for a single-nucleotide polymorphism (SNP) identified in the 5'-flanking region of the gene, using polymerase chain reaction-restriction fragment length analysis.

RESULTS

A G-to-C transition was identified at position -173 of the MIF gene. The presence of a C at -173 creates an activator protein 4 transcription factor binding site. Allele and genotype frequencies differed significantly between the patients and controls for the MIF-173 polymorphism. Individuals possessing a MIF-173*C allele have an increased risk of systemic-onset JIA (36.8% versus 20.3%) (odds ratio 2.3, 95% confidence interval 1.34-3.86; P = 0.0005).

CONCLUSIONS

This is the first report of a SNP in the MIF gene. This polymorphism is associated with systemic-onset JIA.

Neutral matrix metalloproteinases (MMPs) are responsible for the pathological features of rheumatoid arthritis (RA) such as degradation of cartilage. We herein show the up-regulation of MMP-1 (interstitial collagenase) and MMP-3 (stromelysin) mRNAs of cultured synovial fibroblasts retrieved from rheumatoid arthritis (RA) patients in response to macrophage migration inhibitory factor (MIF). The elevation of MMP-1 and MMP-3 mRNA was dose-dependent and started at 6 h post-stimulation by MIF, reached the maximum level at 24 h, and was sustained at least up to 36 h. Interleukin (IL)-1beta mRNA was also up-regulated by MIF. These events were preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1, a common inhibitor of these proteases, was slightly up-regulated by MIF. Similarly, mRNA up-regulation of MMP-1 and MMP-3 was observed in the synovial fibroblasts of patients with osteoarthritis. However, their expression levels were much lower than those of RA synovial fibroblasts. The mRNA up-regulation by MIF was inhibited by the tyrosine kinase inhibitors genestein and herbimycin A, as well as the protein kinase C inhibitors staurosporine and H-7. On the other hand, the inhibition was not seen after the addition of the cyclic AMP-dependent kinase inhibitor, H-8. The mRNA up-regulation of MMPs was also inhibited by curcumin, an inhibitor of transcription factor AP-1, whereas interleukin-1 receptor antagonist, an IL-1 receptor antagonist, failed to inhibit the mRNA up-regulation. Considering these results, it is suggested that 1) MIF plays an important role in the tissue destruction of rheumatoid joints via induction of the proteinases, and 2) MIF up-regulates MMP-1 and MMP-3 via tyrosine kinase-, protein kinase C-, and AP-1- dependent pathways, bypassing IL-1beta signal transduction.

Even tough differentiated spermatozoa are unable of transcriptional or translational activity; the sperm surface undergoes major modifications in macromolecules composition during the transit along the male reproductive tract. This is the result of sequential, well orchestrated interactions between the male reproductive tract secretions and the transiting male gamete. This is particularly true when spermatozoa transit along the epididymis. The epididymis is a long convoluted tubules in which the spermatozoa leaving the testis have to transit. The unraveled epididymal tubule can be as long as 80 m in stallion, and the transit time of spermatozoa is of 3-12 days depending on the species. The epididymis is usually divided in three segments: the caput (proximal part), the corpus, and cauda. While the cauda epididymides acts as a sperm reservoir, the caput and corpus are responsible for sperm maturation. This means that, under androgen control, the epididymal epithelium secretes proteins that will interact sequentially with sperm surface. Some of the sperm proteins acquired during maturation along the excurrent duct behave as integral membrane proteins. In fact, some epididymal originating proteins are glycosylphosphatidylinositol (GPI)-anchored to the sperm plasma membrane. Our laboratory has shown that some of these proteins are secreted in an apocrine manner by the epididymal epithelium and are associated to exosomes, called epididymosomes. Epididymosomes are rich in sphingomyelin and are characterized by a high cholesterol/phospholipids ratio. Many proteins are associated to epididymosomes, some of which are selectively transferred to spermatozoa during the epididymal transit. We have identified some of these exosomes associated proteins transferred to the maturing spermatozoa. These include two enzymes involved in the polyol pathway: an aldose reductase and a sorbitol dehydrogenase. A cytokine named MIF (macrophage migration inhibitory factor) is another protein associated to exosomes who is transferred to spermatozoa during the epididymal transit. We hypothesized that both the polyol pathway and MIF secreted in an apocrine fashion by the epididymal epithelium modulate sperm motility during the transit along the male reproductive tract. Finally, P25b, belonging to a family of sperm surface proteins (P26h/P34H) necessary for the binding to the surface of the egg, is also acquired through the interaction between epididymosomes and the male gamete. In vitro studies have defined the conditions of protein transfer when epididymal spermatozoa are co-incubated with epididymosomes. The transfer of selected proteins to specific membrane domains of spermatozoa is saturable, temperature and pH-dependent, being optimal at pH 6.5. The presence of zinc in the incubation medium, but not of calcium neither magnesium, significantly increases the efficiency of protein transfer. These results show that exosomes play a role in sperm epididymal maturation which is an essential event to produce male gametes with optimal fertilizing ability.

AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

Neutral matrix metalloproteinases (MMPs) play an important role in bone matrix degradation accompanied by bone remodeling. We herein show for the first time that macrophage migration inhibitory factor (MIF) up-regulates MMP-13 (collagenase-3) mRNA of rat calvaria-derived osteoblasts. The mRNA up-regulation was seen at 3 h in response to MIF (10 microg/ml), reached the maximum level at 6-12 h, and returned to the basal level at 36 h. MMP-13 mRNA up-regulation was preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1 and MMP-9 (92-kDa type IV collagenase) were also up-regulated, but to a lesser extent. The MMP-13 mRNA up-regulation was significantly suppressed by genistein, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Similarly, a selective mitogen-activated protein kinase (MAPK) kinase (MEK)1/2 inhibitor (PD98059) and c-jun/activator protein (AP)-1 inhibitor (curcumin) suppressed MMP-13 mRNA up-regulation induced by MIF. The mRNA levels of c-jun and c-fos in response to MIF were also inhibited by PD98059. Consistent with these results, MIF stimulated phosphorylation of tyrosine, autophosphorylation of Src, activation of Ras, activation of extracellular signal-regulated kinases (ERK) 1/2, a MAPK, but not c-Jun N-terminal kinase or p38, and phosphorylation of c-Jun. Osteoblasts obtained from calvariae of newborn JunAA mice, defective in phosphorylation of c-Jun, or newborn c-Fos knockout (Fos -/- ) mice, showed much less induction of MMP-13 with the addition of MIF than osteoblasts obtained from wild-type or littermate control mice. Taken together, these results suggest that MIF increases the MMP-13 mRNA level of rat osteoblasts via the Src-related tyrosine kinase-, Ras-, ERK1/2-, and AP-1-dependent pathway.

Macrophage migration-inhibitory factor (MIF) is an upstream regulator of innate immunity and a potential molecular link between inflammation and cancer. The unusual structural homology between MIF and certain tautomerases, which includes both a conserved substrate-binding pocket and a catalytic N-terminal proline (Pro1), has fueled speculation that an enzymatic reaction underlies MIF's biologic function. To address the functional role of the MIF tautomerase activity in vivo, we created a knock-in mouse in which the endogenous mif gene was replaced by one encoding a tautomerase-null, Pro1->>Gly1 MIF protein (P1G-MIF). While P1G-MIF is completely inactive catalytically, it maintains significant, albeit reduced, binding to its cell surface receptor (CD74) and to the intracellular binding protein JAB1/CSN5. P1G-MIF knock-in mice (mif(P1G/P1G)) and cells derived from these mice show a phenotype in assays of growth control and tumor induction that is intermediate between those of the wild type (mif(+/+)) and complete MIF deficiency (mif(-)(/)(-)). These data provide genetic evidence that MIF's intrinsic tautomerase activity is dispensable for this cytokine's growth-regulatory properties and support a role for the N-terminal region in protein-protein interactions.

BACKGROUND

Injury to the brain induces dramatic local changes in gene expression, cellular morphology and behavior. Activation of microglial cells occurs as an early event after central nervous system (CNS) injury, but it has not been determined whether such activation plays a causal role in neuronal death. We have investigated this question using an excitotoxin-mediated brain injury model system, in conjunction with an endogenous peptide factor (macrophage/microglial inhibiting factor, MIF) that ablates microglial contribution to the cascade.

RESULTS

Using MIF, we inhibited the microglial activation that normally follows excitotoxic injury. In cell culture studies, we found that such inhibition blocked the rapid release of microglia-derived tissue plasminogen activator (tPA), an extracellular serine protease made by both neurons and microglia, which we had previously identified as mediating a critical step in excitotoxin-induced neuronal death. Finally, infusion of MIF into the mouse brain prior to excitotoxic insult resulted in the protection of neurons from cell death.

CONCLUSIONS

Our results demonstrate that microglia undertake a neurotoxic role when excitotoxic injury occurs in the CNS. They also suggest that the tPA released from microglia has a critical role in triggering neurodegeneration.

Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disease of unknown etiology. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is operative in innate and adaptive immunity and important in immune-mediated diseases such as rheumatoid arthritis and atherosclerosis. The functional relevance of MIF in systemic autoimmune diseases such as SLE is unknown. Using the lupus-prone MRL/lpr mice, we aim to examine the expression and function of MIF in this murine model of systemic autoimmune disease. These experiments revealed that renal MIF expression was significantly higher in MRL/lpr mice compared with nondiseased control mice (MRL/MpJ), and MIF was also markedly up-regulated in skin lesions of MRL/lpr mice. To examine the effect of MIF on development of systemic autoimmune disease, we generated MRL/lpr mice with a targeted disruption of the MIF gene (MIF(-/-)MRL/lpr), and compared their disease manifestations to MIF(+/+)MRL/lpr littermates. MIF(-/-)MRL/lpr mice exhibited significantly prolonged survival, and reduced renal and skin manifestations of SLE. These effects occurred in the absence of major changes in T and B cell markers or alterations in autoantibody production. In contrast, renal macrophage recruitment and glomerular injury were significantly reduced in MIF(-/-)MRL/lpr mice, and this was associated with reduction in the monocyte chemokine MCP-1. Taken together, these data suggest MIF as a critical effector of organ injury in SLE.

Although it is known that dendritic cells (DCs) produce cytokines, there is little information about how cytokine synthesis is regulated during DC development. A range of cytokine mRNA/proteins was analyzed in immature (CD86-) or mature (CD86+) murine bone marrow (BM)- derived DCs. Highly purified, flow-sorted, immature DCs exhibited higher amounts of interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor beta1 (TGF-beta1), and macrophage migration inhibitory factor (MIF) mRNA/protein than mature DCs. After differentiation, DC up-regulated the levels of IL-6 and IL-15 mRNA/protein and synthesized de novo mRNA/protein for IL-12p35, IL-12p40, and IL-18. Although immature BM-derived DCs did not stimulate naive allogeneic T cells, mature DCs elicited a mixed population of T helper (Th) 1 (mainly) and Th2 cells in 3d-mixed leukocyte reactions. CD86+ BM DCs switched to different cytokine patterns according to whether they were terminally differentiated by lipopolysaccharide (LPS) or CD40 ligation. Although both stimuli increased IL-6, IL-12p40, IL-15, and TNF-alpha mRNA/protein levels, only LPS up-regulated transcription of IL-1alpha, IL-1beta, IL-12p35, and MIF genes. Although LPS and CD40 cross-linking increased the T-cell allostimulatory function of BM DCs, only LPS stimulation shifted the balance of naive Th differentiation to Th1 cells, a mechanism dependent on the up-regulation of IL-12p35 and not of IL-23. These results demonstrate that, depending on the stimuli used to terminally mature BM DCs, DCs synthesize a different pattern of cytokines and exhibit distinct Th cell-driving potential.