Showing papers by "Richard Bucala published in 2009"

Pure Hemozoin Is Inflammatory In Vivo and Activates the NALP3 Inflammasome via Release of Uric Acid

Abstract: The role of proinflammatory cytokine production in the pathogenesis of malaria is well established, but the identification of the parasite products that initiate inflammation is not complete. Hemozoin is a crystalline metabolite of hemoglobin digestion that is released during malaria infection. In the present study, we characterized the immunostimulatory activity of pure synthetic hemozoin (sHz) in vitro and in vivo. Stimulation of naive murine macrophages with sHz results in the MyD88-independent activation of NF-κB and ERK, as well as the release of the chemokine MCP-1; these responses are augmented by IFN-γ. In macrophages prestimulated with IFN-γ, sHz also results in a MyD88-dependent release of TNF-α. Endothelial cells, which encounter hemozoin after schizont rupture, respond to sHz by releasing IL-6 and the chemokines MCP-1 and IL-8. In vivo, the introduction of sHz into the peritoneal cavity produces an inflammatory response characterized by neutrophil recruitment and the production of MCP-1, KC, IL-6, IL-1α, and IL-1β. MCP-1 and KC are produced independently of MyD88, TLR2/4 and TLR9, and components of the inflammasome; however, neutrophil recruitment, the localized production of IL-1β, and the increase in circulating IL-6 require MyD88 signaling, the IL-1R pathway, and the inflammasome components ICE (IL-1β-converting enzyme), ASC (apoptosis-associated, speck-like protein containing CARD), and NALP3. Of note, inflammasome activation by sHz is reduced by allopurinol, which is an inhibitor of uric acid synthesis. These data suggest that uric acid is released during malaria infection and may serve to augment the initial host response to hemozoin via activation of the NALP3 inflammasome.

Cardiac macrophage migration inhibitory factor inhibits JNK pathway activation and injury during ischemia/reperfusion

Abstract: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that also modulates physiologic cell signaling pathways. MIF is expressed in cardiomyocytes and limits cardiac injury by enhancing AMPK activity during ischemia. Reperfusion injury is mediated in part by activation of the stress kinase JNK, but whether MIF modulates JNK in this setting is unknown. We examined the role of MIF in regulating JNK activation and cardiac injury during experimental ischemia/reperfusion in mouse hearts. Isolated perfused Mif-/- hearts had greater contractile dysfunction, necrosis, and JNK activation than WT hearts, with increased upstream MAPK kinase 4 phosphorylation, following ischemia/reperfusion. These effects were reversed if recombinant MIF was present during reperfusion, indicating that MIF deficiency during reperfusion exacerbated injury. Activated JNK acts in a proapoptotic manner by regulating BCL2-associated agonist of cell death (BAD) phosphorylation, and this effect was accentuated in Mif-/- hearts after ischemia/reperfusion. Similar detrimental effects of MIF deficiency were observed in vivo following coronary occlusion and reperfusion in Mif-/- mice. Importantly, excess JNK activation also was observed after hypoxia-reoxygenation in human fibroblasts homozygous for the MIF allele with the lowest level of promoter activity. These data indicate that endogenous MIF inhibits JNK pathway activation during reperfusion and protects the heart from injury. These findings have clinical implications for patients with the low-expression MIF allele.

A Tautomerase-Null Macrophage Migration-Inhibitory Factor (MIF) Gene Knock-In Mouse Model Reveals That Protein Interactions and Not Enzymatic Activity Mediate MIF-Dependent Growth Regulation

Abstract: Macrophage migration-inhibitory factor (MIF) is a widely expressed cytokine and upstream regulator of the immune response (23). Immunoneutralization and genetic knockout studies have established a central position for MIF in the host response to infection and tissue invasion (5, 9, 15). MIF's importance in human disease also has been revealed by the association of high-expression MIF alleles with clinical severity of different autoimmune disorders (18). An important role for MIF in tumorigenesis and in the contribution of inflammation to cancer development also has been proposed (7, 20). Different tumor types express high levels of MIF, and clinical studies have shown that MIF production correlates with tumor aggressiveness and metastatic potential (1, 22, 27). Studies using genetically engineered MIF-deficient cells and mice show that MIF contributes to the development of the malignant phenotype by several mechanisms, including enhancement of cell cycle progression by sustained mitogen-activated protein kinase (MAPK) activation (28, 30), decreased proteasomal protein degradation (33) leading to altered expression of key cell cycle-regulatory proteins (15, 21, 35), and tumor promotion by neoangiogenesis (10, 48). Importantly, MIF also inhibits the proapoptotic and cell cycle-regulatory function of the p53 tumor suppressor, thereby allowing for the accumulation of oncogenic mutations (20, 32). MIF's role in tumor progression additionally is supported by human genetic studies, and a recent report has described an association between high-expression MIF alleles and incidence of prostate cancer, which is a tumor type in which recurrent inflammation is considered to have an etiologic role (27). Information regarding MIF structure and function has emerged only in the last few years. The mammalian protein is encoded by a unique gene, and the solution of MIF's three-dimensional crystal structure revealed that the protein defines a new structural superfamily (41, 42). Notably, MIF shows structural but not sequence homology with two prokaryotic enzymes, 4-oxalocrotonate tautomerase and 5-carboxy-methyl-2-hydroxymuconate isomerase, which act in the pathway of aromatic amino acid catabolism. Subsequent studies have shown that MIF tautomerizes model substrates such as d-dopachrome and p-hydroxyphenylpyruvate, although the measured enzymatic rate constants do not support a physiologic role for these reactions (36, 37). The d stereoisomer of dopachrome also does not occur in nature, but the l steroisomer is a substrate for the melanotic encapsulation response, which constitutes an invertebrate host defense mechanism against microbial invasion. Accordingly, some investigators have proposed that the catalytic activity of MIF is vestigial and reflects the protein's ancestral origin in innate immunity (6). Others have argued that MIF's highly conserved substrate binding pocket and catalytic N-terminal proline (Pro1), which are shared with the prokaryotic isomerases, provide strong evidence for an intrinsic, enzymatic function for mammalian MIF (44). In vitro studies that have sought to address the precise relationship between MIF's catalytic activity and its biological function have not been conclusive. Site-directed mutagenesis approaches that have replaced MIF's catalytic, N-terminal proline with inactive residues have confirmed that Pro1 and a surrounding hydrophobic pocket mediate tautomerase activity (2). Most studies have shown a preservation of proinflammatory function for catalytically inactive, recombinantly produced MIF proteins in vitro (2, 19); however, this finding has not been consistent (34, 43), and conclusions may be limited by the semiquantitative nature of the assays employed. It also should be noted that a commercially available recombinant human MIF bears a mutation in the carboxy-terminal oligomerization domain (Asn106→Ser106) that may influence bioactivity (4, 12). Recent studies also suggest that the MIF N-terminal region makes critical contacts with CD74, a cell surface receptor that mediates the cytokine's signal transduction activity (24, 38). To address the role of the MIF tautomerase activity in vivo, we created a knock-in mouse in which the endogenous mif gene is replaced by a tautomerase-null, Pro1→Gly1 MIF protein (P1G-MIF). We reasoned that, if the tautomerase activity was necessary for MIF biologic function, a genetically defined P1G-MIF mouse (mifP1G/P1G), or cells derived from such a mouse, would show a phenotype identical to that of the MIF knockout mouse (mif−/−). Alternatively, if the tautomerase activity was dispensable, then P1G-MIF mice should show the same phenotype as the wild type (mif+/+). An intermediate level of activity would support a structural role for Pro1, but not the tautomerase activity per se, in protein-protein interactions necessary for MIF effector function.

Discovery of human macrophage migration inhibitory factor (MIF)-CD74 antagonists via virtual screening.

Abstract: Macrophage migration inhibitory factor (MIF) is a cytokine that is involved in the regulation of inflammation as well as cell proliferation and differentiation. Deactivation of MIF by antibodies or inhibition of MIF binding to its receptor, CD74, attenuates tumor growth and angiogenesis. To discover small-molecule inhibitors of MIF’s biological activity, virtual screening was performed by docking 2.1 million compounds into the MIF tautomerase active site. After visual inspection of 1200 top-ranked MIF-ligand complexes, 26 possible inhibitors were selected and purchased and 23 of them were assayed. The in vitro binding assay for MIF with CD74 revealed that 11 of the compounds have inhibitory activity in the micromolar regime, including four compounds with IC50 values below 5 μM. Inhibition of MIF tautomerase activity was also established for many of the compounds with IC50 values as low as 0.5 μM; Michaelis−Menten analysis was performed for two cases and confirmed the competitive inhibition.

The Golgi-associated protein p115 mediates the secretion of macrophage migration inhibitory factor.

Abstract: Macrophage migration inhibitory factor (MIF) is a leaderless protein that is secreted from cells by a specialized, nonclassical export pathway. The release of MIF nevertheless is regulated and its production in response to different inflammatory, mitogenic, and hormonal stimuli plays an important role in diverse physiologic and pathologic processes. We report herein the identification of the Golgi complex-associated protein p115 as an intracellular binding partner for MIF. MIF interacts with p115 in the cytoplasm and the stimulated secretion of MIF results in the accumulation of both proteins in supernatants, which is consistent with MIF release from cells in conjunction with p115. The depletion of p115 from monocytes/macrophages decreases the release of MIF but not other cytokines following inflammatory stimulation or intracellular bacterial infection. Notably, the small molecule MIF inhibitor 4-iodo-6-phenylpyrimidine inhibits MIF secretion by targeting the interaction between MIF and p115. These data reveal p115 to be a critical intermediary component in the regulated secretion of MIF from monocytes/macrophages.

The influence of macrophage migration inhibitory factor gene polymorphisms on outcome from community-acquired pneumonia

Abstract: The cytokine, macrophage migration inhibitory factor (MIF), is encoded in a functionally polymorphic locus and subjects with high-expression MIF alleles are at an increased risk of inflammatory disease. Severe sepsis is the leading cause of death in intensive care units, and the prevailing hypothesis is that an excessive innate response contributes to its pathogenesis. To assess if MIF alleles influence the clinical course of infection, we conducted a case-control study to assess susceptibility and a parallel inception cohort study of community-acquired pneumonia (CAP) to assess risk of severe sepsis and 90-d mortality. Two distinct polymorphisms in the MIF promoter were analyzed: a G/C transition at -173 and a CATT repeat at -794. The frequency of both polymorphisms was similar in the CAP cohort (n=1739) and controls (n=639); however, the 90-d mortality was lower for the high-expression C allele (P=0.003). This association remained significant after adjusting for demographics, comorbid conditions, and disease severity score [hazard ratio=0.64 (0.44-0.91), P=0.01]. The hazard ratio was similar in different geographic subcohorts, and the association remained significant after adjusting for false discovery. These data indicate that polymorphisms associated with higher MIF expression may have a beneficial effect in community-acquired pneumonia.

MIF (Macrophage Migration Inhibitory Factor) Promoter Polymorphisms and Susceptibility to Severe Malarial Anemia

Abstract: Background Severe malarial anemia (SMA) resulting from Plasmodium falciparum infections is one of the leading causes of childhood mortality in sub-Saharan Africa. The innate immune mediator, macrophage migration inhibitory factor (MIF) plays a critical role in the pathogenesis of SMA.

Human cytomegalovirus paralyzes macrophage motility through down-regulation of chemokine receptors, reorganization of the cytoskeleton, and release of macrophage migration inhibitory factor.

Abstract: Macrophages contribute to host defense and to the maintenance of immune homeostasis. Conversely, they are important targets of human cytomegalovirus (HCMV), a herpesvirus that has evolved many strategies to modulate the host immune response. Because an efficient macrophage trafficking is required for triggering an adequate immune response, we investigated the effects exerted by HCMV infection on macrophage migratory properties. By using endotheliotropic strains of HCMV, we obtained high rates of productively infected human monocyte-derived macrophages (MDM). Twenty-four hours after infection, MDM showed reduced polar morphology and became unable to migrate in response to inflammatory and lymphoid chemokines, bacterial products and growth factors, despite being viable and metabolically active. Although chemotactic receptors were only partially affected, HCMV induced a dramatic reorganization of the cytoskeleton characterized by rupture of the microtubular network, stiffness of the actin fibers, and collapse of the podosomes. Furthermore, supernatants harvested from infected MDM contained high amounts of macrophage migration inhibitory factor (MIF) and were capable to block the migration of neighboring uninfected MDM. Because immunodepletion of MIF from the conditioned medium completely restored MDM chemotaxis, we could show for the first time a functional role of MIF as an inhibitor of macrophage migration in the context of HCMV infection. Our findings reveal that HCMV uses different mechanisms to interfere with movement and positioning of macrophages, possibly leading to an impairment of antiviral responses and to an enhancement of the local inflammation.

A New Class of Isothiocyanate-Based Irreversible Inhibitors of Macrophage Migration Inhibitory Factor

Abstract: Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflamma- tory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-basedirreversibleinhibitorsofMIF.Modificationbybenzylisothiocyanate(BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomeriza- tion state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.

Structural and kinetic analyses of macrophage migration inhibitory factor active site interactions.

Abstract: Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We ...

Gadolinium-containing magnetic resonance image contrast agent promotes fibrocyte differentiation.

Abstract: Gadolinium-containing magnetic resonance imaging (MRI) contrast agents such as Omniscan are associated with nephrogenic systemic fibrosis (NSF). To determine if Omniscan can affect the differentiation of monocytes into fibroblast-like cells called fibrocytes that are found in the fibrotic lesions of NSF, peripheral blood mononuclear cells (PBMCs) from NSF patients, hemodialysis patients without NSF, and healthy, renally sufficient controls were exposed to Omniscan in a standardized in vitro fibrocyte differentiation protocol. When added to PBMCs, the gadolinium-containing MRI contrast agent Omniscan generally had little effect on fibrocyte differentiation. However, 10(-8) to 10(-3) mg/mL Omniscan reduced the ability of the fibrocyte differentiation inhibitor serum amyloid P (SAP) to decrease fibrocyte differentiation in PBMCs from 15 of 17 healthy controls and one of three NSF patients. Omniscan reduced the ability of SAP to decrease fibrocyte differentiation from purified monocytes, indicating that the Omniscan effect does not require the presence of other cells (such as T cells) in the PBMCs. Omniscan also reduced the ability of a different fibrocyte differentiation inhibitor, interleukin-12, to decrease fibrocyte differentiation. These data suggest that Omniscan interferes with the regulatory action of signals that inhibit the differentiation of monocytes to fibrocytes. J. Magn. Reson. Imaging 2009;30:1284-1288. (c) 2009 Wiley-Liss, Inc.

Essential Role for Macrophage Migration Inhibitory Factor in Gastritis Induced by Helicobacter pylori

Abstract: Macrophage migration inhibitory factor (MIF) is an upstream regulator of immune and inflammatory responses; however, its role in Helicobacter pylori (HP)-associated gastritis remains unknown. We infected MIF knockout (KO) and wild-type mice with SS1 HP and found that 2 weeks after infection, MIF and its receptor CD74 were markedly up-regulated in wild-type mice. This up-regulation preceded the up-regulation of both tumor necrosis factor-α and intercellular adhesion molecule-1, as well as the development of moderate gastritis at 8 weeks, as determined by a significant infiltration of neutrophils, T cells, and macrophages. In contrast, KO mice were protected against HP-induced gastritis by preventing the up-regulation of CD74 and Th1-mediated immune injury, including a reduction in the Th1 transcriptional factor T-bet and the expression of interferon-γ. Additionally, inhibition of skin delayed type hypersensitivity reactions to HP antigens in KO mice also suggested a critical role for MIF in cell-mediated injury. A regulatory role for MIF in Th1-immune responses was further demonstrated by the finding that antigen-primed CD4+ T cells lacking MIF failed to differentiate into the Th1 phenotype; these cells were instead promoted to Th2 differentiation after challenge with HP antigen in vitro. Results from this study indicated that inhibition of HP-induced innate immune responses and Th1-mediated immune injury may be the key mechanisms by which KO mice failed to develop gastritis after HP infection.

Intraluminal blockade of cell-surface CD74 and glucose regulated protein 78 prevents substance P-induced bladder inflammatory changes in the rat.

Abstract: 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.

DNA vaccination against macrophage migration inhibitory factor improves atopic dermatitis in murine models.

Abstract: Background Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has been implicated in the pathogenesis of AD. Recently, we developed a novel DNA vaccine that generates neutralizing endogenous anti-MIF antibodies. Objective This study explores the preventive and therapeutic effects of this MIF-DNA vaccine in mouse models of AD. Methods Two different AD model mice (DS-Nh and NC/Nga) received MIF-DNA vaccination to analyze preventive and therapeutic effects, as assessed by clinical skin scores, histologic findings, and serum IgE levels. Results In murine models of AD, MIF-DNA vaccination prevented the occurrence of the AD skin phenotype. Furthermore, administration of MIF-DNA vaccine to mice that had already developed AD produced a rapid improvement in AD skin manifestation. There were reduced histologic signs of inflammation and lower serum IgE levels in treated mice compared with those seen in control animals. Finally, passive transfer of IgG from MIF-DNA vaccinated mice to AD mice also produced a significant therapeutic effect. These results demonstrate that MIF-DNA vaccination not only prevents the development of AD but also improves the symptoms of pre-existing AD. Conclusion Taken together, the induction of an anti-MIF autoantibody response using MIF-DNA vaccination appears to be a useful approach in the treatment of AD.

Modeling Informatively Missing Genotypes in Haplotype Analysis.

Abstract: It is common to have missing genotypes in practical genetic studies. The majority of the existing statistical methods, including those on haplotype analysis, assume that genotypes are missing at random—that is, at a given marker, different genotypes and different alleles are missing with the same probabilities. In our previous work, we demonstrated that the violation of this assumption may lead to serious bias in haplotype frequency estimates and haplotype association analysis. We proposed a general missing data model to simultaneously characterize missing data patterns across a set of two or more biallelic markers. We proved that haplotype frequencies and missing data probabilities are identifiable if and only if there is linkage disequilibrium between these markers under the general missing data model. In this study, we extend our work to multi-allelic markers and observe a similar finding. Simulation studies on the analysis of haplotypes consisting of two markers illustrate that our proposed model can ...

Macrophage migration inhibitory factor and multiple organ dysfunction syndrome

Abstract: During severe sepsis, organ dysfunction is an important predictor of poor outcomes. Animal and human studies suggest that macrophage migration inhibitory factor (MIF) plays a detrimental role in organ dysfunction in severe sepsis. However, MIF is a key mediator of the innate immune response and is necessary for resistance to certain infections. We provide a broad overview of the beneficial and detrimental effects of MIF in sepsis. The interplay between MIF and glucocorticoids, which may play a critical role in sepsis, is also examined.

Modulateurs de mif

Abstract: L’invention concerne de nouveaux composes heterocycliques, de nouvelles compositions pharmaceutiques et de nouveaux procedes de traitement qui modulent les niveaux d’expression de MIF et traitent les troubles associes a des niveaux d’expression de MIF eleves ou bas.