A MIF-Derived Cyclopeptide that Inhibits MIF Binding and Atherogenic Signaling via the Chemokine Receptor CXCR2.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine and atypical chemokine with a key role in inflammatory diseases including atherosclerosis. Key atherogenic functions of MIF are mediated by noncognate interaction with the chemokine receptor CXCR2. The MIF N-like loop comprising the sequence 47-56 is an important structural determinant of the MIF/CXCR2 interface and MIF(47-56) blocks atherogenic MIF activities. However, the mechanism and critical structure-activity information within this sequence have remained elusive. Here, we show that MIF(47-56) directly binds to CXCR2 to compete with MIF receptor activation. By using alanine scanning, essential and dispensable residues were identified. Moreover, MIF(cyclo10), a designed cyclized variant of MIF(47-56), inhibited key inflammatory and atherogenic MIF activities in vitro and in vivo/ex vivo, and exhibited strongly improved resistance to proteolytic degradation in human plasma in vitro, thus suggesting that it could serve as a promising basis for MIF-derived anti-atherosclerotic peptides.

A self-inhibitory interaction within Nup155 and membrane binding are required for nuclear pore complex formation.

Nuclear pore complexes (NPCs) are gateways through the nuclear envelope. How they form into a structure containing three rings and integrate into the nuclear envelope remains a challenging paradigm for coordinated assembly of macro-complexes. In vertebrates, the cytoplasmic and nucleoplasmic rings of NPCs are mostly formed by multiple copies of the Nup107-Nup160 complex, whereas the central, or inner ring is composed of Nup53, Nup93, Nup155 and the two paralogues Nup188 and Nup205. Inner ring assembly is only partially understood. Using in vitro nuclear assembly reactions, we show that direct pore membrane binding of Nup155 is crucial for NPC formation. Replacing full-length Nup155 with its N-terminal ß-propeller allows assembly of the outer ring components to the NPC backbone that also contains Nup53. However, further assembly, especially recruitment of the Nup93 and Nup62 complexes, is blocked. Self-interaction between the N- and C-terminal domains of Nup155 has an auto-inhibitory function that prevents interaction between the N-terminus of Nup155 and the C-terminal region of Nup53. Nup93 can overcome this block by binding to Nup53, thereby promoting formation of the inner ring and the NPC.

Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin.

The response of the skin to harmful environmental agents is shaped decisively by the status of the immune system. Keratinocytes constitutively express and secrete the chemokine-like mediator, macrophage migration inhibitory factor (MIF), more strongly than dermal fibroblasts, thereby creating a MIF gradient in skin. By using global and epidermis-restricted Mif-knockout (Mif-/- and K14-Cre+/tg; Miffl/fl) mice, we found that MIF both recruits and maintains antigen-presenting cells in the dermis/epidermis. The reduced presence of antigen-presenting cells in the absence of MIF was associated with accelerated and increased formation of nonmelanoma skin tumors during chemical carcinogenesis. Our results demonstrate that MIF is essential for maintaining innate immunity in skin. Loss of keratinocyte-derived MIF leads to a loss of control of epithelial skin tumor formation in chemical skin carcinogenesis, which highlights an unexpected tumor-suppressive activity of MIF in murine skin.-Brocks, T., Fedorchenko, O., Schliermann, N., Stein, A., Moll, U. M., Seegobin, S., Dewor, M., Hallek, M., Marquardt, Y., Fietkau, K., Heise, R., Huth, S., Pfister, H., Bernhagen, J., Bucala, R., Baron, J. M., Fingerle-Rowson, G. Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin.

The Effect of Lipoaspirates on Human Keratinocytes.

BACKGROUND: One increasingly important trend in plastic, reconstructive, and aesthetic surgery is the use of fat grafts to improve cutaneous wound healing. In clinical practice, lipoaspirates (adipose tissue harvested by liposuction) are re-injected in a procedure called lipofilling. Previous studies, however, mainly evaluated the regenerative effect of isolated adipocytes, adipose-derived stem cells, and excised en bloc adipose tissue on keratinocytes, whereas no study to date has examined the effect of lipoaspirates. OBJECTIVES: The authors aimed to investigate differences in the regenerative property of en bloc adipose tissue and lipoaspirates on keratinocytes. METHODS: Human keratinocytes, lipoaspirates, and en bloc adipose tissue from 36 healthy donors were isolated. In vitro proliferation, differentiation, migration, stratification, and wound healing of keratinocyte monolayers were measured. Furthermore, secreted levels of VEGF, bFGF, IGF-1, MMP-9, and MIF were detected by ELISA. RESULTS: Migration, proliferation, and wound healing of keratinocytes were increased by lipoaspirates. Interestingly, the effect of lipoaspirates on keratinocyte proliferation was significantly higher than by en bloc adipose tissue after 5 days. The differentiation of keratinocytes was equally attenuated by lipoaspirates and en bloc adipose tissue. Stratification of keratinocyte layers was enhanced by lipoaspirates and en bloc fat when compared to controls. Lipoaspirates secrete higher levels of bFGF, whereas higher levels of VEGF and IGF-1 are released by en bloc adipose tissue. CONCLUSION: We show that lipoaspirates and en bloc adipose tissue have a regenerative effect on keratinocytes. One reason for the higher effect of lipoaspirates on keratinocyte proliferation may be the secretion of different cytokines.

Cardioprotection through S-nitros(yl)ation of macrophage migration inhibitory factor.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a structurally unique inflammatory cytokine that controls cellular signaling in human physiology and disease through extra- and intracellular processes. Macrophage migration inhibitory factor has been shown to mediate both disease-exacerbating and beneficial effects, but the underlying mechanism(s) controlling these diverse functions are poorly understood. METHODS AND RESULTS: Here, we have identified an S-nitros(yl)ation modification of MIF that regulates the protective functional phenotype of MIF in myocardial reperfusion injury. Macrophage migration inhibitory factor contains 3 cysteine (Cys) residues; using recombinant wtMIF and site-specific MIF mutants, we have identified that Cys-81 is modified by S-nitros(yl)ation whereas the CXXC-derived Cys residues of MIF remained unaffected. The selective S-nitrosothiol formation at Cys-81 led to a doubling of the oxidoreductase activity of MIF. Importantly, S-nitrosothiol-MIF formation was measured both in vitro and in vivo and led to a decrease in cardiomyocyte apoptosis in the reperfused heart. This decrease was paralleled by a S-nitrosothiol-MIF- but not Cys81 serine (Ser)-MIF mutant-dependent reduction of infarct size in an in vivo model of myocardial ischemia/reperfusion injury. CONCLUSIONS: S-nitros(yl)ation of MIF is a pivotal novel regulatory mechanism, providing enhanced activity resulting in increased cytoprotection in myocardial reperfusion injury.

Macrophage migration inhibitory factor is a potential inducer of endothelial progenitor cell mobilization after flap operation.

BACKGROUND: Endothelial progenitor cells (EPCs) promote angiogenesis and play an important role in tissue revascularization and wound healing. Yet, the exact stimuli and mechanisms for the mobilization remain understood poorly. Macrophage migration inhibitory factor (MIF), which is a structurally unique pleiotropic cytokine, has been suggested to play a role in EPC recruitment and thus was a target of this study. METHODS: This study included 20 patients who underwent flap operation. Subjects were divided into 3 groups according to the pattern of flap applied. The number of circulating EPCs and serum levels of MIF or CXCL12 were determined at different time intervals. In vitro chemotaxis experiments using Transwell devices were carried out to test whether MIF promotes the chemotactic migration of EPCs. To underscore functionally the chemotactic potential of MIF toward EPCs in flap patients, the chemotactic effects of serum samples from all groups were also examined in the presence and absence of monoclonal anti-macrophage migration inhibitory factor and anti-CXCL12 antibodies on EPC recruitment using in vitro migration chambers. RESULTS: In flap patients, the number of circulating EPCs and serum levels of MIF but not CXCL12 serum levels were increased markedly compared with preoperative levels at day 1 after operation, especially in the group of free microvascular flaps. Serum levels of CXCL12 in the flap patients were increased only significantly compared with the healthy control group. An analysis between EPCs and MIF revealed a significant correlation, whereas no correlation was observed for CXCL12. MIF exerted a dose-dependent, prochemotactic effect on isolated human EPCs, and serum samples from all flap patients promoted EPC migration. Importantly, this effect was blocked partially by anti-macrophage migration inhibitory factor and to a weaker extent by anti-CXCL12 antibodies. CONCLUSION: We conclude that MIF plays an important role in the mobilization of EPCs, which is dependent on the degree of ischemia. Enhancement by MIF of chemotactic EPCs migration in vitro underpins its proposed in vivo function.

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.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

Macrophage migration inhibitory factor (MIF) promotes fibroblast migration in scratch-wounded monolayers in vitro.

MIF was recently redefined as an inflammatory cytokine, which functions as a critical mediator of diseases such as septic shock, rheumatoid arthritis, atherosclerosis, and cancer. MIF also regulates wound healing processes. Given that fibroblast migration is a central event in wound healing and that MIF was recently demonstrated to promote leukocyte migration through an interaction with G-protein-coupled receptors, we investigated the effect of MIF on fibroblast migration in wounded monolayers in vitro. Transient but not permanent exposure of primary mouse or human fibroblasts with MIF significantly promoted wound closure, a response that encompassed both a proliferative and a pro-migratory component. Importantly, MIF-induced fibroblast activation was accompanied by an induction of calcium signalling, whereas chronic exposure with MIF down-regulated the calcium transient, suggesting receptor desensitization as the underlying mechanism.

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.

A novel drug-eluting stent coated with an integrin-binding cyclic Arg-Gly-Asp peptide inhibits neointimal hyperplasia by recruiting endothelial progenitor cells.

OBJECTIVES: Novel stents loaded with an integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) were analyzed for their potential to limit coronary neointima formation and to accelerate endothelialization by attracting endothelial progenitor cells (EPCs). BACKGROUND: Re-endothelialization is important for healing after arterial injury. METHODS: Effects of cRGD on EPC number, recruitment in flow, and invasion were analyzed in vitro. A durable polymer coating containing 67 microg cRGD per stent was developed for Guidant Tetra stents. Twelve cRGD-loaded polymer, 12 unloaded polymer, and 12 bare metal stents were deployed in porcine coronary arteries. Quantification of cRGD in peri-stent tissue was established by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Histomorphometry and immunostaining were performed after 4 and 12 weeks. Recruitment of labeled porcine EPCs was assessed 7 days after intracoronary infusion. RESULTS: The cRGD clearly supported the outgrowth, recruitment, and migration of EPCs in vitro. At 4 weeks, there was no difference for mean neointimal area and percent area stenosis in the cRGD-loaded, polymer, or bare metal stent group. At 12 weeks, neointimal area (2.2 +/- 0.3 mm2) and percent area stenosis (33 +/- 5%) were significantly reduced compared with polymer stents (3.8 +/- 0.4 mm2, 54 +/- 6%; p = 0.010) or bare metal stents (3.8 +/- 0.3 mm2, 53 +/- 3%; p < 0.001). The HPLC/MS confirmed cRGD tissue levels of 1 to 3 mug/stent at 4 weeks, whereas cRGD was not detectable at 12 weeks. Staining for CD34 and scanning electron microscopy indicated enhanced endothelial coverage on cRGD-loaded stents at 4 weeks associated with a significant increase in the early recruitment of infused EPCs. CONCLUSIONS: Stent coating with cRGD may be useful for reducing in-stent restenosis by accelerating endothelialization.