Sandra Kraemer

A functional heteromeric MIF receptor formed by CD74 and CXCR4.

MINT‐7234499: CXCR4 (uniprotkb:P61073) and CD74 (uniprotkb:P04233) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Structural determinants of MIF functions in CXCR2-mediated inflammatory and atherogenic leukocyte recruitment

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 MIFs 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.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Selenium and Its Supplementation in Cardiovascular Disease—What do We Know?

The trace element selenium is of high importance for many of the body’s regulatory and metabolic functions. Balanced selenium levels are essential, whereas dysregulation can cause harm. A rapidly increasing number of studies characterizes the wide range of selenium dependent functions in the human body and elucidates the complex and multiple physiological and pathophysiological interactions of selenium and selenoproteins. For the majority of selenium dependent enzymes, several biological functions have already been identified, like regulation of the inflammatory response, antioxidant properties and the proliferation/differentiation of immune cells. Although the potential role of selenium in the development and progression of cardiovascular disease has been investigated for decades, both observational and interventional studies of selenium supplementation remain inconclusive and are considered in this review. This review covers current knowledge of the role of selenium and selenoproteins in the human body and its functional role in the cardiovascular system. The relationships between selenium intake/status and various health outcomes, in particular cardiomyopathy, myocardial ischemia/infarction and reperfusion injury are reviewed. We describe, in depth, selenium as a biomarker in coronary heart disease and highlight the significance of selenium supplementation for patients undergoing cardiac surgery.

Ribosomal Protein S19 Interacts with Macrophage Migration Inhibitory Factor and Attenuates Its Pro-inflammatory Function

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (KD = 1.3 × 10-6 m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF.

MIF-chemokine receptor interactions in atherogenesis are dependent on an N-loop-based 2-site binding mechanism

Macrophage migration inhibitory factor (MIF) is a cytokine that mediates inflammatory diseases. MIF promotes atherogenic leukocyte recruitment through a promiscuous, yet highly affine, interaction with CXCR2 and CXCR4. Binding to CXCR2 is dependent on a pseudo‐(E)LR motif in MIF, but a second interaction site has been elusive. Here we identified an N‐like loop in MIF, suggesting that MIF binding to CXCR2 follows the 2‐site binding mode of bona fide chemokines. For MIF, the model predicts interactions between the N‐like loop and the CXCR2 N domain (site 1) and pseudo‐(E)LR and extracellular loops (ELs) of CXCR2 (site 2). Applying biophysical and peptide array analysis, we demonstrated an interaction between MIF and the CXCR2 N domain, which was pseudo‐(E)LR independent. Peptide array analysis also indicated that the pseudo‐(E)LR motif is responsible for MIF binding to EL2 and 3. Notably, peptides MIF‐(40–49) and MIF‐(47–56), representing N‐like‐loop‐derived peptides, but not a scrambled control peptide, significantly blocked MIF/CXCR2 binding, MIF‐mediated monocyte arrest under flow on aortic endothelial cells in vitro (IC50: 1.24X10 “6 M), and MIF‐dependent monocyte adhesion to atherosclerotic mouse carotid arteries in vivo. Thus, the N‐like loop in MIF is critical for MIFs noncognate interaction with CXCR2 and proatherogenic functions. The 2‐site binding model that explains chemokine receptor activation also applies to MIF.—Kraemer, S., Lue, H., Zernecke, A., Kapurniotu, A., Andreetto, E., Frank, R., Lennartz, B., Weber, C., Bernhagen, J. MIF‐chemokine receptor interactions in atherogenesis are dependent on an N‐loop‐based 2‐site binding mechanism. FASEB J. 25, 894–906 (2011). www.fasebj.org

Interaction of MIF Family Proteins in Myocardial Ischemia/Reperfusion Damage and Their Influence on Clinical Outcome of Cardiac Surgery Patients.

Abstract Aims: Cardiac surgery involves myocardial ischemia/reperfusion (I/R) with potentially deleterious consequences. Macrophage migration inhibitory factor (MIF) is a stress-regulating chemokine-like cytokine that protects against I/R damage, but functional links with its homolog, d-dopachrome tautomerase (MIF-2), and the circulating soluble receptor CD74 (sCD74) are unknown. In this study, we investigate the role of MIF, MIF-2, sCD74, and MIF genotypes in patients scheduled for elective single or complex surgical procedures such as coronary artery bypass grafting or valve replacement. Results: MIF and MIF-2 levels significantly increased intraoperatively, whereas measured sCD74 decreased correspondingly. Circulating sCD74/MIF complexes were detectable in 50% of patients and enhanced MIF antioxidant activity. Intraoperative MIF levels were independently associated with a reduced risk for the development of atrial fibrillation (AF) (odds ratio 0.99 [0.98–1.00]; p=0.007). Circulating levels of MIF-2, but not MIF, were associated with an increased frequency of organ dysfunction and predicted the occurrence of AF (area under the curve [AUC]=0.663; p=0.041) and pneumonia (AUC=0.708; p=0.040). Patients with a high-expression MIF genotype exhibited a reduced incidence of organ dysfunction compared with patients with low-expression MIF genotypes (3 vs. 25; p=0.042). Innovation: The current study comprehensively highlights the kinetics and clinical relevance of MIF family proteins and the MIF genotype in cardiac surgery patients. Conclusion: Our findings suggest that increased MIF levels during cardiac surgery feature organ-protective properties during myocardial I/R, while the soluble MIF receptor, sCD74, may enhance MIF antioxidant activity. In contrast, high MIF-2 levels are predictive of the development of organ dysfunction. Importantly, we provide first evidence for a gene–phenotype relationship between variant MIF alleles and clinical outcome in cardiac surgery patients. Antioxid. Redox Signal. 00, 000–000.

Assessment of macrophage migration inhibitory factor in humans: protocol for accurate and reproducible levels

The analytical validation of a possible biomarker is the first step in the long translational process from basic science to clinical routine. Although the chemokine-like cytokine macrophage migration inhibitory factor (MIF) has been investigated intensively in experimental approaches to various disease conditions, its transition into clinical research is just at the very beginning. Because of its presence in preformed storage pools, MIF is the first cytokine to be released under various stimulation conditions. In the first proof-of-concept studies, MIF levels correlated with the severity and outcome of various disease states. In a recent small study with acute coronary syndrome patients, elevation of MIF was described as a new factor for risk assessment. When these studies are compared, not only MIF levels in diseased patients differ, but also MIF levels in healthy control groups are inconsistent. Blood MIF concentrations in control groups vary between 0.56 and 95.6 ng/ml, corresponding to a 170-fold difference. MIF concentrations in blood were analyzed by ELISA. Other than the influence of this approach due to method-based variations, the impact of preanalytical processing on MIF concentrations is unclear and has not been systematically studied yet. Before large randomized studies are performed to determine the impact of circulating MIF on prognosis and outcome and before MIF is characterized as a diagnostic marker, an accurate protocol for the determination of reproducible MIF levels needs to be validated. In this study, the measurement of MIF in the blood of healthy volunteers was investigated focusing on the potential influence of critical preanalytical factors such as anticoagulants, storage conditions, freeze/thaw stability, hemolysis, and dilution. We show how to avoid pitfalls in the measurement of MIF and that MIF concentrations are highly susceptible to preanalytical factors. MIF serum concentrations are higher than plasma concentrations and show broader ranges. MIF concentrations are higher in samples processed with latency than in those processed directly and strongly correlate with hemoglobin in plasma. Neither storage temperature nor storage length or dilution or repeated freezing and thawing influenced MIF concentrations in plasma. Preanalytical validation of MIF is essential. In summary, we suggest using plasma and not serum samples when determining circulating MIF and avoiding hemolysis by processing samples immediately after blood drawing.

Hetero-Oligomerization of Chemokine Receptors: Diversity and Relevance for Function

The G protein-coupled receptor (GPCR) family of membrane receptors encompasses over 1000 members, representing the largest known receptor family, with a variety of structurally different ligands. GPCRs are favorite targets for drug development in numerous diseases. Chemokine receptors are an important GPCR sub-class and are known to play a crucial role in the regulation of multiple physiological and various pathophysiological processes, including inflammation, atherosclerosis, cancer, and viral infections. Chemokine receptor activation is controlled by some 50 chemokine ligands which often act in a redundant and overlapping manner, enabling for a complex regulatory system together controlling and fine-tuning the specificity and spatio-temporal properties of the response. Recent findings have indicated that additionally the organization of chemokine receptors on the cell surface could be critical for driving their biological effects. In fact, chemokine receptors have increasingly been found to organize into homo- or hetero-oligomeric complexes, in part in a ligand-inducible manner, resulting in complex networks and crosstalk with other orthogonal signaling complexes. There has even been evidence for heterologous complex formation between chemokine receptors and non-chemokine receptor G protein-coupled receptors (GPCRs), and even non-GPCRs. However, the functional consequences of this kind of oligomerization have remained poorly understood, even for the chemokine receptor homo-oligomers. Yet, there is growing evidence that targeting homo- and/or hetero-oligomerization of chemokine receptors might be beneficial for the development of novel and specific therapeutics. In the present article, we highlight the multi-faceted complexity of chemokine receptor structures with a focus on their hetero-oligomerization properties.

The Role of Macrophage Migration Inhibitory Factor in Anesthetic-Induced Myocardial Preconditioning

Introduction Anesthetic-induced preconditioning (AIP) is known to elicit cardioprotective effects that are mediated at least in part by activation of the kinases AMPK and PKCε as well as by inhibition of JNK. Recent data demonstrated that the pleiotropic cytokine macrophage migration inhibitory factor (MIF) provides cardioprotection through activation and/or inhibition of kinases that are also known to mediate effects of AIP. Therefore, we hypothesized that MIF could play a key role in the AIP response. Methods Cardiomyocytes were isolated from rats and subjected to isoflurane preconditioning (4 h; 1.5 vol. %). Subsequently, MIF secretion and alterations in the activation levels of protective kinases were compared to a control group that was exposed to ambient air conditions. MIF secretion was quantified by ELISA and AIP-induced activation of protein kinases was assessed by Western blotting of cardiomyocyte lysates after isoflurane treatment. Results In cardiomyocytes, preconditioning with isoflurane resulted in a significantly elevated secretion of MIF that followed a biphasic behavior (30 min vs. baseline: p = 0.020; 24 h vs. baseline p = 0.000). Moreover, quantitative polymerase chain reaction demonstrated a significant increase in MIF mRNA expression 8 h after AIP. Of note, activation of AMPK and PKCε coincided with the observed peaks in MIF secretion and differed significantly from baseline. Conclusions These results suggest that the pleiotropic mediator MIF is involved in anesthetic-induced preconditioning of cardiomyocytes through stimulation of the protective kinases AMPK and PKCε.