Extracellular MIF, but not its homologue D-DT, promotes fibroblast motility independently of its receptor complex CD74/CD44.

Macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (D-DT) are widely expressed pro-inflammatory cytokines with chemokine-like functions that coordinate a wide spectrum of biological activities, such as migration. Here, we biotin-tagged intracellular MIF/D-DT in vivo to identify important cytosolic interactors and found a plethora of actin cytoskeleton-associated proteins. Although the receptor complex between CD74 and CD44 (CD74/CD44) is essential for signalling transduction in fibroblasts via extracellular MIF/D-DT, our interactome data suggested direct effects. We, thus, investigated whether MIF/D-DT can modulate cell migration independently of CD74/CD44. To distinguish between receptor- and non-receptor-mediated motility, we used fibroblasts that are either deficient or that express CD74/CD44 proteins, and treated them with recombinant MIF/D-DT. Interestingly, only MIF could stimulate chemokinesis in the presence or absence of CD74/CD44. The pro-migratory effects of MIF depended on lipid raft/caveolae-mediated but not clathrin-mediated endocytosis, on its tautomerase activity and, probably, on its thiol protein oxidoreductase activity. As MIF treatment restrained actin polymerisation in vitro, our findings establish a new intracellular role for MIF/D-DT in driving cell motility through modulation of the actin cytoskeleton.

Blocking Macrophage Migration Inhibitory Factor Protects Against Cisplatin-Induced Acute Kidney Injury in Mice.

Macrophage migration inhibitory factor (MIF) is elevated in patients with acute kidney injury (AKI) and is suggested as a potential predictor for renal replacement therapy in AKI. In this study, we found that MIF also plays a pathogenic role and is a therapeutic target for AKI. In a cisplatin-induced AKI mouse model, elevated plasma MIF correlated with increased serum creatinine and the severity of renal inflammation and tubular necrosis, whereas deletion of MIF protected the kidney from cisplatin-induced AKI by largely improving renal functional and histological injury, and suppressing renal inflammation including upregulation of cytokines such as interleukin (IL)-1ß, tumor necrosis factor-alpha (TNF-α), IL-6, inducible nitric oxide synthase (iNOS), MCP-1, IL-8, and infiltration of macrophages, neutrophils, and T cells. We next developed a novel therapeutic strategy for AKI by blocking the endogenous MIF with an MIF inhibitor, ribosomal protein S19 (RPS19). Similar to the MIF-knockout mice, treatment with RPS19, but not the mutant RPS19, suppressed cisplatin-induced AKI. Mechanistically, we found that both genetic knockout and pharmacological inhibition of MIF protected against AKI by inactivating the CD74-nuclear factor κB (NF-κB) signaling. In conclusion, MIF is pathogenic in cisplatin-induced AKI. Targeting MIF with an MIF inhibitor RPS19 could be a promising therapeutic potential for AKI.

Galectin-1 enhances TNFα-induced inflammatory responses in Sertoli cells through activation of MAPK signalling.

Galectin-1 (Gal-1) is a pleiotropic lectin involved in the modulation of immune responses. Using a model of rat experimental autoimmune orchitis (EAO), we investigated the role of Gal-1 in testicular inflammation. EAO is characterized by leukocytic infiltrates in the interstitium, damage of spermatogenesis and production of inflammatory mediators like TNFα and MCP1 causing infertility. In normal rat testis Gal-1 was mainly expressed in Sertoli cells and germ cells. In the inflamed testis, Gal-1 expression was significantly downregulated most likely due to germ cell loss. Analyses of lectin binding and expression of glucosaminyl- and sialyltransferases indicated that the glycan composition on the cell surface of Sertoli and peritubular cells becomes less favourable for Gal-1 binding under inflammatory conditions. In primary Sertoli cells Gal-1 expression was found to be upregulated after TNFα challenge. Pretreatment with Gal-1 synergistically and specifically enhanced TNFα-induced expression of MCP1, IL-1α, IL-6 and TNFα in Sertoli cells. Combined stimulation of Sertoli cells with Gal-1 and TNFα enhanced the phosphorylation of MAP kinases as compared to TNFα or Gal-1 alone. Taken together, our data show that Gal-1 modulates inflammatory responses in Sertoli cells by enhancing the pro-inflammatory activity of TNFα via stimulation of MAPK signalling.

Resistance to apoptosis and autophagy leads to enhanced survival in Sertoli cells.

STUDY QUESTION: What is the underlying mechanism of Sertoli cell (SC) resistance to cell death? SUMMARY ANSWER: High expression of prosurvival B-cell lymphoma-2 (BCL2) proteins and inhibition of apoptosis and autophagy prolongs SC survival upon exposure to stress stimuli. WHAT IS KNOWN ALREADY: In human and in experimental models of orchitis, tolerogenic SC survive stress conditions, while germ cells undergo massive apoptosis. In general, non-dividing highly differentiated cells tend to resist stress conditions for a longer time by favoring activation of prosurvival mechanisms and inhibition of cell death pathways. STUDY DESIGN, SIZE, DURATION: In this cross sectional study, conditions stimulating apoptosis and autophagy were used to induce cell death in primary rat SC. Primary rat peritubular cells (PTC) and immortalized rat 93RS2 SC were used as controls. Each cell isolation was counted as one experiment (n = 1), and each experiment was repeated three to six times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsy samples from infertile or subfertile patients and testis samples from rats with experimental autoimmune orchitis were used for immunohistological analysis. Primary SC were isolated from 19-day-old male Wistar rats. To maintain cell purity, cells were cultured in serum-free medium for apoptosis experiments and in medium supplemented with 1% serum for autophagy analyses. To induce apoptosis, cells were stimulated with staurosporine, borrelidin, cisplatin and etoposide for 4 or 24 h. Caspase three activation was examined by immunoblotting and enzymatic activity assay. Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester followed by flow cytometric analysis. Cytochrome c release was monitored by immunofluorescence. Cell viability was determined using the methylthiazole tetrazolium assay. To monitor autophagy flux, cells were deprived of nutrients using Hank's balanced salt solution for 1, 2 and 3 h. Formation of autophagosomes was analyzed by using immunoblotting, immunofluorescence labeling and ultrastructural analyses. Relative mRNA levels of genes involved in the regulation of apoptosis and autophagy were evaluated. Extracellular high mobility group box protein one was measured as a marker of necrosis using ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: SC survive the inflammatory conditions in vivo in human testis and in experimental autoimmune orchitis. Treatment with apoptosis inducing chemotherapeutics did not cause caspase three activation in isolated rat SC. Moreover, mitochondrial membrane potential and mitochondrial localization of cytochrome c were not changed by treatment with staurosporine, suggesting a premitochondrial blockade of apoptosis in SC. Expression levels of prosurvival BCL2 family members were significantly higher in SC compared to PTC at both mRNA and protein levels. Furthermore, after nutrient starvation, autophagy signaling was initiated in SC as observed by decreased levels of phosphorylated UNC- 51-like kinase -1 (ULK1). However, levels of light chain 3 II (LC3 II) and sequestosome1 (SQSTM1) remained unchanged, indicating blockade of the autophagy flux. Lysosomal activity was intact in SC as shown by accumulation of LC3 II following administration of lysosomal protease inhibitors, indicating that inhibition of autophagy flux occurs at a preceding stage. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we have used primary SC from prepubertal rats. Caution should be taken when translating our results to adult animals, where crosstalk with other testicular cells and hormonal factors may also play a role in regulating survival of SC. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that inhibition of autophagy and apoptosis following exposure to extrinsic stress stimuli promotes SC survival, and is a possible mechanism to explain the robustness of SC in response to stress. Cell death resistance in SC is crucial for the recovery of spermatogenesis after chemotherapy treatment in cancer patients. Additionally, understanding the molecular mechanisms of SC survival unravels valuable target proteins, such as BCL2, that may be manipulated therapeutically to control cell viability depending on the context of the disease. STUDY FUNDING AND COMPETING INTEREST(S): This study was funded by the Deutsche Forschungsgemeinschaft (DFG) Grant BH93/1-1, and by the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) funded by the DFG and Monash University. The support of the Medical Faculty of Justus-Liebig University of Giessen is gratefully acknowledged. The authors declare no conflict of interest.

Differential activation of inflammatory pathways in testicular macrophages provides a rationale for their subdued inflammatory capacity.

Spermatogenic cells express cell-specific molecules with the potential to be seen as "foreign" by the immune system. Owing to the time difference between their appearance in puberty and the editing of the lymphocyte repertoire around birth, local adaptations of the immune system coined immune privilege are required to confer protection from autoattack. Testicular macrophages (TM) play an important role in maintaining testicular immune privilege and display reduced proinflammatory capacity compared with other macrophages. However, the molecular mechanism underlying this macrophage phenotype remained elusive. We demonstrate that TM have a lower constitutive expression of TLR pathway-specific genes compared with peritoneal macrophages. Moreover, in TM stimulated with LPS, the NF-κB signaling pathway is blocked due to lack of IκBα ubiquitination and, hence, degradation. Instead, challenge of TM with LPS or polyinosinic-polycytidylic acid induces MAPK, AP-1, and CREB signaling pathways, which leads to production of proinflammatory cytokines such as TNF-α, although at much lower levels than in peritoneal macrophages. Pretreatment of TM with inhibitors for MAPKs p38 and ERK1/2 suppresses activation of AP-1 and CREB signaling pathways and attenuates LPS-induced TNF-α and IL-10 secretion. High levels of IL-10 production and activation of STAT3 by LPS stimulation in TM indicate a regulatory macrophage phenotype. Our results suggest that TM maintain testicular immune privilege by inhibiting NF-κB signaling through impairment of IκBα ubiquitination and a general reduction of TLR cascade gene expression. However, TM do maintain some capacity for innate immune responses through AP-1 and CREB signaling pathways.

Ribosomal protein S19 is a novel therapeutic agent in inflammatory kidney disease.

RPS19 (ribosomal protein S19), a component of the 40S small ribosomal subunit, has recently been identified to bind the pro-inflammatory cytokine macrophage MIF (migration inhibitory factor). In vitro experiments identify RPS19 as the first endogenous MIF inhibitor by blocking the binding of MIF to its receptor CD74 and MIF functions on monocyte adherence to endothelial cells. In the present study, we sought to establish whether recombinant RPS19 can exert anti-inflammatory effects in a mouse model of anti-GBM (glomerular basement membrane) GN (glomerulonephritis) in which MIF is known to play an important role. Accelerated anti-GBM GN was induced in C57BL/6J mice by immunization with sheep IgG followed 5 days later by administration of sheep anti-mouse GBM serum. Groups of eight mice were treated once daily by intraperitoneal injection with 6 mg of RPS19/kg of body weight or an irrelevant control protein (human secretoglobin 2A1), or received no treatment, from day 0 until being killed on day 10. Mice that received control or no treatment developed severe crescentic anti-GBM disease on day 10 with increased serum creatinine, declined creatinine clearance and increased proteinuria. These changes were associated with up-regulation of MIF and its receptor CD74 activation of ERK (extracellular-signal-regulated kinase) and NF-κB (nuclear factor κB) signalling, prominent macrophage and T-cell infiltration, as well as up-regulation of Th1 [T-bet and IFNγ (interferon γ)] and Th17 [STAT3 (signal transducer and activator of transcription 3) and IL (interleukin)-17A] as well as IL-1ß and TNFα (tumour necrosis factor α). In contrast, RPS19 treatment largely prevented the development of glomerular crescents and glomerular necrosis, and prevented renal dysfunction and proteinuria (all P<0.001). Of note, RPS19 blocked up-regulation of MIF and CD74 and inactivated ERK and NF-κB signalling, thereby inhibiting macrophage and T-cell infiltration, Th1 and Th17 responses and up-regulation of pro-inflammatory cytokines (all P<0.01). These results demonstrate that RPS19 is a potent anti-inflammatory agent, which appears to work primarily by inhibiting MIF signalling.

COP9 signalosome interacts ATP-dependently with p97/valosin-containing protein (VCP) and controls the ubiquitination status of proteins bound to p97/VCP.

Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.

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 (K(D) = 1.3 x 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.

Regulatory cytokine expression and interstitial fluid formation in the normal and inflamed rat testis are under leydig cell control.

Leydig cells have been implicated in several inflammation-related responses of the testis. Specifically, these cells produce the proinflammatory cytokines interleukin-1 (IL-1) and IL-6, stimulate macrophage recruitment, and promote interstitial fluid formation. In addition, the immunoregulatory cytokines macrophage migration inhibitory factor (MIF), transforming growth factor-beta1 (TGFbeta1), and interferon-gamma (IFNgamma) are constitutively expressed by testicular cells, including the Leydig cells. In the present study, the contribution of the Leydig cell to testicular inflammatory responses was examined in adult male rats treated with the Leydig cell-specific toxin, ethane dimethane sulfonate (EDS). Intratesticular testosterone levels were modulated by subcutaneous testosterone implants. After 10 days, animals received an injection of lipopolysaccharide (LPS) to induce an inflammatory response, or saline alone, and were killed 3 hours later. Both depletion of Leydig cells by EDS and LPS treatment caused a decrease in collected testicular interstitial fluid to about 35% of control levels, but the effects were not additive. Maintenance of intratesticular testosterone reversed the interstitial fluid decline following EDS treatment and partially prevented the LPS-induced effect. MIF, TGFbeta1, and IFNgamma were expressed in both the normal and inflamed testis at similar levels. In contrast, EDS treatment caused a significant decline in expression of all 3 cytokines, which was prevented by the testosterone implants. These data indicate that 1) expression of TGFbeta1, MIF, and IFNgamma in the testis is not dependent on the presence of intact Leydig cells but is under direct testosterone control and 2) the decline in testicular interstitial fluid during inflammation involves the Leydig cells, acting via both androgens and nonandrogenic secretions. These data provide further support for a significant role for the Leydig cell in modulating the testicular response to inflammation.