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.

Macrophage migration inhibitory factor promotes renal injury induced by ischemic reperfusion.

Macrophage migration inhibitory factor (MIF) is pleiotropic cytokine that has multiple effects in many inflammatory and immune diseases. This study reveals a potential role of MIF in acute kidney injury (AKI) in patients and in kidney ischemic reperfusion injury (IRI) mouse model in MIF wild-type (WT) and MIF knockout (KO) mice. Clinically, plasma and urinary MIF levels were largely elevated at the onset of AKI, declined to normal levels when AKI was resolved and correlated tightly with serum creatinine independent of disease causes. Experimentally, MIF levels in plasma and urine were rapidly elevated after IRI-AKI and associated with the elevation of serum creatinine and the severity of tubular necrosis, which were suppressed in MIF KO mice. It was possible that MIF may mediate AKI via CD74/TLR4-NF-κB signalling as mice lacking MIF were protected from AKI by largely suppressing CD74/TLR-4-NF-κB associated renal inflammation, including the expression of MCP-1, TNF-α, IL-1ß, IL-6, iNOS, CXCL15(IL-8 in human) and infiltration of macrophages, neutrophil, and T cells. In conclusion, our study suggests that MIF may be pathogenic in AKI and levels of plasma and urinary MIF may correlate with the progression and regression of AKI.

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.

Uropathogenic Escherichia coli virulence factor hemolysin A causes programmed cell necrosis by altering mitochondrial dynamics.

Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections. In this study, UPEC strains harboring hemolysin A (HlyA) did not induce programmed cell death pathways by the activation of caspases. Instead, the UPEC pore-forming toxin HlyA triggered an increase in mitochondrial Ca2+ levels and manipulated mitochondrial dynamics by causing fragmentation of the mitochondrial network. Alterations in mitochondrial dynamics resulted in severe impairment of mitochondrial functions by loss of membrane potential, increase in reactive oxygen species production, and ATP depletion. Moreover, HlyA caused disruption of plasma membrane integrity that was accompanied by extracellular release of the danger-associated molecules high-mobility group box 1 (HMGB1) and histone 3 (H3). Our results indicate that UPEC induced programmed cell necrosis by irreversibly impairing mitochondrial function. This finding suggests a strategy devised by UPEC at the onset of infection to escape early innate immune response and silently propagate inside host cells.-Lu, Y., Rafiq, A., Zhang, Z., Aslani, F., Fijak, M., Lei, T., Wang, M., Kumar, S., Klug, J., Bergmann, M., Chakraborty, T., Meinhardt, A., Bhushan, S. Uropathogenic Escherichia coli virulence factor hemolysin A causes programmed cell necrosis by altering mitochondrial dynamics.

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.

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+ regulatory T-cells.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are able to inhibit proliferation and cytokine production in effector T-cells and play a major role in immune responses and prevention of autoimmune disease. A master regulator of Treg cell development and function is the transcription factor Foxp3. Several cytokines, such as TGF-ß and IL-2, are known to regulate Foxp3 expression as well as methylation of the Foxp3 locus. We demonstrated previously that testosterone treatment induces a strong increase in the Treg cell population both in vivo and in vitro. Therefore we sought to investigate the direct effect of androgens on expression and regulation of Foxp3. We show a significant androgen-dependent increase of Foxp3 expression in human T-cells from women in the ovulatory phase of the menstrual cycle but not from men and identify a functional androgen response element within the Foxp3 locus. Binding of androgen receptor leads to changes in the acetylation status of histone H4, whereas methylation of defined CpG regions in the Foxp3 gene is unaffected. Our results provide novel evidence for a modulatory role of androgens in the differentiation of Treg cells.

Autoantibodies against protein disulfide isomerase ER-60 are a diagnostic marker for low-grade testicular inflammation.

STUDY QUESTION: Is there a non-invasive biomarker for the diagnosis of testicular inflammatory lesions? SUMMARY ANSWER: In sera from infertile azoospermic patients with histologically confirmed low-grade testicular inflammation, significantly elevated titers of autoantibodies against disulfide isomerase family A, member 3 (ER-60) were found. WHAT IS KNOWN ALREADY: Infection and inflammation of the genital tract are supposed to be responsible for up to 15% of cases among infertile males. However, specific seminal or serological markers are not available to assess subacute or chronic inflammatory conditions in the testis. STUDY DESIGN, SIZE, DURATION: This study consisted of the identification of autoantibodies for testicular antigens in sera of patients with low-grade testicular inflammation, validation of candidates, development of an ELISA for the most promising target antigen and measurement of autoantibodies titers in healthy normozoospermic men (n = 20); male blood donors (n = 14); men with impaired semen quality without (n = 14) or with (n = 26) symptoms of genital tract infection/inflammation; azoospermic men with histologically confirmed testicular inflammatory lesions (n = 16); men after pharmacotherapy of genital tract infection/inflammation (n = 15) and men with acute epididymo-orchitis (n = 30). PARTICIPANTS/MATERIALS, SETTING, METHODS: Proteins in lysates of normal testicular tissue were separated by high-resolution 2D gel electrophoresis and probed with sera of 13 patients with histologically confirmed chronic testicular inflammation. There were 14 proteins that immunoreacted with a majority of these sera and could be identified by mass spectrometry. Of these 14 proteins, disulfide isomerase family A, member 3 (ER-60), transferrin and chaperonin containing TCP1 complex, subunit 5 (epsilon) (CCT5) were considered as specific. Since ER-60 reacted with 92% of patient sera, an ER-60-autoantibody ELISA was developed. MAIN RESULTS AND THE ROLE OF CHANCE: The newly established ELISA detected significantly elevated titers of autoantibodies against ER-60 in the sera from infertile men with histologically confirmed chronic testicular inflammation (median 8.6; P < 0.01) compared with the control groups. Moreover, elevated levels of anti-ER-60 titers were detected in patients suffering from acute epididymo-orchitis (median 3.3; P < 0.05) as compared with healthy normozoospermic men (median 2.13; P < 0.001), male blood donors with unknown fertility status (median 2.72; P < 0.01), patients with impaired semen quality but no infection/inflammation (median 2.59; P < 0.001) and patients with symptoms of genital tract infections and/or inflammation (median 2.18; P < 0.001). Significantly lower levels of anti-ER-60 antibodies were measured in sera from patients after application of anti-inflammatory pharmacotherapy (median 1.9; P < 0.01) compared with those with histologically confirmed chronic testicular inflammation. The cut-off value of the assay was set to 6.6 U/ml based on a calculated sensitivity of 100% and a specificity of 81.2%. LIMITATIONS, REASONS FOR CAUTION: The results obtained in this study showed statistically significant elevated titers of ER-60 antibodies in sera from patients with histologically confirmed testicular inflammatory lesions and from a few patients with acute epididymo-orchitis. However, the number of serum samples tested was limited. Severe testicular damage seen in azoospermic patients could represent a bias towards ER-60 reactivity, while the assay does not allow for different etiologies of the lesions to be distinguished. Due to ethical reasons, the prevalence of testicular inflammatory lesions among controls and non-azoospermic men cannot be studied at the histological level. WIDER IMPLICATIONS OF THE FINDINGS: Measurement of ER-60 autoantibody titers in serum could be a novel non-invasive marker for the diagnosis of asymptomatic testicular inflammation causing male fertility disturbances. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a grant of the Deutsche Forschungsgemeinschaft (ME 1323/4-4) and the Translational Science Fund (Wirtschafts-und Strukturbank Hessen-WI Bank). M.F., A.P., W.W., H.-C.S. and A.M. are supported by the LOEWE focus group 'MIBIE' (Male infertility during infection and inflammation). The ER-60 ELISA is protected by a patent to the Justus-Liebig-University of Giessen with A.M. and M.F. as inventors (patent no. DE 10 2008 053 503). T.Z. as employee of the DRG Company was responsible for the ELISA development.

Expression pattern of estrogen receptors α and ß and G-protein-coupled estrogen receptor 1 in the human testis.

Estrogen signaling is considered to play an important role in spermatogenesis, spermiogenesis and male fertility. Estrogens can act via the two nuclear estrogen receptors ESR1 (ERα) and ESR2 (ERß) or via the intracellular G-protein-coupled estrogen receptor 1 (GPER, formerly GPR30). Several reports on the localization and expression of all three receptors in the human testis have been published but are controversial particularly in case of ERα. Contrary to previous studies, we decided therefore to evaluate expression of all three receptors in the testis by a number of different methods and in comparison with MCF-7 cells. Using qPCR, we could show that mRNA expression of ERα is considerably lower and expression of ERß and GPER much higher in the testis than in MCF-7 cells. RT-PCR after laser-assisted microdissection of tubular and interstitial compartments from normal and Sertoli cell only syndrome testes plus in situ hybridization and immunohistochemical analyses of the same samples demonstrated that there is very low expression of ERα in germ cells and in single interstitial cells, very high expression of ERß in germ cells and Sertoli cells and high expression of GPER in interstitial cells and less in Sertoli cells.

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.

Uropathogenic E. coli induce different immune response in testicular and peritoneal macrophages: implications for testicular immune privilege.

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1ß, IL-6 downregulated) and TM (IL-1ß, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells.

Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory T cell expansion.

Despite the immune-privileged status of the male genital tract, infection and inflammation of the male genital tract are important etiological factors in male infertility. A common observation in clinical and experimental orchitis as well as in systemic infection and inflammation are decreased levels of testosterone. Emerging data point to an immunosuppressive role of testosterone. In our study, we substituted testosterone levels in experimental autoimmune orchitis (EAO) in rat by s.c. testosterone implants. EAO development was reduced to 17% when animals were treated with low-dose testosterone implants (3 cm long, EAO+T3) and to 33% when rats were supplied with high-dose testosterone implants (24 cm, EAO+T24) compared with 80% of animals developing disease in the EAO control group. In the testis, testosterone replacement in EAO animals prevented the accumulation of macrophages and significantly reduced the number of CD4(+) T cells with a strong concomitant increase in the number of regulatory T cells (CD4(+)CD25(+)Foxp3(+)) compared with EAO control. In vitro testosterone treatment of naive T cells led to an expansion of the regulatory T cell subset with suppressive activity and ameliorated MCP-1-stimulated chemotaxis of T lymphocytes in a Transwell assay. Moreover, expression of proinflammatory mediators such as MCP-1, TNF-α, and IL-6 in the testis and secretion of Th1 cytokines such as IFN-γ and IL-2 by mononuclear cells isolated from testicular draining lymph nodes were decreased in the EAO+T3 and EAO+T24 groups. Thus, our study shows an immunomodulatory and protective effect of testosterone substitution in the pathogenesis of EAO and suggests androgens as a new factor in the differentiation of regulatory T cells.

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.

Heterogeneous nuclear ribonucleoprotein h1, a novel nuclear autoantigen.

BACKGROUND: Serum samples from patients with autoimmune connective tissue diseases that show a finely speckled antinuclear antibody (ANA) on indirect immune-fluorescence often have antibodies against unknown nuclear target antigens. To search for such autoantigens we applied a proteomic approach using sera from patients with a high ANA titer (>or=640) and finely speckled fluorescence but in whom no antibodies to extractable nuclear antigens (ENA) could be identified. METHODS: Using an immunoproteomics approach we identified heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) as a novel nuclear target of autoantibody response. RESULTS: Recombinant rat hnRNP H1 reacted in Western blot analyses with 48% of 93 sera from patients with primary Sjögren syndrome and with 5.2% of 153 sera from patients with other connective tissue diseases (diseased controls). For comparison, the diagnostic sensitivity and specificity of anti-Sjögren syndrome A (SSA) antibodies for primary Sjögren syndrome in the same patient cohort were 88.2% and 76.3%, respectively. Interestingly, 5 of 11 primary Sjögren syndrome patients with no anti-SSA or anti-SSB antibodies had anti-hnRNP H1 antibodies. Anti-hnRNP H1 antibodies were preabsorbed by hnRNP H1, as demonstrated by indirect immunofluorescence. In an evaluation of the presence of anti-hnRNP H1 antibodies in 188 consecutive samples submitted to the clinical laboratory with positive ANA (titer >or=160), anti-hnRNP H1 antibodies were found in 3 of 7 (2 primary and 5 secondary) Sjögren syndrome patients and in 8.3% of the diseased controls. CONCLUSIONS: HnRNP H1 is a newly discovered autoantigen that could become an additional diagnostic marker.

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.

Uropathogenic Escherichia coli block MyD88-dependent and activate MyD88-independent signaling pathways in rat testicular cells.

Uropathogenic Escherichia coli (UPEC) is the most common etiological cause of urogenital tract infections and represents a considerable cause of immunological male infertility. We examined TLR 1-11 expression profiles in testicular cells and the functional response to infection with UPEC. All testicular cell types expressed mRNAs for at least two TLRs and, in particular, synthesis of TLR4 was induced in testicular macrophages (TM), Sertoli cells (SC), peritubular cells (PTC), and peritoneal macrophages (PM) after UPEC exposure. Even though MyD88-dependent pathways were activated as exemplified by phosphorylation of mitogen-activated protein kinases in TM, SC, PTC, and PM and by the degradation of IkappaBalpha and the nuclear translocation of NF-kappaB in PTC and PM, treatment with UPEC did not result in secretion of the proinflammatory cytokines IL-1alpha, IL-6, and TNF-alpha in any of the investigated cells. Moreover, stimulated production of these cytokines by nonpathogenic commensal E. coli or LPS in PM was completely abolished after coincubation with UPEC. Instead, in SC, PTC, TM, and PM, UPEC exposure resulted in activation of MyD88-independent signaling as documented by nuclear transfer of IFN-related factor-3 and elevated expression of type I IFNs alpha and beta, IFN-gamma-inducible protein 10, MCP-1, and RANTES. We conclude that in this in vitro model UPEC can actively suppress MyD88-dependent signaling at different levels to prevent proinflammatory cytokine secretion by testicular cells. Thus, testicular innate immune defense is shifted to an antiviral-like MyD88-independent response.

Macrophage migration inhibitory factor does not modulate co-activation of androgen receptor by Jab1/CSN5.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory immune modulator that plays an important role in the regulation of innate and adaptive immune responses. MIF signaling involves CD74/CD44 membrane receptor complexes, the chemokine receptors CXCR2 and 4 as well as uptake by non-receptor mediated endocytosis. Endocytosed or endogenous MIF interacts with Jun activation domain-binding protein 1 (Jab1), originally described as transcriptional co-activator for the transcription factor AP-1, that is also known as subunit 5 of the COP9 signalosome (CSN5). Since Jab1/CSN5 also functions as a co-activator for a number of steroid hormone receptors (SHRs), it had been speculated that MIF could modulate Jab1/CSN5-SHR interactions. Here we show (i) that fluorescently labeled MIF is internalized by NIH 3T3 cells within minutes, (ii) compromises the induction of phospho-c-Jun levels by TNFalpha and PMA and, hence, is biologically active, but (iii) is not able to interfere with co-activation by Jab1/CSN5 of the androgen receptor.

Immunohistochemical analysis of secretoglobin SCGB 2A1 expression in human ocular glands and tissues.

Human secretoglobin (SCGB) 2A1 (or lipophilin C, lacryglobin, mammaglobin B) is a small protein of unknown function that forms heterodimers with secretoglobin 1D1 (lipophilin A) in tears. SCGB 2A1 is homologous to mammaglobin (mammaglobin A) and the C3 component of prostatein, the major secretory protein of the rat ventral prostate. Androgen-dependent expression of SCGB 2A1 has been observed in the prostate. Besides identification of SCGB 2A1 in the tear proteome only its mRNA had been detected in the lacrimal gland. Here, we report expression of SCGB 2A1 in all ocular glands and in the keratinized stratified squamous epithelium of the eyelid as well as in the stratified epithelium of the conjunctiva and in the orbicularis oculi muscle. Almost all of these tissues are also known to express the androgen receptor. Therefore, we conclude that presence of the androgen signalling machinery could be the main general determinant of SCGB 2A1 expression. Implications of the presence in tear fluid of an androgen-regulated secretoglobin, which most likely binds hydrophobic ligands, for tear film lipid layer formation and function is discussed.