Glycosidic intermediates identified in 1H MR spectra of intact tumour cells may contribute to the clarification of aspects of glycosylation pathways

The glycosylation process, through the addition of carbohydrates, is a major post‐translational modification of proteins and glycolipids. Proteins may be glycosylated in either the secretory pathway leading to N‐linked or O‐linked glycoproteins or as nucleocytoplasmic glycosylation that targets only single proteins involving a single β‐linked N‐acetylglucosamine. In both cases, the key precursors are the uridine diphospho‐N‐acetylhexosamines synthesised by the hexosamine biosynthetic pathway. Furthermore, uridine diphospho‐N‐acetylglucosamine participates in the biosynthesis of sialic acid. In this work, we propose MRS for the detection of uridine diphospho‐N‐acetylhexosamines visible in high‐resolution MR spectra of intact cells from different human tumours. Signals from the nucleotide and amino sugar moieties, including amide signals observed for the first time in whole cells, are assigned, also taking advantage of spectral changes that follow cell treatment with ammonium chloride. Finally, parallel changes in uridine diphospho‐N‐acetylhexosamines and glutamine pools, observed after pH changes induced by ammonium chloride in the different tumour cell lines, may provide more details on the glycosylation processes. Copyright © 2010 John Wiley & Sons, Ltd.     

Autophagy is involved in regulating the immune response of dendritic cells to influenza A (H1N1) pdm09 infection

Autophagy can mediate antiviral immunity. However, it remains unknown whether autophagy regulates the immune response of dendritic cells (DCs) to influenza A (H1N1) pdm09 infection. In this study, we found that infection with the H1N1 virus induced DC autophagy in an endocytosis‐dependent manner. Compared with autophagy‐deficient Beclin‐1^+/? mice, we found that bone‐marrow‐derived DCs from wild‐type mice (WT BMDCs) presented a more mature phenotype on H1N1 infection. Wild‐type BMDCs secreted higher levels of interleukin‐6 (IL‐6), tumour necrosis factor‐ α (TNF‐α), interferon‐β (IFN‐β), IL‐12p70 and IFN‐γ than did Beclin‐1^+/? BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs exhibited increased activation of extracellular signal‐regulated kinase, Jun N‐terminal kinase, p38, and nuclear factor‐κB as well as IFN regulatory factor 7 nuclear translocation. Blockade of autophagosomal and lysosomal fusion by bafilomycin A1 decreased the co‐localization of H1N1 viruses, autophagosomes and lysosomes as well as the secretion of IL‐6, TNF‐α and IFN‐β in H1N1‐infected BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs were more efficient in inducing allogeneic CD4⁺ T‐cell proliferation and driving T helper type 1, 2 and 17 cell differentiation while inhibiting CD4⁺ Foxp3⁺ regulatory T‐cell differentiation. Moreover, WT BMDCs were more efficient at cross‐presenting the ovalbumin antigen to CD8⁺ T cells. We consistently found that Beclin‐1^+/? BMDCs were inferior in their inhibition of H1N1 virus replication and their induction of H1N1‐specific CD4⁺ and CD8⁺ T‐cell responses, which produced lower levels of IL‐6, TNF‐α and IFN‐β in vivo. Our data indicate that autophagy is important in the regulation of the DC immune response to H1N1 infection, thereby extending our understanding of host immune responses to the virus.     

Indication for a role of regulatory T cells for the advent of influenza A (H1N1)-related pneumonia

Regulatory T cells (T_regs) have an anti‐inflammatory role. A former study in a limited number of patients found that absolute counts of T_regs increase when infection by the new influenza H1N1 virus is complicated with pneumonia. These results generate the question if H1N1‐related pneumonia is associated with a state of hypo‐inflammation. A total of 135 patients were enrolled with blood sampling within less than 24 h from diagnosis; 23 with flu‐like syndrome; 69 with uncomplicated H1N1‐infection; seven with bacterial pneumonia; and 36 with H1N1‐related pneumonia. T_regs and CD14/HLA‐DR co‐expression were estimated by flow cytometry; concentrations of tumour necrosis factor‐alpha (TNF‐α), of interleukin (IL)‐6 and of soluble triggering receptor expressed on myeloid cells‐1 (sTREM‐1) by an enzyme immunoassay; those of procalcitonin (PCT) by immuno‐time‐resolved amplified cryptate technology assay. Expression of human leucocyte antigen D‐related (HLA‐DR) on monocytes was similar between groups; absolute T_reg counts were greater among patients with H1N1‐related pneumonia than flu‐like syndrome or H1N1‐uncomplicated infection. Serum TNF‐α of patients with bacterial pneumonia was greater than those of other groups, but IL‐10 was similar between groups. Serum PCT was greater among patients with H1N1‐related pneumonia and sTREM‐1 among those with H1N1‐related pneumonia. Regression analysis revealed that the most important factors related with the advent of pneumonia were the existence of underlying illnesses (P = 0·006) and of T_regs equal to or above 16 mm³ (P = 0·013). It is concluded that the advent of H1N1‐related pneumonia is related to an early increase of the absolute T_reg counts. This increase is probably not part of a hypo‐inflammatory state of the host.     

Preclinical study of treatment response in HCT-116 cells and xenografts with (1) H-decoupled (31) P MRS

The topoisomerase I inhibitor, irinotecan, and its active metabolite SN‐38 have been shown to induce G₂/M cell cycle arrest without significant cell death in human colon carcinoma cells (HCT‐116). Subsequent treatment of these G₂/M‐arrested cells with the cyclin‐dependent kinase inhibitor, flavopiridol, induced these cells to undergo apoptosis. The goal of this study was to develop a noninvasive metabolic biomarker for early tumor response and target inhibition of irinotecan followed by flavopiridol treatment in a longitudinal study. A total of eleven mice bearing HCT‐116 xenografts were separated into two cohorts where one cohort was administered saline and the other treated with a sequential course of irinotecan followed by flavopiridol. Each mouse xenograft was longitudinally monitored with proton (¹H)‐decoupled phosphorus (³¹P) magnetic resonance spectroscopy (MRS) before and after treatment. A statistically significant decrease in phosphocholine (p = 0.0004) and inorganic phosphate (p = 0.0103) levels were observed in HCT‐116 xenografts following treatment, which were evidenced within twenty‐four hours of treatment completion. Also, a significant growth delay was found in treated xenografts. To discern the underlying mechanism for the treatment response of the xenografts, in vitro HCT‐116 cell cultures were investigated with enzymatic assays, cell cycle analysis, and apoptotic assays. Flavopiridol had a direct effect on choline kinase as measured by a 67% reduction in the phosphorylation of choline to phosphocholine. Cells treated with SN‐38 alone underwent 83 ± 5% G₂/M cell cycle arrest compared to untreated cells. In cells, flavopiridol alone induced 5 ± 1% apoptosis while the sequential treatment (SN‐38 then flavopiridol) resulted in 39 ± 10% apoptosis. In vivo ¹H‐decoupled ³¹P MRS indirectly measures choline kinase activity. The decrease in phosphocholine may be a potential indicator of early tumor response to the sequential treatment of irinotecan followed by flavopiridol in noninvasive and/or longitudinal studies. Copyright © 2011 John Wiley & Sons, Ltd.     

(1H) MRS studies of signals from mobile lipids and from lipid metabolites: comparison of the behavior in cultured tumor cells and in spheroids

(1)H magnetic resonance studies on MCF-7 and HeLa cells were undertaken to reveal differences in lipid and lipid metabolite signals during the growth in culture. High intensity mobile lipid (ML) signals were found during the first days in culture, while afterwards the same signals declined and started increasing again at confluence and at late confluence. At the same time, signals from the lipid metabolite phosphocholine decreased in intensity while signals from glycerophosphocholine in MCF-7 and from choline in HeLa increased as cells approached confluence. Spectral parameters from actively proliferating and non-proliferating cells were used to classify cells with respect to the proliferative conditions by means of a multivariate statistical analysis. Furthermore, it was shown that polyunsaturation of mobile lipid chains was lower in the confluent group with respect to the actively proliferating cells. The examination of spectra from suspensions of MCF-7 spheroids with diameter smaller than 500 microm suggests that cells in spheroids are in condition of lipid metabolism similar to that of confluent cultured cells.     

Non-invasive detection of hippocampal sclerosis: correlation between metabolite alterations detected by (1)H-MRS and neuropathology

We assessed (1)H-MRS as a screening tool for detection of hippocampal sclerosis in patients with temporal lobe epilepsy (TLE). (1)H-MRS was carried out in the hippocampus of 23 patients with unilateral TLE. Metabolite alterations detected by (1)H-MRS correlated with degree of segmental neuronal cell loss and amount of astrogliosis. Positive correlation was found between total N-Acetylaspartate (tNAA) reduction and neuronal density in hippocampal CA1 (P < 0.001), CA3 (P = 0.015), and CA4 subfields (P = 0.031) and the dentate gyrus (P = 0.006). Neuronal cell loss in CA1 turned out to be the most predictive and only significant variable for tNAA reduction (P = 0.027). The association between myo-inositol (m-Ins) and astroglial glial fibrillary acidic protein (GFAP) expression revealed significantly increased m-Ins concentrations associated with diffuse astrogliosis (m-Ins = 6.4 +/- 1.1 institutional units) compared with gliosis restricted to isolated sectors of the hippocampus (i.e. hilus) (m-Ins = 5.2 +/- 1.2 institutional units) (P = 0.039). A negative correlation was found between m-Ins and neuronal loss in the CA4 subfield of the hippocampus (P = 0.028). Our results support (1)H-MRS as a suitable non-invasive method for preoperative identification of hippocampal sclerosis in patients with TLE. The extent of tNAA reduction correlates with hippocampal neuronal cell density. Furthermore, m-Ins is associated with the extent of hippocampal astrogliosis.     

A defective proviral DNA with a 2.6-kb deletion of human immunodeficiency virus type 1 (HIV-1) in a persistently HIV-1 infected cell clone

A cell line (H2-5) producing defective doughnut-shaped particles of human immunodeficiency virus type 1 (HIV-1) was found to contain proviral DNA with a large deletion of 2558 bases, corresponding to the 3 half ofpol gene, the entirevif andvpr genes, and the 5 terminal of thetat gene.     

时空扫描方法在北京市甲型H1N1流感聚集性探测中的应用

目的 探讨北京市2009年甲型H1N1流感发病时空分布特征,为传染病预防控制提供理论依据.方法 利用SaTScan8.0软件进行时空扫描分析,通过ArcGIS8.3软件呈现甲型H1N1流感时空聚集区域.结果 按照7天、14天、30天,50％的时间周期进行单纯时间扫描分析,结果显示甲型H1N1流感发病在9月4日-12月1...     

Linear discriminant analysis of brain tumour (1)H MR spectra: a comparison of classification using whole spectra versus metabolite quantification

(1)H MRS is an attractive choice for non-invasively diagnosing brain tumours. Many studies have been performed to create an objective decision support system, but there is not yet a consensus as to the best techniques of MRS acquisition or data processing to be used for optimum classification. In this study, we investigate whether LCModel analysis of short-TE (30 ms), single-voxel tumour spectra provide a better input for classification than the use of the original spectra. A total of 145 histologically diagnosed brain tumour spectra were acquired [14 astrocytoma grade II (AS2), 15 astrocytoma grade III (AS3), 42 glioblastoma (GBM), 41 metastases (MET) and 33 meningioma (MNG)], and linear discriminant analyses (LDA) were performed on the LCModel analysis of the spectra and the original spectra. The results consistently suggest improvement in classification when the LCModel concentrations are used. LDA of AS2, MNG and high-grade tumours (HG, comprising GBM and MET) correctly classified 94% using the LCModel dataset compared with 93% using the spectral dataset. The inclusion of AS3 reduced the accuracy to 82% and 78% for LCModel analysis and the original spectra, respectively, and further separating HG into GBM and MET gave 70% compared with 60%. Generally MNG spectra have profiles that are visually distinct from those of the other tumour types, but the classification accuracy was typically about 80%, with MNG with substantial lipid/macromolecule signals being classified as HG. Omission of the lipid/macromolecule concentrations in the LCModel dataset provided an improvement in classification of MNG (91% compared with 76%). In conclusion, there appears to be an advantage to performing pattern recognition on the quantitative analysis of tumour spectra rather than using the whole spectra. However, the results suggest that a two-step LDA process may help in classifying the five tumour groups to provide optimum classification of MNG with high lipid/macromolecule contributions which maybe misclassified as HG.     

Association of prostate cancer with rapid N-acetyltransferase 1 (NAT1*10) in combination with slow N-acetyltransferase 2 acetylator genotypes in a pilot case-control study

N-acetyltransferase-1 (NAT1) and N-acetyltransferase-2 (NAT2) are important in the metabolism of aromatic and heterocyclic amine carcinogens that induce prostate tumors in the rat. We investigated the association of genetic polymorphisms in NAT1 and NAT2, alone and in combination, with human prostate cancer. Incident prostate cancer cases and controls in a hospital-based case-control study were frequency-matched for age, race, and referral pattern. The frequency of slow acetylator NAT1 genotypes (NAT1*14, *15, *17) was 5.8% in controls but absent in cases. In contrast, in comparison with all other NAT1 genotypes the putative rapid acetylator NAT1 genotype (NAT1*10) was significantly higher in prostate cancer cases than controls (OR, 2.17; 95% CI, 1.08-4.33; P = 0.03). Combinations of NAT1*10 with NAT2 slow acetylator genotypes (OR, 5.08; 95% CI, 1.56-16.5; P = 0.008) or with NAT2 very slow (homozygous NAT2*5) acetylator genotypes (OR, 7.50; 95% CI, 1.55-15.4; P = 0.016) further increased prostate cancer risk. The results of this small pilot study suggest increased susceptibility to prostate cancer for subjects with combinations of NAT1*10 and slow (particularly very slow) NAT2 acetylator genotypes. This finding should be investigated further in larger cohorts and in other ethnic populations.     

Longitudinal (1) H MRS assessment of the thalamus in a Coriaria lactone-induced rhesus monkey status epilepticus model

Neurophysiological, biochemical and anatomical evidence implicates the thalamus as playing a role in epileptic seizures. Until recently, however, longitudinal characterization of in vivo thalamus dynamics had not been reported. In this study, we investigated the metabolism in the thalamus to identify the changes that occur following Coriaria lactone (CL)‐induced status epilepticus (SE) and to observe whether the epileptiform discharges could present a difference between the left and right thalami. Five rhesus monkeys underwent whole‐brain MRI and single‐voxel MRS on a Siemens Trio Tim 3‐T MR scanner with a 12‐channel head coil. Spectra were processed using LCModel. Scans were performed in five animals before SE and at 1, 7, 21 and 42 days after the onset of SE. Statistical analysis of the data obtained demonstrated no significant difference in the bilateral thalamus of healthy macaques. Our MRS data showed symmetrical distributions of N‐acetylaspartate in the right and left thalami after SE (p = 0.003). In addition, this longitudinal study demonstrated elevated glutamate/glutamine (p < 0.05) and reduced myo‐inositol (p < 0.05) in the bilateral thalamus 1 day after SE, and all metabolites approached their baseline levels by the fifth scan. Our results demonstrate that metabolic changes occur in the thalamus during CL‐induced SE in rhesus monkeys. The various metabolic changes may indicate that the left thalamus is more vulnerable to epileptic strike. Copyright © 2012 John Wiley & Sons, Ltd.     

Delivery of antigen in allogeneic cells preferentially generates CD(4+) Th1 cells

We have examined the possibility of evoking antigen-specific T cell immune response by using allogeneic cells as a source of adjuvant and also as a vehicle to deliver antigen. The mice were immunized with different preparations of antigen-pulsed allogeneic and syngeneic splenocytes. It was observed during the study that the animals immunized with antigen-pulsed mitomycin C treated allogeneic cells elicited antigen specific CD(4+) Th1 cell response. Predominant release of IL-2, interferon (IFN)-gamma and IgG2a-isotype also occurred. In contrast, mice immunized with antigen-pulsed syngeneic cells chiefly enhanced the production of interleukin (IL)-4 and IgG1-isotype. Further, allogeneic macrophages induced better T cell response than B cells or splenocytes and prominently induced the expression of B7-1 and B7-2. Immunization with antigen-pulsed macrophages provided better recall responses compared to B cells. This was manifested by the high LFA-1alpha and low CD45RB expression on T cells. Because it is already known that mitomycin C-treated cells undergo apoptosis and dendritic cells engulf apoptotic cells, we therefore propose that generation of T cell response using antigen-pulsed allogeneic cells may be due to the engulfment of these cells by dendritic cells, which may then process and present antigen entrapped in allogeneic cells to activate naive CD(4+) T cells and differentiate them to Th1 cells. This study therefore provides a rational basis for manipulating antigen-specific responses by immunizing with antigen-pulsed allogeneic cells.     

Characteristics of hospitalized children with 2009 pandemic influenza A (H1N1): a multicenter study in Korea

The majority of Korean patients with pandemic influenza A (H1N1) during the 2009 epidemic were under 20 yr of age. The limited data on the clinical characteristics of these children led us to conduct a case note-based investigation of children admitted to 6 university hospitals with 2009 H1N1 influenza. A total of 804 children was enrolled. The median age was 5 yr; 63.8% were males; and 22.4% had at least one chronic underlying disease. Ninety-five of the patients (11.8%) were critically ill and they suffered more from shortness of breath, dyspnea and lymphopenia than the other patients. Among all the patients, 98.8% were treated with antivirals and 73% received treatment within 48 hr of illness onset. All the enrolled patients are alive and appear to have had good outcomes, probably due to the early intervention and antiviral treatment. This study deals with hospitalized children whose diagnoses of influenza A (H1N1) were confirmed, and therefore provides important new information about the clinical patterns of children with influenza A (H1N1) in Korea.     

IgG1 antimycobacterial antibodies can reverse the inhibitory effect of pentoxifylline on tumour necrosis factor alpha (TNF-alpha) secreted by mycobacterial antigen-stimulated adherent cells

Chronic inflammation associated with cachexia, weight loss, fever and arthralgia is the hallmark of advanced mycobacterial diseases. These symptoms are attributed to the chronic stimulation of tumour necrosis factor (TNF)-alpha. Mycobacterial components directly stimulate adherent cells to secrete TNF-alpha. We have shown recently that IgG1 antimycobacterial antibodies play a role in augmenting TNF-alpha in purified protein derivative (PPD)-stimulated adherent cells from non-BCG-vaccinated donors. We now show that IgG1 antibodies can also augment TNF-alpha expression in stimulated adherent cells obtained from BCG-vaccinated donors and this augmentation is not linked to interleukin (IL)-10 secretion. In addition IgG1 antimycobacterial antibodies can reverse the effect of TNF-alpha blockers such as pentoxifylline and thalidomide. These studies therefore have clinical implications for anti-inflammatory drug treatments which are used increasingly to alleviate symptoms associated with chronic inflammation.     

Islet-1 在乙酰化调控网络中特异性辅助C3H10T1/2细胞向心肌样细胞分化

 目的 筛选并分析转染Islet-1慢病毒载体的C3H10T1/2细胞转化为心肌样细胞过程中与Islet-1 相互作用的组蛋白乙酰化酶（HATs）和组蛋白去乙酰化酶（HDACs）,明确Islet-1在C3H10T1/2细胞分化为心肌样细胞乙酰化调控网络中的关键枢纽作用。方法 培养转染Islet-1慢病毒载体的C3H10T1/2细胞,观察细胞形态。免疫荧光和免疫印迹检测Islet-1的表达部位和最高表达时间点。免疫共沉淀沉淀与Islet-1结合的蛋白。免疫印迹验证Islet-1 相互作用的HATs和HDACs。结果 诱导组细胞形态出现心肌样细胞改变。各组Islet-1主要在胞浆表达。诱导组Islet-1表达量在诱导后3周最高（0.782±0.015）。诱导组Islet-1表达量显著高于空白对照组和C3H10组（分别为0.819±0.026,0.127±0.006和0.126±0.001）（P<0.05）,免疫共沉淀技术可行。与Islet-1相互作用的HATs和HDACs有GCN5、P300/CBP和HDAC4。结论 Islet-1 与GCN5、P300/CBP和HDAC4相互作用特异性辅助C3H10T1/2细胞向心肌样细胞分化。     

Mechanisms of hypotonic inhibition of the sodium, proton exchanger type 1 (NHE1) in a biliary epithelial cell line (Mz-Cha-1)

Aim: To elucidate the cellular events that results in inhibition of Na⁺, H⁺ exchanger type 1 (NHE1) by hypotonicity. Methods: Intracellular pH (pH_i) was measured in biliary epithelial cells, with the pH‐sensitive fluorochrome 2′,7′‐bis‐(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF) using a spectrophotometer. Regulatory volume decrease (RVD) was analysed from confocal images. Changes in NHE1 membrane content were visualized by confocal laser scanning microscopy after transfection of Mz‐Cha‐1 cells with a NHE1–cMyc fusion protein. Results: In Mz‐Cha‐1 cells hypotonicity (?80 mmol L^?1 NaCl) inhibited endogenous Na⁺, H⁺ exchange. Tyrosine and serine kinase inhibitors were incapable to prevent inhibition. As several signalling pathways influence Na⁺, H⁺ exchange, we tested the effect of the Ca⁺⁺, Calmodulin, protein kinase C or the cAMP, protein kinase A system on inhibition of Na⁺, H⁺ exchange by hypotonic challenge, but neither system was involved. In contrast, cytoskeleton did influence the effect of hypotonicity. Inhibition of microtubule polymerization by colchicine prevented inhibition of NHE1, and also restored Na⁺, H⁺ exchange kinetics. Specific inhibition of Src kinases with PP2, attenuated pH_i recovery rate from 1.93 ± 0.16 pH units min^?1 (normotonic environment) to 1.02 ± 0.50 pH units min^?1 (hypotonic environment). Membrane staining of NHE1–cMyc fusion protein was maintained after hypotonic exposure in colchicine pre‐treated cells as was RVD. Microfilament inhibition by cytochalasin preserved NHE1 activity. Inhibition of phosphatidylinositol‐3′‐kinase was unable to restore Na⁺, H⁺ exchange activity. Conclusion: We conclude that regulation of Na⁺, H⁺ exchange during RVD is mediated by cytoskeletal elements. This receptor independent pathway is regulated by Src.     

Phosphorylated 4E-binding protein 1 (p-4E-BP1): a novel prognostic marker in human astrocytomas

Korkolopoulou P, Levidou G, El‐Habr E A, Piperi C, Adamopoulos C, Samaras V, Boviatsis E, Thymara I, Trigka E‐A, Sakellariou S, Kavantzas N, Patsouris E & Saetta A A
(2012) Histopathology  61, 293–305 Phosphorylated 4E‐binding protein 1 (p‐4E‐BP1): a novel prognostic marker in human astrocytomas Aims: To investigate the significance of the mammalian target of rapamycin (mTOR) pathway in astrocytic tumours, published information in this context being limited, especially regarding phosphorylated 4E‐binding protein (p‐4E‐BP) 1. Methods and results: Paraffin‐embedded tissue from 111 patients with astroglial tumours (grades II–IV) was investigated for the association of phosphorylated mTOR (p‐mTOR) signalling components with phosphorylated extracellular signal‐related kinase 1/2 (p‐ERK1/2) and phosphorylated AKT (p‐AKT) expression, clinicopathological features, angiogenesis, isocitrate dehydrogenase 1 (IDH1)‐R132H, and survival. Expression was also quantified by western blot analysis in 12 cases and in three primary glioma cell cultures following rapamycin treatment. p‐mTOR expression correlated with p‐4E‐BP1 expression and marginally with p‐p70S6K expression. p‐4E‐BP1 expression increased with tumour grade. Rapamycin induced a decline in phosphorylation levels of all three proteins. Nuclear p‐AKT and cytoplasmic p‐ERK1/2 immunoexpression correlated with p‐4E‐BP1 expression, whereas cytoplasmic p‐AKT expression correlated with p‐p70S6K expression. All three proteins were associated with increased angiogenesis but not with IDH1‐R132H expression status. p‐mTOR adversely affected overall and disease‐free survival in univariate analysis. In multivariate survival analysis, the presence of p‐4E‐BP1 predicted shortened overall survival in the entire cohort and glioblastomas. Conclusions: mTOR signalling components are differentially involved in the acquisition of a more aggressive and angiogenic phenotype in astrocytic tumours. Moreover, p‐4E‐BP1 emerges as a novel prognostic marker, which might aid in the selection of patients who are more likely to benefit from therapy with mTOR inhibitors.     

Osteoprotegerin (OPG) acts as an endogenous decoy receptor in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of fibroblast-like synovial cells

We examined the role of osteoprotegerin (OPG) on tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in rheumatoid fibroblast-like synovial cells (FLS). OPG protein concentrations in synovial fluid from patients with rheumatoid arthritis (RA) correlated with those of interleukin (IL)-1beta or IL-6. A similar correlation was present between IL-1beta and IL-6 concentrations. Rheumatoid FLS in vitro expressed both death domain-containing receptors [death receptor 4 (DR4) and DR5] and decoy receptors [decoy receptor 1 (DcR1) and DcR2]. DR4 expression on FLS was weak compared with the expression of DR5, DcR1 and DcR2. Recombinant TRAIL (rTRAIL) rapidly induced apoptosis of FLS. DR5 as well as DR4 were functional with regard to TRAIL-mediated apoptosis induction in FLS; however, DR5 appeared be more efficient than DR4. In addition to soluble DR5 (sDR5) and sDR4, OPG administration significantly inhibited TRAIL-induced apoptogenic activity. OPG was identified in the culture supernatants of FLS, and its concentration increased significantly by the addition of IL-1beta in a time-dependent manner. Neither IL-6 nor tumour necrosis factor (TNF)-alpha increased the production of OPG from FLS. TRAIL-induced apoptogenic activity towards FLS was reduced when rTRAIL was added without exchanging the culture media, and this was particularly noticeable in the IL-1beta-stimulated FLS culture; however, the sensitivity of FLS to TRAIL-induced apoptosis itself was not changed by IL-1beta. Interestingly, neutralization of endogenous OPG by adding anti-OPG monoclonal antibody (MoAb) to FLS culture restored TRAIL-mediated apoptosis. Our data demonstrate that OPG is an endogenous decoy receptor for TRAIL-induced apoptosis of FLS. In addition, IL-1beta seems to promote the growth of rheumatoid synovial tissues through stimulation of OPG production, which interferes with TRAIL death signals in a competitive manner.