M. Jakstadt

Osteoarthritis synovial fluid activates pro-inflammatory cytokines in primary human chondrocytes.

PurposeTwo of the most common joint diseases are rheumatoid arthritis (RA) and osteoarthritis (OA). Cartilage degradation and erosions are important pathogenetic mechanisms in both joint diseases and have presently gained increasing interest. The aim of the present study was to investigate the effects of the synovial fluid environment of OA patients in comparison with synovial fluids of RA patients on human chondrocytes in vitro.MethodsPrimary human chondrocytes were incubated in synovial fluids gained from patients with OA or RA. The detection of vital cell numbers was determined by histology and by using the Casy Cell Counter System. Cytokine and chemokine secretion was determined by a multiplex suspension array.ResultsMicroscopic analysis showed altered cell morphology and cell shrinkage following incubation with synovial fluid of RA patients. Detection of vital cells showed a highly significant decrease of vital chondrocyte when treated with RA synovial fluids in comparison with OA synovial fluids. An active secretion of cytokines such as vascular endothelial growth factor (VEGF) of chondrocytes treated with OA synovial fluids was observed.ConclusionsSignificantly increased levels of various cytokines in synovial fluids of RA, and surprisingly of OA, patients were shown. Activation of pro-inflammatory cytokines of human chondrocytes by synovial fluids of OA patient supports a pro-inflammatory process in the pathogenesis of OA.

A Pronounced Inflammatory Activity Characterizes the Early Fracture Healing Phase in Immunologically Restricted Patients

Immunologically restricted patients such as those with autoimmune diseases or malignancies often suffer from delayed or insufficient fracture healing. In human fracture hematomas and the surrounding bone marrow obtained from immunologically restricted patients, we analyzed the initial inflammatory phase on cellular and humoral level via flow cytometry and multiplex suspension array. Compared with controls, we demonstrated higher numbers of immune cells like monocytes/macrophages, natural killer T (NKT) cells, and activated T helper cells within the fracture hematomas and/or the surrounding bone marrow. Also, several pro-inflammatory cytokines such as Interleukin (IL)-6 and Tumor necrosis factor α (TNFα), chemokines (e.g., Eotaxin and RANTES), pro-angiogenic factors (e.g., IL-8 and Macrophage migration inhibitory factor: MIF), and regulatory cytokines (e.g., IL-10) were found at higher levels within the fracture hematomas and/or the surrounding bone marrow of immunologically restricted patients when compared to controls. We conclude here that the inflammatory activity on cellular and humoral levels at fracture sites of immunologically restricted patients considerably exceeds that of control patients. The initial inflammatory phase profoundly differs between these patient groups and is probably one of the reasons for prolonged or insufficient fracture healing often occurring within immunologically restricted patients.

High-sensitivity immunofluorescence staining: a comparison of the liposome procedure and the FASER technique on mGR detection.

Flow cytometry has become a widely-used and powerful tool for the characterization of cells according to their expression of specific proteins. However, sensitivity of this method is still limited since conventionally labeled antibodies can be conjugated with at maximum 1–10 dye molecules. This fact resulted in the need to develop new techniques in order to identify molecules which are expressed in very low but functionally relevant amounts. In the past, we have successfully used a liposome-based high-sensitivity immunofluorescence technique to measure the expression of low abundant membrane bound glucocorticoid receptors (mGR) on different cell types. The use of this technique allows the detection of as few as 50–100 antigen molecules per cell which is due to a 100-fold to 1000-fold increase in fluorescence signal intensity compared with conventional methods. The higher sensitivity is achieved since thousands of dye molecules can be enclosed in liposomes. Another modern high-sensitivity immunofluorescence staining method is the purchasable Fluorescence Amplification by Sequential Employment of Reagents (FASER) procedure. Here, we aimed at comparing sensitivity and specificity of these two techniques for the detection of the mGR. Our data demonstrate the FASER technique to be more sensitive and also more specific for the detection of mGR as compared to the liposome technique. However, both methods have advantages and disadvantages which are discussed in detail.

OP0086 Alterations of immune cellular circadian rhythms in rheumatoid arthritis

Background The circadian variation of clinical symptoms and the underlying variation of cytokine and hormone levels in rheumatoid arthritis (RA) are well described and have already led to the successful application of chronotherapy with prednisone (Buttgereit et al., Lancet, 2008). Much less is known about the circadian rhythms of different immune cell populations in RA. Objectives In this pilot study we investigated molecular, cellular and humoral circadian parameters in postmenopausal female RA patients in comparison to healthy control subjects. Methods Blood samples from postmenopausal female patients with active RA (DAS 28 ≥4.2) (n=5) and postmenopausal female healthy controls (n=5) were collected every 2 hours for 24 hours and analysed by flow cytometry and multiplex suspension array of 28 cytokines. Clock gene expression of isolated CD14+ monocytes was analysed by quantitative RT-PCR. Endogenous circadian rhythm dynamics of macrophages were determined by means of a Bmal1-promotor driven luciferase reporter construct. COSINOR analysis was used for statistical analysis of the groups. Results Expression of the clock gene RevErbα in CD14+ monocytes showed a significant circadian expression pattern in both RA patients and healthy controls subjects, whereas the clock genes Per2 and Per3 were not expressed in a circadian manner in RA patients but in healthy controls only. The amplitude of the endogenous circadian rhythm of macrophages tended to be lower in RA patients than in healthy controls, whereas period length was not altered. In flow cytometric analysis of surface marker expression of blood cells we found a significant circadian rhythm in RA patients and healthy subjects for the frequency of CD3-CD56+ natural killer (NK) cells, Interleukin-8 Receptor (IL-8R) expressing CD4+ T helper and CD8+ cytotoxic T cells, and CXCR4 expressing CD4+ T helper and CD8+ cytotoxic cells. A significant circadian rhythm was not detectable in RA patients but in healthy controls only for CD3+CD56+ NK T cells. In contrast, a significant circadian expression of IL-8R+ monocytes was found in RA patients only but not in healthy subjects. Of note, CCR7 did not at all show a circadian expression. A significant circadian cytokine expression was detected only for MCP-1 in healthy controls. Conclusions This is the first indication of alterations of clock gene expression and endogenous circadian rhythms in immune cells of RA patients. Traffic of peripheral blood cells shows circadian variation in RA patients and healthy controls with characteristic peak phases, especially in NK cells and chemokine receptor expressing cells. NKT and other cells may lose their normal circadian rhythm in RA, whereas IL-8R expression on monocytes may be established as new “inflammatory” circadian rhythm in RA patients. These findings provide new aspects of RA chronobiology and may have therapeutic implications. Disclosure of Interest C. Spies: None Declared, T. Gaber: None Declared, P. Hoff: None Declared, J. Mazuch: None Declared, B. Maier: None Declared, M. Hahne: None Declared, C. Strehl: None Declared, C. Tran: None Declared, N. Soboleva: None Declared, A. Stoehr: None Declared, M. Wagegg: None Declared, M. Fangradt: None Declared, M. Jakstadt: None Declared, D. Huscher: None Declared, G.-R. Burmester: None Declared, J. Detert: None Declared, A. Kramer: None Declared, F. Buttgereit Grant/Research support from: This study was supported by Horizon Pharma AG, Reinach, Switzerland and Merck KGaA, Darmstadt, Germany. Dr. Buttgereit reports receiving consultancy fees, honoraria and travel expenses from Merck Serono, Horizon Pharma (formerly Nitec Pharma) Mundipharma Int Ltd and grant support from Merck Serono and Horizon Pharma., Consultant for: see above, Speakers Bureau: see above

THU0119 Impact of Amino-Pva Coated Nanoparticles on Viability and Cytokine Secretion of Human Immune Cells Obtained from Healthy Donors and Patients with Rheumatoid Arthritis

Background Nanotechnology has developed into a key technology of the 21st century. Over the recent years, the number of nanotechnical products has received an enormous boost. Furthermore, nanotechnology in medical applications provides new opportunities for diagnostic and therapeutic interventions in a variety of human diseases. Especially in rheumatoid arthritis (RA) an early diagnosis is still missing, although the treatment is most effective when started early. However, safety aspects still represent crucial problems for the further development of nanotechnology based products. Therefore, the focus of our work here was to identify putative effects of amino-PVA-coated super paramagnetic iron oxide nanoparticles (SPION) on human immune cell functions. Objectives We analysed the effects of various concentrations of PVA coated SPION on cell viability and cell activation with regard to cytokine secretion in a whole blood assay. Methods Venous blood, obtained from 18 healthy donors (HD) or 19 patients suffering from RA was collected. Whole blood was diluted 1:1 with RPMI 1640 culture medium in deep-well-plates, and cells were stimulated with LPS (1μg/ml), PHA (5μg/ml), PVA-SPION (1μg/ml, 10μg/ml, 100μg/ml, 1000μg/ml) or left untreated for 20h at 37°C. Cells were analysed for viability by flow cytometry, and supernatants were collected for quantification of cytokines via Bio-Plex® suspension array system. Cell viability was analysed by a combined staining for cell specific surface markers (CD3; CD14; CD15; CD19) and apoptotic and dead cells using Annexin V and 7AAD. As a second approach, intracellular IL1β was analysed to identify the main producers of IL1β. Therefore, cellular transport was blocked by adding Brefeldin A to the whole blood 3h prior to analysis. Results For both HD and RA, we did not find any significant influence on cell viability as induced by PVA-SPION at concentrations <1000μg/ml. However, when analysing the impact of PVA-SPION on cytokine secretion, we demonstrated a significant dose dependent increase of several secreted cytokines in the cell culture supernatant (such as IL1β, IL4, IL6, IL8 or MIP1b for both HD and RA samples). When analysing IL1β secretion in more detail, we found a PVA-SPION concentration dependent increase in the percentage of IL1β positive cells. Furthermore, we observed that the major cell populations producing IL1β after incubation with PVA-SPION are CD15 positive granulocytes as well as CD14 positive monocytes. Conclusions PVA-SPION at concentrations up to 1000μg/ml do not increase the frequencies of apoptotic or dead human immune cells but do induce a PVA-SPION dose dependent cytokine secretion. We conclude that PVA-SPION represent a promising diagnostic and possibly also a therapeutic tool in rheumatic diseases, but prior to clinical use the impact of PVA-SPION on other human immune cells and the induction of cytokine secretion has to be further evaluated. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.1379

AB0026 Pathophysiological hypoxia affects both redox state and il-2 signaling of human cd4+ t cells and concomitantly impairs survival and proliferation

Background Rheumatoid arthritis (RA) is a chronic inflammatory disease of diarthrodial joints, characterized by the infiltration of immune cells, alterations of cellular redox state and pathophysiological local tissue hypoxia (<1% O2). Objectives We investigated the impact of different hypoxia levels on survival, proliferation, cytokine secretion, intracellular energy and redox state of quiescent and mitogen stimulated human CD4+ T cells. Methods Isolated human CD4+ T cells were exposed to normoxia (18% O2), physiological hypoxia (5% O2) and pathophysiological hypoxia (<1% O2), respectively and cultured with or without PHA stimulation at defined periods of time (6h, 24, 48, and 72h). Cells were then analyzed by measuring IL-2R signaling (CD25 expression, phosphoSTAT5a), proliferation and the production of intracellular ROS (iROS) using flow cytometry. In addition, caspase-3/7 activity and ATP levels were determined by luminometric assays. Cellular oxidative damage and cytokine release were analyzed by ELISA based quantification of protein carbonylation (ROS damage) and IL2 production. Results We found pathophysiological hypoxia (<1% O2) to decrease significantly CD4+ T cell survival after mitogenic stimulation. This effect was neither due to induced caspase-3/7-mediated apoptosis nor to increased ATP consumption or increased ATP depletion. However, the ability of stimulated T cells to proliferate was both delayed and suppressed under these hypoxic conditions, despite increased expression of CD25 and unchanged secreted IL-2 amounts. Hypoxia was also found to modify iROS levels in stimulated T cells over time, but without producing measurable effects on oxidative protein damage as compared to normoxia. In contrast, both normoxia (18% O2) and physiological hypoxia (5% O2) did not decrease CD4+ T cell survival and proliferation after mitogenic stimulation. Conclusions We conclude that pathophysiological hypoxia (<1% O2) but not physiological hypoxia (5% O2) affects T cell proliferation and viability via disturbed IL-2R signaling downstream of STAT5a phosphorylation. However, the decreased T cell proliferation was not linked to an impaired cellular energy homeostasis. We suggest iROS links early events in T cell stimulation to the inhibition of the lymphoproliferative response under hypoxic conditions. The level of iROS may therefore act as a mediator of immune functions leading to down-regulation of long-term T cell activity in inflamed tissues. Disclosure of Interest None Declared

OP0271 Differential regulatory functions of HIF-1α and HIF-2α during angiogenesis of human microvascular endothelial cells (HMECS)

Background Hypoxia and angiogenesis are features of inflamed and injured tissues. The transcription factors Hypoxia inducible factor (HIF)-1α and (HIF)-2α control cellular metabolic response to decreased oxygen tension thereby promoting angiogenesis and having implications on the pathogenesis of RA. Objectives We focused on the effects of HIF-1α and HIF-2α on angiogenesis and developed a human microvascular endothelial cells (HMEC) lentiviral based knockdown system for both transcription factors allowing us to analyze angiogenesis of HMECs under hypoxia in the absence of HIF-1α or HIF-2α, respectively. Methods Specific knockdown of HIF-1α or HIF-2α was achieved using lentiviral-based shRNA technology. The reduction of HIF-1α or HIF-2α was confirmed on transcriptional and translational level by realtime RT-PCR and Western blot. Angiogenesis of transduced HMECs in comparison to scrambled cells was studied by investigating both tubuli and node formation under hypoxia (<1% O2). Expression of hypoxia driven genes HIF1A, HIF2A, VEGFA and IL8 was quantified by realtime RT-PCR. Multiplex suspension array technology was used to measure the concentrations of secreted VEGF and IL8. Results The successful knockdown of HIF-1α and HIF-2α was confirmed by demonstrating considerably reduced gene expression levels of HIF1A and HIF2A by up to 71% (p=0.0222) under normoxic and hypoxic conditions. As a consequence, strongly reduced HIF-1α and HIF-2α protein levels were detected by Western blot. Targeting of HIF-1α led to a significantly decreased node formation (1.6-fold change under hypoxia, p=0.0067) with similar effects by trend on tubuli formation. The HIF-2α knockdown also led to a significantly decreased tubuli formation (1.7-fold change, p=0.0444) with similar effects by trend on node formation. Furthermore, HIF-1α targeted cells did not show any significant decrease in the gene expressions of VEGFA and IL8 but surprisingly raised cytokine levels of IL8 under hypoxia (1.4-fold change, p=0.021) compared to the control. In contrast, targeting of HIF-2α gave rise to reduced levels of secreted VEGF and IL8 with a significant suppression of IL8 gene expression under normoxia (2.1-fold change, p=0.0101). Conclusions Our findings show essential and overlapping functions of HIF-1α and HIF-2α with regard to their regulatory potential of angiogenesis in HMECs. Both transcription factors have the same impact on VEGF but differ in their effects on IL8 expression. These findings provide new insights into basic principles of angiogenesis in inflamed tissues and are therefore considered to be of clinical importance. Disclosure of Interest None Declared

THU0088 The Bioenergetic Role of HIF-1 and HIF-2 During Angiogenesis of Human Microvascular Endothelial Cells

Background Hypoxia and angiogenesis are features of inflamed and injured tissues. The transcription factors Hypoxia inducible factor (HIF)-1 and (HIF)-2 regulate the cellular and metabolic responses to reduced oxygen tensions thereby promoting angiogenesis with implications on the pathogenesis of rheumatoid arthritis (RA). Objectives To knockdown either HIF-1α or HIF-2α in human microvascular endothelial cells (HMEC) in order to investigate resulting effects on angiogenesis and bioenergetics under hypoxic versus normoxic conditions. Methods Specific knockdown of either HIF-1α or HIF-2α was achieved using lentiviral-based shRNA technology. Angiogenesis of transduced HMECs was studied by investigating both tubuli and node formation under hypoxic (<1% O2) versus normoxic (~18% O2) conditions. Expression of hypoxia driven genes involved in the metabolic response to hypoxia (GAPDH, PGK, GLUT1, LDHA) was quantified by realtime RT-PCR. The bioenergetic status of the cells was quantified via ATP/ADP measurements. Results Knockdown of HIF-1α resulted in a loss of both hypoxia induced node (p=0.007) and tubuli formation (p=0.09). Also HIF-2α knockdown was followed by a significant reduction of hypoxia induced formation of tubuli (p=0.04). Focussing on bioenergetic aspects, we found hypoxia to significantly induce PGK (p=0.0004), LDHA (p=0,039) and GAPDH (p=0.049) in control cells. Interestingly, knockdown of HIF-1α and HIF-2α, respectively, did not affect the hypoxic induction of PGK and LDHA expression. In both HIF-1α (p=0.01) and HIF-2α (p=0.13) knockdown cells, hypoxia was still capable of inducing GAPDH, but the effect was considerably less pronounced in HIF-1α knockdown cells. Hypoxia did not significantly up-regulate GLUT1 (encoding the glucose transporter 1), neither in control nor in HIF-1α or HIF-2α knockdown cells. However, the knockdown of HIF-2α resulted in significantly decreased expression levels of GLUT1 under hypoxia (p=0,0039). We also found the ATP/ADP ratio to be similar in control, HIF-1α knockdown and HIF-2α knockdown cells under normoxia. Under hypoxic conditions, however, HIF-1α knockdown cells showed a significantly reduced ATP/ADP ratio (p<0.05) – indicating that less ATP is available – compared to HIF-2α knockdown cells. Conclusions HIF-1α and HIF-2α are both key regulators of angiogenesis. However, they do differ in their potency to regulate cellular energy metabolism. This leads us to conclude that HIF-2α does directly influence angiogenesis via regulating the synthesis of proangiogenic factors (as previously shown), whereas HIF-1α affects angiogenesis via effects on cellular energy metabolism as indicated by the reduced expression of GAPDH and the diminished ATP/ADP ratio. These findings provide new insights into regulation of angiogenesis in inflamed (hypoxic) tissues and are, therefore, considered to be of clinical relevance in RA. Disclosure of Interest None Declared

THU0127 Functional activity of membrane-bound glucocorticoid receptors

Background Glucocorticoids are among the most commonly used anti-inflammatory and immunosuppressive drugs in the treatment of rheumatic diseases. They exert their anti-inflammatory and immunosuppressive effects primarily via the cytosolic glucocorticoid receptor (cGR), but also via rapid, specific and unspecific non-genomic actions. The latter actions are hypothesized to be in part mediated by membrane-bound glucocorticoid receptors (mGR), which are up-regulated in monocytes from patients with inflammatory diseases such as RA, SLE and AS. We have previously shown that the human GR gene encodes the expression of both the cGR and the mGR protein. We also showed that the activation of the mGR resulted in rapid signalling via (de)phosphorylation events. Objectives Here, we investigated more in detail the mechanisms of this rapid mGR signalling, and the subsequent effects on the genome. Methods Human monocytes (obtained from healthy donors) were stimulated with LPS (2μg/ml) for 24 hours in order to increase mGR expression up to 70%. Subsequently, cells were treated with membrane impermeable dexamethasone bound to BSA (10-9M) (DEX-BSA), DEX (10-8M) or BSA (10-9M) alone. The incubation time was 20 minutes for kinome analysis and 3 h 20 min for genome analysis. Using the PepChip™ array technique, we identified kinases solely triggered by DEX-BSA. These kinases were classified according to their molecular and biological functions via the Panther database. In order to analyse mGR-mediated effects on gene expression, we analysed lysates of the cells treated with DEX-BSA, DEX or BSA alone by DNA microarray. The results obtained were validated by RT-qPCR. Functional analysis of the data was performed using the Panther database. Results The analysis of the kinome of LPS stimulated human monocytes demonstrated DEX-BSA to induce rapid (de)phosphorylation events of proteins belonging to signalling cascades. We identified a number of MAP-kinases, including p38-MAPK, protein kinase A and C as well as casein-kinase 2 which have to be considered as possible upstream kinases being responsible for the modification of peptide-substrates. Increased phosphorylation of p38-MAPK due to DEX-BSA treatment was observed in phosphoprotein detection Bio-Plex assay as well as in Western blot analysis. Focusing on the functional activity of the mGR, genome analysis of LPS stimulated human monocytes revealed that DEX-BSA also alters gene expression. Validation of these findings via RT-qPCR confirmed that predominantly cell surface receptor linked signal transduction pathways, intracellular signalling cascades and processes involved in the metabolism of proteins, nucleosides, nucleotides and nucleic acids are affected. Conclusions The human mGR is functionally active as shown by rapid effects on the kinome as well as on the genome. These effects need to be further investigated in order to identify mGR signalling pathways and their targeted genes more in detail. Disclosure of Interest None Declared

THU0022 The Role of HIF-1 and HIF-2 During Angiogenesis and Metabolic Adaptation of Human Microvascular Endothelial Cells Towards Hypoxia

Background Chronically inflamed tissues as well as injured tissues are characterized by local hypoxia and – as a consequence – enhanced angiogenesis. The two transcription factors Hypoxia inducible factor (HIF)-1 and (HIF)-2 regulate the cellular and metabolic responses to pathophysiologically reduced oxygen tension thereby promoting both angiogenesis and metabolic adaptation of endothelial cells with implications on the pathogenesis of rheumatoid arthritis (RA). Objectives To knockdown either HIF-1α or HIF-2α in human microvascular endothelial cells (HMEC), respectively, in order to investigate the impact of HIF-1 and HIF-2 on the angiogenic and metabolic transcriptome under hypoxic versus normoxic conditions. Methods Specific knockdown of either HIF-1α or HIF-2α was achieved using lentiviral-based shRNA technology. Angiogenic and metabolic transcriptome of transduced HMECs (and untransduced control cells) was studied by performing gene expression studies (Agilent Human Whole Genome 60K Microarrays) under normoxic (18% O2) vs. hypoxic (1% O2) conditions. Obtained data were analyzed in silico by the classification of significantly? regulated genes (≥2-fold change, p<0.01) into angiogenic and metabolic processes using Panther database. Results In comparison to normoxia the incubation of untransduced HMECs at 1% O2 resulted in 73 regulated angiogenesis related genes in 11 different pathways and 17 regulated cellular metabolism related genes in 9 different pathways, respectively. In both HIF-1α and HIF-2α knockdown cells, hypoxia was still capable of inducing a differential gene expression pattern, but the effect was much less pronounced if compared with cells without knockdown. Analysis of effects on angiogenesis related processes showed (such as i.e. angiogenesis, VEGF pathway, HIF activation, EGF receptor pathway) that 74% of the differentially expressed genes are controlled by both HIF-1 and HIF-2, respectively. Another 14% of the regulated genes are dependent on the presence of HIF-1, among them i.e. the genes GRB2, PDGFRB, PLD, WNT5A and MMP3. The remaining 12% of regulated genes are under control of HIF-2, among them i.e. the genes DLL3, HSP27beta2, NOTCH4, PKC and MMP1. The differentially regulated genes encoding proteins/enzymes involved in the cellular metabolism (i.e. glycolysis, ATP synthesis, TCA cycle) were found to be to 80% controlled by both HIF-1 and HIF-2, respectively. The remaining 20% are dependent on the presence of HIF-1, among them the genes GRB2, FOXJ1, FOXQ1 and Cyt C. Conclusions HIF-1α and HIF-2α are both key regulators of the adaptation of endothelial cells towards hypoxia with overlapping functions. However, they do differ in their capacity to regulate cellular energy metabolism and angiogenesis. This leads us to conclude that HIF-1α affects angiogenesis via indirect effects on cellular energy metabolism as indicated by the regulation of metabolic transcriptome to one fifth. HIF-2α does more influence angiogenesis directly via regulating the synthesis of proangiogenic factors (as has been previously shown). These findings provide new insights into the divergent regulation of angiogenesis in inflamed (hypoxic) tissues by HIF-1 and HIF-2 and are, therefore, considered to be of clinical relevance in RA. Disclosure of Interest None Declared