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and Ingenuity Pathway Analysis (IPA). In vitro proteasomal ... Conclusions: We have combined quantitative label-free proteomics with Ingenu- ity Pathway ...
Sixth International Congress on Spondyloarthropathies

Poster Presentations

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ANTIGEN SPONDYLITIS MONOCYTES SHOW UPREGULATION OF PROTEINS INVOLVED IN ANTIGEN PRESENTATION

FREQUENCY AND PHENOTYPE OF TH17 CELLS IN AS PATIENTS AND HEALTHY CONTROLS

P. Bowness1, C. Wright1, S. McGowan1, S. Taylor1, K. diGleria1, M. Edelman2, H.Kramer2, B.Kessler2 1 WIMM, Human Immunology Unit, Oxford; 2Henry Wellcome Centre for Human Genetics, Human Immunology Unit, Oxford, UK

H. Shen1, J. Goodall1, H. Gaston1, H. Shen2 1 Addenbrooke’s Hospital, Medicine, Cambridge, UK; 21st Hospital of China Medical University, Rheumatology, Shen Yang, China Introduction: T helper cells that produce IL-17 (Th17 cells) have been described as a new lineage of CD4+ T cells and are thought to have key roles in inflammatory arthritis. Ankylosing spondylitis (AS) is a chronic inflammatory disease in which elevated serum levels of IL-17 have been reported. This study, we compared the frequencies of Th17 cells in AS and healthy controls, and studied the phenotype, chemokine receptor expression and other cytokines produced by Th17 cells. Materials and Methods: 8-color flow cytometry was used to analyze surface phenotype, cytokine production, and chemokine receptor expression of PBMCderived T cells from 20 AS and 16 healthy people. We also measured by ELISA secretion of IL-17 into culture supernatants by PBMC. Results: (1) The percentages of IL-17+CD4+ T cells and IL-22+CD4+T cells were increased in PBMC of AS as compared with healthy controls. The ratio of IL17+CD4+ T cells to IFN-γ +CD4+ T cells was much higher in AS than in healthy controls ; conversely, the ratio of IL-10+CD4+T cells to IL-17+CD4+ T cells in AS was much lower. (2) IL-17 concentrations in AS supernatants were significantly higher in compared to healthy controls by ELISA. There was a correlation between the percentages of IL-17+CD4+T cells and the amounts of IL-17 in culture supernatants. (3) All Th17 cells were CD4+ and CD45RO+. Most of the Th17 cells expressed both CCR6 and CCR4. However, not all of the Th17 cells expressed the IL-23 receptor. (4) A significant proportion of cells that produced IL-17 also produced IL-22 and IFN-γ but not IL-10. Conclusions: This study has shown that Th17 cells are significantly more prevalent in PBMC in AS. This supports the hypothesis that IL-17 producing cells contribute to the pathogenesis of AS.

Introduction: Ankylosing Spondylitis (AS) is an autoimmune inflammatory disease of unknown aetiology. Transgenic rat studies have implicated Monocytes. We wished to quantify differences in protein expression in monocytes between patients with Ankylosing Spondylitis (AS), Rheumatoid Arthritis (RA) and healthy controls. Methods: The protein expression of CD14 bead purified AS, RA and control monocytes was studied by 2D gel electrophoresis and by quantitative label-free expression profiling. Tryptic digestion of monocyte proteins was followed by nano ultra-performance liquid chromatography coupled to ESI MS/MS mass spectrometry. Data sets were analysed using Waters Expression Profiling System (WEPS) and Ingenuity Pathway Analysis (IPA). In vitro proteasomal digests of extended HLA-B27 epitopes were carried out in the presence or absence of the proteasome activator complex PA28. Results: 2D gel electrophoresis identified the immunoprotasome prtein PA28 as upregulated in some AS patient’s monocytes compared to healthy controls. AS and RA monocytes differed in protein expression the healthy controls using IPA. The most significant pathways for both the AS and RA pools, based on numbers of differentially expressed proteins were the Vascular Endothelial Growth Factor (AS:25, RA:24), leukocyte extravasation (AS:39, RA:35), NF-κB (AS:27,25), Integrin (AS:32, RA:36), Jak-Stat (AS:15, RA:16) and Toll-like Receptor signalling pathways (AS:11, RA:10). The only pathway in which a marked difference was observed in the number of differentially expressed proteins between the AS and RA patients was the Proteasome Ubiquitin Pathway. PA28 enhanced generation of HLA-B27 peptide epitopes in vitro. Conclusions: We have combined quantitative label-free proteomics with Ingenuity Pathway Analysis to characterize differential protein expression in AS monocytes. AS monocytes shows significant changes in protein expression compared to matched RA and healthy controls. Our results support a role for proteasome-dependent proteolysis in AS monocytes. We further demonstrate the utility of novel proteomic techniques in investigating inflammatory rheumatic diseases.

P 12 INTERLEUKIN 17 ACTIVATION IN SMALL INTESTINE IN RHEUMATOID ARTHRITIS AND ANKYLOSING SPONDYLITIS S. Aittomaki1, V. Hölttä1, H.M. Salo1, L. Paimela2, L. Halme3, M. Leirisalo-Repo4, O. Vaarala1 1 National Public Health Institute, Viral Diseases and Immunology, Helsinki; 2Orton Hospital, Invalid Foundation, Helsinki; Helsinki University Central Hospital, 3 Dept. of Surgery and 4Dept. of Medicine Division of Rheumatology, Helsinki, Finland

P 10 IL-23 IS NOT ELEVATED IN THE SERUM OR UPREGULATED IN THE PERIPHERAL BLOOD MONOCYTES (PBMCS) OF ANKYLOSING SPONDYLITIS (AS) PATIENTS

Introduction: The purpose of the study was to investigate the involvement of interleukin 17 (IL-17) in rheumatoid arthritis (RA). Methods: We studied IL-17 mRNA expression and the number of IL-17 positive cells in duodenal biopsy samples from 12 patients with RA with real-time reverse transcriptase polymerase chain reaction and immunohistochemistry, respectively. 3 duodenal and 3 ileal biopsy samples from patients with ankylosing spondylitis (AS) were analysed for IL-17 mRNA expression. We also measured IL-17 mRNA expression in synovial fluid and peripheral blood derived cells as well as soluble IL-17 concentration in synovial fluid and peripheral blood in patients with RA. Results: The expression of IL-17 mRNA in duodenal biopsy samples from patients with RA was increased (p