IL-15 and its role in rheumatoid arthritis

Background: IL-15 is involved in all phases of rheumatoid arthritis. Recently we have shown that rheumatoid arthritis synovial fibroblasts (RASF) express both IL-15 and functional IL-15 receptor [1]. Objective: The aim of present study was to identify pathways that are regulated by autocrine IL-15 (IL-15R) in RASF. Methods: RASF were transfected with plasmid encoding IL-15R antagonist (CRB-15, Cardion AG) or control constructs. RNA from transient transfectants were used for Microarray analysis. The differential expression of genes obtained by microarray analysis was verified by SYBR Green real-time PCR. The expression of IL-15Rα, cell proliferation and the expression of p16 and p21 were evaluated in stably transfected cells. Results: The IL-15R antagonist produced by transfected RASF blocked the endogenous IL-15/IL-15Rα interaction, which resulted in an inhibition of cell proliferation (45 ± 10%) via an increase of the expression of p16. In addition, we found that inhibition of IL-15Rα induced the expression of mRNA for FGFR-3. Since two isoforms of FGFR-3 have been identified (FGFR-3b and FGFR-3c) [2], we tested the effect of IL-15Rα inhibition on their expression. In contrast to FGFR-3b, the level of mRNA for FGFR-3c was strongly increased in cells transfected with the IL-15R antagonist (4.71 ± 2.5 in transient transfectants and 6.1 ± 1 fold in stable transfectants). FGFR-3c isoform binds specifically FGF-9, but also FGF-2 [2]. Besides FGFR-3, FGF-2 that is abundant in RA joints binds to FGFR-1. In vitro studies revealed that FGFR-1 transmits a potent mitogenic signal, whereas FGFR-3 usually has no stimulatory effect or inhibits cell proliferation. In contrast to FGFR-3c, blocking of IL-15Rα did not change the mRNA expression for FGFR-1 in RASF. Moreover, we checked whether FGF-2 affects the expression of IL-15Rα. Indeed, FGF-2 strongly decreased the spontaneous and tumor necrosis factor alpha-triggered expression of IL-15Rα at the mRNA and protein levels. Conclusion: Our findings raise the possibility of a negative loop between FGF-2/FGFR-3c and IL-15/IL-15R signaling in RASF. Moreover, the activation of RASF by FGFs could depend on the ratio of FGFR-1/FGFR-3 expression, which is controlled by the endogenous IL-15/IL-15R system

The inhibitor of differentiation-2 promotes synovial fibroblast-dependent osteoclastogenesis in rheumatoid arthritis

Objectives: Despite indirect evidence suggesting that low oxygen levels might occur in the rheumatoid arthritis (RA) synovium, direct proof of the presence of hypoxia in the arthritic synovium as well as the relevance of low oxygen levels for joint destruction is lacking. The aim of this study was to analyse the distribution of hypoxia in arthritic joints and to evaluate the molecular effects of the hypoxic environment on the phenotype of RA synovial fibroblasts (SF).<p></p> Methods: The hypoxia marker EF-5 was applied in mice with the collagen-induced arthritis (CIA). Expression profile analysis with hypoxic and normoxic SF was performed using subtractive hybridization and microarray. The expression of the inhibitor of differentiation-2 (Id-2), CD68 (macrophage marker) and prolyl hydroxylase (fibroblast marker) was evaluated by immunohistochemistry on synovial tissues from RA, osteoarthritis patients and CIA mice. To evaluate the function of Id-2 in SF, cells were transfected with the pcDNA3.1 containing cDNA for Id-2 or Id-2-specific siRNA or mock controls. The expression of Id-2 and genes regulated by Id-2 in transfected SF was evaluated by SYBR Green real-time PCR and western blot. SF stably transfected with Id-2 were cocultured with bone marrow cells in a transwell system. The expression of the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin were measured by real-time PCR. The development of osteoclasts was evaluated by visualization of the activity of tartrate-resistant acid phosphatase.<p></p> Results: Using the hypoxia marker EF-5 we found that in mice with CIA, synovial cells invading bone and cartilage are exposed to reduced oxygen levels. Expression profile studies identified Id-2 as being upregulated under low oxygen conditions. In addition, IL-1beta stimulation increased the expression of Id-2 in these cells. Histological studies of RA synovium and CIA synovium showed strong expression of Id-2 in SF at sites of synovial invasion into bone. Overproduction of Id-2 in SF by stable transfection triggered the expression of several genes promoting osteoclastogenesis, including BMP-2, PTHrP, Wnt5a and vascular endothelial growth factor. Conversely, the suppression of endogenous Id-2 led to the downregulation of the expression of these molecules. Consistent with these findings coculture of Id-2 transfected SF with bone marrow cells increased the expression of the osteoclast differentiation factor RANKL, and decreased the expression of the osteoclast inhibitory factor osteoprotegerin in bone marrow stromal cells, which was followed by an increase in the number of osteoclasts.<p></p> Conclusion: Taken together, our data provide evidence that hypoxia is present at sites of synovial invasion in RA and that Id-2 induced by hypoxia contributes at these sites to joint destruction by promoting SF-dependent osteoclastogenesis

Changes in MiRNA-5196 expression as a potential biomarker of anti-TNF-α therapy in rheumatoid arthritis and ankylosing spondylitis Patients

In this study, we analysed the expression level of sera circulating miRNA-5196 in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients before and after tumor necrosis factor (TNF)-α therapy as biomarkers predicting positive treatment outcome. We enrolled 10 RA patients, 13 AS patients, and 12 healthy individuals in the study. The expression of miRNA-5196 was measured by real-time polymerase chain reaction before and after anti-TNF-α therapy. Disease activity of RA patients was assessed using disease activity score 28 (DAS28), whereas ankylosing spondylitis DAS (ASDAS) was used in AS patients. MiRNA-5196 expression was significantly higher in patients with RA and AS before TNF-α therapy than in those following anti-TNF-α therapy and healthy controls. Changes in miRNA-5196 expression positively correlated with delta DAS28 or delta ASDAS, respectively, following TNF-α therapy. In contrast, changes in C-reactive protein (CRP) levels in RA and AS patients did not positively correlate with DAS28 or ASDAS changes. Receiver-operating characteristic analysis showed better diagnostic accuracy of miRNA-5196 expression both in RA (area under curve (AUC) = 0.87, p = 0.055) and AS patients (AUC = 0.90, p = 0.050) compared to CRP levels in RA (AUC = 0.75, p = 0.201) and AS patients (AUC = 0.85, p = 0.086) upon biologic therapy treatment. Finding novel biomarkers, including miRNA-5196 which allow to predict and monitor anti-TNF-α response, would be of clinical value especially during the early phase of RA or AS development

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion