Juvenile Idiopathic Arthritis Synovial Fluid Research Articles

The immune landscape of the inflamed joint defined by spectral flow cytometry.

Cellular phenotype and function are altered in different microenvironments. For targeted therapies it is important to understand site-specific cellular adaptations. Juvenile Idiopathic Arthritis (JIA) is characterised by autoimmune joint inflammation, with frequent inadequate treatment responses. To comprehensively assess the inflammatory immune landscape, we designed a 37-parameter spectral flow cytometry panel delineating mononuclear cells from JIA synovial fluid (SF) of autoimmune inflamed joints, compared to JIA and healthy control blood. Synovial monocytes and NK cells (CD56bright) lack Fc-receptor CD16, suggesting antibody-mediated targeting may be ineffective. B cells and DCs, both in small frequencies in SF, undergo maturation with high 4-1BB, CD71, CD39 expression, supporting T cell activation. SF effector and regulatory T cells were highly active with newly described co-receptor combinations that may alter function, and suggestion of metabolic reprogramming via CD71, TNFR2 and PD-1. Most SF effector phenotypes, as well as an identified CD4-Foxp3+ T cell population, were restricted to the inflamed joint, yet specific SF-predominant CD4+Foxp3+ Treg subpopulations were increased in blood of active but not inactive JIA, suggesting possible recirculation and loss of immunoregulation at distal sites. This first comprehensive dataset of the site-specific inflammatory landscape at protein level will inform functional studies and the development of targeted therapeutics to restore immunoregulatory balance and achieve remission in JIA.

Differentially Expressed Inflammation-Regulating MicroRNAs in Oligoarticular Juvenile Idiopathic Arthritis.

To evaluate microRNA expression in synovial fluid (SF), plasma, and leukocytes from patients with juvenile idiopathic arthritis (JIA). MicroRNA expression in pooled JIA plasma and SF was assessed by absolute quantitative droplet digital PCR array. The results were validated in individual patient samples. MicroRNA content in leukocytes and extracellular vesicles was evaluated by real-time PCR in JIA blood and SF. Blood microRNA expression was compared with healthy controls (HCs). Principal component analysis was used to profile JIA plasma and SF microRNAs, and the potential biological consequences of microRNA dysregulation were investigated by pathway analysis. MiR-15a-5p and miR-409-3p levels were higher in JIA plasma than in HC plasma. JIA SF contained elevated levels of miR-21-5p, miR-27a-3p, miR-146b-5p, miR-155-5p, and miR-423-5p, and decreased miR-192-5p and miR-451a, compared to JIA plasma. Extracellular vesicle analysis demonstrated variable encapsulation among selected microRNAs, with only miR-155-5p being represented substantially in extracellular vesicles. SF leukocytes also had higher expression of miR-21-5p, miR-27a-3p, miR-146b-5p, and miR-155-5p, and lower expression of miR-409-3p and miR-451a, relative to blood. No differences were observed between JIA and HC blood leukocytes. Clusters of microRNAs were commonly altered in JIA joint fluid and leukocytes compared to JIA blood samples. In silico analysis predicted that differentially expressed microRNAs in JIA target the transforming growth factor (TGF)-β pathway. The expression of multiple microRNAs is dysregulated in JIA both locally and systemically, which may inhibit the TGF-β pathway. These findings advance our knowledge of JIA immunopathogenesis and may lead to the development of targeted therapies.

P168 An enriched population of tissue-resident CD8 memory T cells in young people with juvenile idiopathic arthritis recapitulate findings from mouse models of inflammatory arthritis flares

Abstract Background/Aims Asymmetrical joint inflammation at presentation and during flares of juvenile idiopathic arthritis (JIA) implicates resident cell populations and the microenvironment in disease. Persistent CD8+ tissue-resident cell populations have been identified from murine models, capable of priming the joint towards an exaggerated inflammatory response, which resist systemic treatment. Clonally-expanded enriched populations of these tissue-resident CD8+ memory T cells (TRM) have been identified in synovial fluid of children with JIA. Methods We integrate and compare scRNA-seq findings from paired synovial fluid mononuclear cells and blood mononuclear cell (PBMC) samples from children with oligoarticular JIA (n = 2), adults with psoriatic arthritis (n = 3) and blood from healthy adult volunteers (n = 4). Results A number of T lymphocyte and myeloid cell populations demonstrated enrichment amongst synovial fluid mononuclear cells (compared to paired PBMCs) that corresponded across arthritic subtypes. A mean of 2.98% of T cells in synovial JIA samples had the CD69+ITGAE+CD8A+ tissue-resident signature compared to a mean of 0.37% in paired JIA PBMCs and 0.24% in other sample types (P <.00001, vs synovial psoriatic samples). These cells recapitulated previously observed traits of tissue-resident CD8+ T cells, including high expression of transcription factor RUNX3, granzymes and the FABP5 fatty acid transporter. In mouse models the chemokine CCL5 was critical for leukocyte recruitment to mediate inflammatory flares and indeed, JIA synovial fluid samples exhibited stronger expression of CCL5 in TRM. Additionally, transcripts for cytokines involved in CD8+ tissue-resident T cell regulation and maintenance were specifically enriched in JIA synovial samples compared to peripheral blood, including IL-15, IL-12, IFN-γ; suggesting an environment conducive to the persistence of this population. Conclusion We confirm the existence of previously-described cell populations in synovial fluid from JIA patients and identify the enrichment of CD8 T cells with a tissue-resident signature. Further study is required to identify the significance or functional implications of these populations. Disclosure C. Bolton: None. C. Rich-Griffin: None. C. Dendrou: None. C. Brown: None. H. Al-Mossawi: None. A. Croft: None. L. Wedderburn: Consultancies; L.R.W. receieved a consultancy fee from Pfizer unrelated to this work. Grants/research support; L.R.W. declares support from AbbVie, GSK, Pfizer, Sobi, and UCB to the CLUSTER Consortium.

A Proteinase 3 Contribution to Juvenile Idiopathic Arthritis-Associated Cartilage Damage.

A full understanding of the molecular mechanisms implicated in the etiopathogenesis of juvenile idiopathic arthritis (JIA) is lacking. A critical role for leukocyte proteolytic activity (e.g., elastase and cathepsin G) has been proposed. While leukocyte elastase’s (HLE) role has been documented, the potential contribution of proteinase 3 (PR3), a serine protease present in abundance in neutrophils, has not been evaluated. In this study we investigated: (1) PR3 concentrations in the synovial fluid of JIA patients using ELISA and (2) the cartilage degradation potential of PR3 by measuring the hydrolysis of fluorescently labeled collagen II in vitro. In parallel, concentrations and collagen II hydrolysis by HLE were assessed. Additionally, the levels of the co-secreted primary granule protein myeloperoxidase (MPO) were assessed in synovial fluid of patients diagnosed with JIA. We report the following levels of analytes in JIA synovial fluid: PR3—114 ± 100 ng/mL (mean ± SD), HLE—1272 ± 1219 ng/mL, and MPO—1129 ± 1659 ng/mL, with a very strong correlation between the PR3 and HLE concentrations (rs = 0.898, p < 1 × 10–6). Importantly, PR3 hydrolyzed fluorescently labeled collagen II as efficiently as HLE. Taken together, these novel findings suggest that PR3 (in addition to HLE) contributes to JIA-associated joint damage.

Innate Lymphoid Cells and T Cells Contribute to the Interleukin‐17A Signature Detected in the Synovial Fluid of Patients With Juvenile Idiopathic Arthritis

ObjectiveEvidence suggests that aberrant function of innate lymphoid cells (ILCs), whose functional and transcriptional profiles overlap with those of Th cell subsets, contributes to immune‐mediated pathologies. To date, analysis of juvenile idiopathic arthritis (JIA) immune pathology has concentrated on the contribution of CD4+ T cells; we have previously identified an expansion of Th17 cells within the synovial fluid (SF) of JIA patients. We undertook this study to extend this analysis to further investigate the role of ILCs and other interleukin‐17 (IL‐17)–producing T cell subsets in JIA.MethodsILCs and CD3+ T cell subsets were defined in peripheral blood mononuclear cells (PBMCs) from healthy adults, healthy children, and JIA patients and in SF mononuclear cells (SFMCs) from JIA patients using flow cytometry. Defined subsets in SFMCs were correlated with clinical measures including physician's global assessment of disease activity on a visual analog scale, number of joints with active disease, and erythrocyte sedimentation rate. Transcription factor and cytokine profiles of sorted ILCs were assessed by quantitative reverse transcriptase–polymerase chain reaction.ResultsGroup 1 ILCs (ILC1s), NKp44− group 3 ILCs (natural cytotoxicity receptor–negative [NCR−] ILC3s), and NKp44+ ILC3s (NCR+ ILC3s) were enriched in JIA SFMCs compared to PBMCs, which corresponded to an increase in transcripts for TBX21, IFNG, and IL17A. Of the ILC subsets, the frequency of NCR− ILC3s in JIA SFMCs displayed the strongest positive association with clinical measures, which was mirrored by an expansion in IL‐17A+CD4+, IL‐17A+CD8+, and IL‐17A+ γδ T cells.ConclusionWe demonstrate that the strength of the IL‐17A signature in JIA SFMCs is determined by multiple lymphoid cell types, including NCR− ILC3s and IL‐17A+CD4+, IL‐17A+CD8+, and IL‐17A+ γδ T cells. These observations may have important implications for the development of stratified therapeutics.

Increased autophagy contributes to the inflammatory phenotype of juvenile idiopathic arthritis synovial fluid T cells

JIA is an autoimmune disease involving disturbed T-cell homeostasis, marked by highly activated effector T cells. Autophagy, a lysosomal degradation pathway, is crucial for maintaining cellular homeostasis by regulating the survival, differentiation and function of a large variety of cells, including T cells. The aim of this study was to examine the rate of autophagy in JIA T cells and to investigate the effect of inhibition of autophagy on the inflammatory phenotype of JIA T cells. Autophagy-related gene expression was analysed in CD4+ T cells from the SF of JIA patients and healthy controls using RNA sequencing. Autophagy was measured by flow cytometry and western blot. The effect of inhibition of autophagy, using HCQ, on the cellular activation status was analysed using flow cytometry and multiplex immunoassay. Autophagy was increased in T cells derived from the site of inflammation compared with cells from the peripheral blood of patients and healthy controls. This increase in autophagy was not induced by JIA SF, but is more likely to be the result of increased cellular activation. Inhibition of autophagy reduced proliferation, cytokine production and activation marker expression of JIA SF-derived CD4+ T cells. These data indicate that autophagy is increased in JIA SF-derived T cells and that targeting autophagy could be a promising therapeutic strategy to restore the disrupted T-cell homeostasis in JIA.

Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients

This study tested the hypothesis that subsets of human T helper cells can orchestrate leukocyte adhesion to synovial fibroblasts (SFbs), thus regulating the retention of leukocytes in the joints of juvenile idiopathic arthritis (JIA) patients. Several cell types, such as monocytes/macrophages, granulocytes, T and B lymphocytes, SFbs and osteoclasts participate in joint tissue damage JIA. Among T cells, an enrichment of classic and non-classic Th1 subsets, has been found in JIA synovial fluid (SF), compared to peripheral blood (PB). Moreover, it has been shown that IL-12 in the SF of inflamed joints mediates the shift of Th17 lymphocytes towards the non-classic Th1 subset. Culture supernatants of Th17, classic and non-classic Th1 clones, have been tested for their ability to stimulate proliferation, and to induce expression of adhesion molecules on SFbs, obtained from healthy donors. Culture supernatants of both classic and non-classic Th1, but not of Th17, clones, were able to induce CD106 (VCAM-1) up-regulation on SFbs. This effect, mediated by tumor necrosis factor (TNF)-α, was crucial for the adhesion of circulating leukocytes on SFbs. Finally, we found that SFbs derived from SF of JIA patients expressed higher levels of CD106 than those from healthy donors, resembling the phenotype of SFbs activated in vitro with Th1-clones supernatants. On the basis of these findings, we conclude that classic and non-classic Th1 cells induce CD106 expression on SFbs through TNF-α, an effect that could play a role in leukocytes retention in inflamed joints.

The transcription factor CREMalpha regulates inflammatory T cell subsets in juvenile idiopathic arthritis (BA13P.115)

Abstract The cAMP response element (CRE) modulator (CREM)α binds to genes promoters with CREs and regulates transcription via a chromatin-dependent mechanism. CREMα is important for the T cell pathophysiology of SLE suppressing IL-2 and CD3ζ transcription, but enhances IL-17. Juvenile idiopathic arthritis (JIA) is an autoimmune disease of unknown origin. Th17 cells have a pathogenic role in arthritis and are not controlled by local FoxP3+ regulatory T cells (Tregs). Pathogenic T cells in the inflamed joints of JIA patients have enhanced expression of IL-17, IFNgamma and CD161. CD161+ CD4+ cells also contain FoxP3+ cells that produce proinflammatory cytokines. We observed enhanced CREM expression in JIA patient synovial fluid (SF) T cells and also after culture of healthy donor PBMCs with JIA SF. We furthermore found enhanced expression of CREM in CD4+CD161+ and in CD4+FoxP3+CD161+ cells, which produce inflammatory cytokines. Vice versa Helios+FoxP3+ cells, which are characteristically stable Tregs and do not express inflammatory cytokines, had lower levels of CREM. JIA SF induced IFNgamma, IL-17 and FoxP3 in T cells during incubation, which could be abrogated by CREM siRNA transfection. Within the FoxP3+ population, CREM siRNA reduced CD161+FoxP3+ cells but not Helios+FoxP3+ cells. We thus suggest CREMα overexpression in T cells contributes to T cell pathophysiology in JIA by regulating percentages of inflammatory CD4+ IL-17+ cells, as well as inflammatory CD161+FoxP3+ cells.

SAT0439 Correlation between Vitamin D Metabolites and TH17 or T-Regulatory Cells as well as Pro-Inflammatory Cytokines in Children with Juvenile Idiopathic Arthritis

BackgroundImbalance between cells secreting IL-17 (Th17) and regulatory T lymphocytes (Tregs) may be a key pathogenic event in development of autoimmnune diseases. Recent studies suggest that vitamin D may have...

EVIDENCE OF A TRANSIENT NATURE OF THE TH17 PHENOTYPE OF CD4+CD161+ T CELLS IN THE SYNOVIAL FLUID FROM PATIENTS WITH JUVENILE IDIOPATHIC ARTHRITIS.

Event Abstract Back to Event EVIDENCE OF A TRANSIENT NATURE OF THE TH17 PHENOTYPE OF CD4+CD161+ T CELLS IN THE SYNOVIAL FLUID FROM PATIENTS WITH JUVENILE IDIOPATHIC ARTHRITIS. Laura Maggi1*, Lorenzo Cosmi1, Veronica Santarlasci1, Manuela Capone1, Valentina Querci1, Francesco Liotta1, Raffaele De Palma2, Rolando Cimaz3, Enrico Maggi1, Sergio Romagnani1 and Francesco Annunziato1 1 University of Florence, Department of Clinical and Experimental Medicine, Italy 2 Second University of Naples, Italy 3 Anna Meyer Children’s Hospital and University of Florence, Rheumatology unit, Italy OBJECTIVE: To investigate the phenotype and function of CD4+ T cells in synovial fluid (SF) from the affected joints of children with oligoarticular-onset juvenile idiopathic arthritis (JIA) and to establish a possible link with disease activity. METHODS: CD4+ T cells were obtained from the peripheral blood (PB) and SF of 23 children with oligoarticular-onset JIA, as well as from the PB of 15 healthy children and were analyzed for CXCR3, CCR6, CD161 expression and for IFN(interferon)- gamma and interleukin-17A (IL-17A) production. RESULTS: The numbers of CD4+CD161+ cells, showing either the type 1 T helper (Th1) or Th17/Th1 phenotype, were higher in SF than in PB of children with JIA. The few Th17 cells from SF of JIA underwent a spontaneous shift to the Th1 phenotype in vitro, whereas Th17 cells from PB of healthy children shifted only in presence of JIA SF; this effect was neutralized by antibody blocked of IL-12 activity. The frequencies of CD4+CD161+ cells, particularly the Th17/Th1 cells, in the JIA SF positively correlated with levels of erythrocyte sedimentation rate and C-reactive protein. CONCLUSION: These findings suggest that a shifting of CD4+CD161+ T cells from Th17 to the Th17/Th1 or Th1 phenotype can occur in the SF of oligoarticular-onset JIA, and indicate that the accumulation of these cells is correlated with parameters of inflammation. Thus, the results support the hypothesis that these cells may play a role in JIA disease activity. Keywords: Th17, Th1 Cells, CD161, JIA, RORC Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Maggi L, Cosmi L, Santarlasci V, Capone M, Querci V, Liotta F, De Palma R, Cimaz R, Maggi E, Romagnani S and Annunziato F (2013). EVIDENCE OF A TRANSIENT NATURE OF THE TH17 PHENOTYPE OF CD4+CD161+ T CELLS IN THE SYNOVIAL FLUID FROM PATIENTS WITH JUVENILE IDIOPATHIC ARTHRITIS.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00958 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Laura Maggi, University of Florence, Department of Clinical and Experimental Medicine, Florence, 50134, Italy, laura.maggi@unifi.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Laura Maggi Lorenzo Cosmi Veronica Santarlasci Manuela Capone Valentina Querci Francesco Liotta Raffaele De Palma Rolando Cimaz Enrico Maggi Sergio Romagnani Francesco Annunziato Google Laura Maggi Lorenzo Cosmi Veronica Santarlasci Manuela Capone Valentina Querci Francesco Liotta Raffaele De Palma Rolando Cimaz Enrico Maggi Sergio Romagnani Francesco Annunziato Google Scholar Laura Maggi Lorenzo Cosmi Veronica Santarlasci Manuela Capone Valentina Querci Francesco Liotta Raffaele De Palma Rolando Cimaz Enrico Maggi Sergio Romagnani Francesco Annunziato PubMed Laura Maggi Lorenzo Cosmi Veronica Santarlasci Manuela Capone Valentina Querci Francesco Liotta Raffaele De Palma Rolando Cimaz Enrico Maggi Sergio Romagnani Francesco Annunziato Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the aggrecan interglobular domain but robust at the aggrecan C‐terminus

To investigate aggrecan degradation in juvenile idiopathic arthritis (JIA). The pattern and abundance of aggrecan fragments in synovial fluid (SF) aspirates from JIA patients were analyzed and compared with aggrecan fragments in SF from patients with other arthritides, children with knee injury, and a knee-healthy reference group. Concentrations of sulfated glycosaminoglycan (sGAG) in SF were measured by Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density-gradient centrifugation, deglycosylated, and analyzed by Western blot using antibodies specific for either aggrecanase-derived ARGS, SELE, and KEEE neoepitopes or the aggrecan G3 domain. The concentration of sGAG in SF from patients with JIA was significantly lower compared with that in SF from patients with osteoarthritis (OA) (P < 0.001), patients with juvenile knee injury (P = 0.006), and knee-healthy controls (P = 0.022). Western blot analysis revealed KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulfate-rich region of aggrecan in patients with JIA. The pattern of aggrecan fragments in JIA patients was not identical to that in pooled OA SF, although there were notable similarities. Surprisingly, aggrecanase-derived ARGS fragments were barely detectable in JIA SF, in marked contrast to levels in OA SF. Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in the chondroitin sulfate-rich region. These results suggest that in JIA, unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event.

Presence of anti-cyclic citrullinated peptide antibody isotypes in juvenile idiopathic arthritis synovial fluid indicates autoantibody production at the site of inflammation

Results Eleven of 47 (23%) JIA SF samples were positive for the different anti-CCP antibody isotypes. IgM anti-CCP antibodies were positive in 9 JIA patients, including 5 oligoarthritis, 3 IgM RF+ and 1 IgM RFpolyarthritis. IgG antiCCP antibodies were positive in 8 JIA patients, including 4 oligoarthritis, 2 IgM RF+, 1 IgM RFpolyarthritis, and 1 enthesitis-related. Two IgM RF+ polyarthritis patients were positive for IgA anti-CCP antibodies, and also positive for all 3 isotypes. Four JIA patients were positive for 2 anti-CCP antibody isotypes, including 1 IgM RFpolyarthritis patient (IgG and IgM) and 3 oligoarthritis patients (2 IgG and IgM, 1 IgA and IgM). Five JIA patients were positive for 1 anti-CCP antibody isotype, including 3 oligoarthritis (2 IgM, 1 IgG), 1 enthesitis-related (IgG), and 1 IgM RF+ polyarthritis patient (IgM). Three JIA patients were positive for the combined IgA/IgG anti-CCP antibody ELISA, including 2 with IgM RF+ polyarthritis and 1 enthesitis-related. Polyarthritis patients demonstrated significantly higher levels of all anti-CCP antibody isotypes compared to the other JIA subtypes (p<0.05). When only evaluating the IgM RF+ polyarthritis patients against the other subtypes, the levels were even more significantly increased (p<0.05). Serum-matched IgG anti-CCP antibody data was available in 27 JIA patients and 5 healthy controls. All 3 IgM RF+ polyarthritis patients were positive for the IgG anti-CCP antibodies in serum and SF. Two JIA patients who were positive for IgG anti-CCP antibodies in SF had a negative result in serum. Date-matched SF and serum IgM and IgA data was available on 12 JIA patients. More JIA patients were positive for IgA anti-CCP antibodies in serum compared to SF, while SF showed increased positivity for IgM anti-CCP antibodies compared to serum.

Evidence of the transient nature of the Th17 phenotype of CD4+CD161+ T cells in the synovial fluid of patients with juvenile idiopathic arthritis

To investigate the phenotype and function of CD4+ T cells in synovial fluid (SF) from the affected joints of children with oligoarticular-onset juvenile idiopathic arthritis (JIA), and to establish a possible link with disease activity. CD4+ T cells were obtained from the peripheral blood (PB) and SF of 23 children with oligoarticular-onset JIA, as well as from the PB of 15 healthy children. The cells were analyzed for the expression of CXCR3, CCR6, and CD161 and for the production of interferon-γ and interleukin-17A (IL-17A). Spectratyping and clonotype analyses were performed to assess different T cell subsets. The numbers of CD4+CD161+ cells showing either the Th1 or the Th17/Th1 phenotype were higher in the SF than in the PB of children with JIA. The few Th17 cells from JIA SF underwent a spontaneous shift to the Th1 phenotype in vitro, whereas Th17 cells from the PB of healthy children shifted only in the presence of JIA SF; this effect was neutralized by antibody blockade of IL-12 activity. Spectratyping and clonotype analyses showed a similar skewing of the T cell receptor V(β) repertoire in both CD161+ Th17 cells and CD161+ Th1 cells derived from the SF of the same JIA patient. The frequencies of CD4+CD161+ cells, particularly the Th17/Th1 cells, in the JIA SF positively correlated with the erythrocyte sedimentation rate and levels of C-reactive protein. These findings suggest that a shifting of CD4+CD161+ T cells from Th17 to the Th17/Th1 or Th1 phenotype can occur in the SF of children with oligoarticular-onset JIA, and indicate that the accumulation of these cells is correlated with parameters of inflammation. Thus, the results support the hypothesis that these cells may play a role in JIA disease activity.

Synovial fluid proteins differentiate between the subtypes of juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases, and no clinically useful prognostic markers to predict disease outcome in children with JIA are currently available. Synovial fluid likely reflects the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether protein expression differs in the different subtypes of JIA. Synovial fluid samples obtained from children with oligoarticular JIA, polyarticular JIA, or systemic JIA were compared. Two-dimensional gel electrophoresis for protein separation and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and quadripole time-of-flight mass spectrometry for protein identification were used for this study. Synovial fluid cells were analyzed by polymerase chain reaction (PCR) for the presence of haptoglobin messenger RNA (mRNA). The synovial fluid proteome of the samples was delineated. The majority of proteins showed overexpression in JIA synovial fluid as compared with noninflammatory control samples. There were 24 statistically significantly differentially expressed spots (>2-fold change; P < 0.05) between the subtypes of JIA. PCR analysis revealed haptoglobin mRNA, suggesting that haptoglobin is locally produced in an inflamed joint in JIA. Despite the similar histologic appearance of inflamed joints in patients with different subtypes of JIA, there are differences in protein expression according to the subtype of JIA. Haptoglobin is differentially expressed between the subtypes of JIA and is locally produced in an inflamed joint in JIA. Haptoglobin and other differentially expressed proteins may be potential biomarkers in JIA.

Relationship between impaired apoptosis of lymphocytes and distribution of dendritic cells in peripheral blood and synovial fluid of children with juvenile idiopathic arthritis

Introduction:The pathogenesis of juvenile idiopathic arthritis (JIA) is not fully understood. Recently the present authors described disturbed apoptosis of JIA lymphocytes in both peripheral blood (PB) and synovial fluid (SF) as well as an abnormal distribution of blood dendritic cells (BDCs) between the PB and SF in this disease. Possible relationships between these events during the development of JIA process are assessed here.Materials and Methods:Lymphocyte apoptosis and BDC counts were assessed in the PB and SF of untreated JIA children. Lymphocyte apoptosis was analyzed by the Annexin-V/propydium iodide assay. Total DC (TDC) number was based on the sum of three BDC subpopulations determined using a panel of monoclonal antibodies against BDC antigens (BDCA): myeloid type 1 (mDC1, BDCA-1+/HLA-DR+/CD19-), myeloid type 2 (mDC2, BDCA-3+/HLA-DR+/CD14-), and plasmacytoid (pDC, BDCA-2+/HLA-DR+/CD123+). Cells were enumerated by the flow cytometric “single-platform” method. The concentration of tumor necrosis factor (TNF)-α and the distribution of particular lymphocyte subtypes in both PB and SF were also investigated.Results:There was significant positive correlation between apoptosis of PB lymphocytes and SF TDC count (p=0.002) as well as SF TNF-α concentration (p=0.007). SF TNF-α levels also correlated with SF TDC count (p=0.003). Moreover, JIA SF was distinctly enriched with CD4+ and CD8+ T lymphocytes and included CD4+/CD25high cells as well. There was significant positive correlation between the number of CD4+/CD25high cells and SF JIA BDC count (p=0.015).Conclusions:These data suggest a possible link between impaired apoptosis of PB/SF lymphocytes and increased recruitment of PB BDCs to SF and other elements of the immune system in JIA, including regulatory CD4+/CD25high cells.

Distribution and clinical significance of blood dendritic cells in children with juvenile idiopathic arthritis

Background:A role for dendritic cells (DC) in the development of adult rheumatoid arthritis has been suggested. To date, this problem has been poorly explored in juvenile idiopathic arthritis (JIA).Objective:To analyse...

Levels of matrix metalloproteinase (MMP)-1 in paired sera and synovial fluids of juvenile idiopathic arthritis patients: relationship to inflammatory activity, MMP-3 and tissue inhibitor of metalloproteinases-1 in a longitudinal study

To measure levels of the collagenases matrix metalloproteinase (MMP)-1 and -13 in the synovial fluid (SF) and serum of patients with juvenile idiopathic arthritis (JIA), and to correlate these measurements with inflammatory activity, levels of the collagenase activator MMP-3 and the tissue inhibitor of metalloproteinases-1 (TIMP-1). Levels of MMP-1, -3, -13 and TIMP-1 were measured in paired SF and serum from 82 JIA patients using enzyme-linked immunsorbent assay and compared between subtypes and patients of different ages and disease durations. These levels were also correlated to the active joint count (AJC) and standard measures of inflammatory activity and therapeutic response, including erythrocyte sedimentation rate (ESR) and platelet count (PLT). MMP-1 was detected in JIA SF and correlated with PLT. MMP-3 levels were high in SF and detectable in serum where they correlated with PLT, ESR and AJC. MMP-13, however, was not detected in SF or serum. No differences were observed between patients grouped by subtype, age or disease duration. MMP-3 contributed the majority of total MMP in SF samples resulting in excess MMP levels over TIMP-1. MMP-1 is up-regulated in SF concordant with inflammatory activity in JIA. This was true for patients in all JIA subtypes and age groups, suggesting that the capability for degradation of type II collagen is present in early disease, and throughout the disease course. MMP-3 may be important in the activation of collagenases and the saturation of exogenous inhibitors. Serum MMP-3 may therefore be a useful, measurable and specific marker of active disease in JIA.

Regulation of peripheral blood and synovial fluid lymphocyte apoptosis in juvenile idiopathic arthritis

Objective: Complex regulatory mechanisms are involved in the induction of apoptosis. Their impairment may play a role in the pathogenesis of several autoimmune diseases. Recently, we have described higher incidences of spontaneous apoptosis of peripheral blood (PB) lymphocytes in children with juvenile idiopathic arthritis (JIA). This study aimed to evaluate the regulatory mechanisms that may be responsible for this phenomenon.Methods: Thirty‐four JIA children were examined and compared with 20 healthy children of similar ages. Expression of regulatory proteins p53, Bax and Bcl‐2 in lymphocytes isolated from PB and synovial fluid (SF) was assessed. Serum and SF levels of interleukin‐15 (IL‐15) were also evaluated.Results: The study showed significantly decreased Bcl‐2 expression in JIA PB lymphocytes, compared to both healthy control (p=0.03) and JIA SF lymphocytes (p=0.005). There were no significant differences found in Bax expression between groups or compartments examined. However, the Bax/Bcl‐2 ratio was nearly two‐fold higher in PB lymphocytes than in SF of JIA patients (p=0.001). p53 expression in PB lymphocytes from both JIA and control children did not statistically differ. In JIA, however, p53 was significantly higher in PB than SF lymphocytes (p=0.016). IL‐15 levels were about 20‐fold higher in JIA SF than in serum from either JIA or healthy children (p<0.0001).Conclusion: The results suggest that a higher incidence of apoptosis of PB lymphocytes observed in JIA may be associated with down‐regulation of Bcl‐2, rather than with changes in expression of Bax and p53. In contrast, the low p53 expression and elevated IL‐15 appear to provide mechanisms responsible for suppression of apoptosis in SF cells from JIA patients.