transforming-growth-factor-beta and Arthritis--Juvenile

Joint inflammation in juvenile rheumatoid arthritis (JRA) is sometimes associated with an autoimmune response to type II collagen (CII), a cartilage-specific protein. To test the hypothesis that down-regulation of autoimmunity to CII can be accomplished in JRA by oral administration of CII, an open-label study of CII was performed in 9 patients with JRA.. Seven rheumatoid factor-negative JRA patients with polyarticular disease and 2 JRA patients with pauciarticular disease (1 with early onset and 1 with late onset) were treated for 3 months with oral bovine CII. Patients were examined for disease activity and underwent routine laboratory testing at monthly intervals. Two of the patients had flares of disease when treatment was discontinued, and these patients were re-treated for an additional 3 months. To test the hypothesis that oral tolerance induces an immune deviation of T cells, peripheral blood mononuclear cells from patients were collected before and after treatment and cultured with CII. Supernatants and RNA were collected and analyzed for the presence of various cytokines.. Eight patient trials met the criteria for clinical improvement outlined by Giannini and coworkers in 1997. None of the patients had any side effects from the treatment. In 6 of the 8 patients who improved, interferon-gamma production decreased after oral CII therapy, correlating with clinical improvement, while 6 patients had increases in levels of transforming growth factor beta3.. These results are encouraging. The possible beneficial effect of oral CII in JRA merits further investigation.

Topics: Administration, Oral; Adolescent; Arthritis, Juvenile; Autoantigens; Autoimmunity; Cells, Cultured; Child; Child, Preschool; Collagen; Cytokines; Female; Humans; Interferon-gamma; Male; RNA, Messenger; T-Lymphocytes; Transforming Growth Factor beta; Treatment Outcome

Topics: Antirheumatic Agents; Arthritis, Juvenile; Epigenesis, Genetic; Humans; Transforming Growth Factor beta

Juvenile Idiopathic Arthritis (JIA) is the most prevalent chronic pediatric rheumatic disorder. In joints of JIA patients, aggressive phenotypic changes in fibroblast-like synoviocytes (FLS) of the synovial lining play a key role in inflammation. MicroRNAs are dysregulated in rheumatoid arthritis and JIA, including miR-27a-3p. However, it is not understood if miR-27a-3p, enriched in JIA synovial fluid (SF) and leukocytes, alters FLS function.. Primary JIA FLS cells were transfected with a miR-27a-3p mimic or a negative control microRNA (miR-NC) and stimulated with pooled JIA SF or inflammatory cytokines. Viability and apoptosis were analyzed by flow cytometry. Proliferation was evaluated using a. MiR-27a-3p was constitutively expressed in FLS. Overexpression of miR-27a-3p caused increased interleukin-8 secretion in resting FLS, and interleukin-6 was elevated in SF-activated FLS compared to miR-NC. Furthermore, stimulation with pro-inflammatory cytokines augmented FLS proliferation in miR-27a-3p-transfected FLS relative to miR-NC. Expression of multiple TGF-β pathway genes was modulated by overexpression of miR-27a-3p.. MiR-27a-3p significantly contributes to FLS proliferation and cytokine production, making it a potential candidate for epigenetic therapy that targets FLS in arthritis.

Topics: Arthritis, Juvenile; Cytokines; Fibroblasts; Humans; MicroRNAs; Phenotype; Synoviocytes; Transforming Growth Factor beta

This study was designed to investigate the pathogenic contributions of fibroblast-like synoviocytes (FLS) to juvenile idiopathic arthritis (JIA) by identifying pathways with dysregulated gene expression in FLS from patients with oligoarticular JIA.. FLS were derived from synovial fluid obtained by arthrocentesis from patients with JIA undergoing intraarticular steroid injections and from orthopedic control patients. Gene expression profiles of the JIA and control FLS were obtained using the Affymetrix platform, with application of Ingenuity Pathway Analysis and Gene Set Enrichment Analysis software to define gene sets in dysregulated pathways and networks of potential pathologic relevance in this disease. Biologically relevant differentially expressed genes were confirmed by RNA and protein analysis.. Exploration of global gene expression profiles of the JIA FLS revealed important dysregulated pathways, including the transforming growth factor β (TGFβ) signaling, as well as endochondral bone formation, cartilage formation, and β-catenin networks. Importantly, bone morphogenetic protein 4 (BMP-4) was significantly overexpressed in the JIA FLS. FLS from patients with oligoarticular JIA exhibit a chondrocyte phenotype, as evidenced by expression of type II collagen and aggrecan.. Dysregulation of the pathways involved in the pathogenesis of oligoarticular JIA were revealed through gene expression profiling. JIA FLS displayed dysregulated TGFβ signaling and exhibited a hypertrophic chondrocyte phenotype. These characteristics, along with contributions from the β-catenin network may have implications for endochondral bone formation and local growth disturbances in oligoarticular JIA. Overexpression of BMP-4 in FLS from patients with oligoarticular JIA in particular may play an important role in disease pathogenesis, with a direct effect on functional outcome and with implications for future treatment.

Topics: Adolescent; Aggrecans; Arthritis, Juvenile; beta Catenin; Bone Morphogenetic Protein 4; Case-Control Studies; Cell Differentiation; Child; Child, Preschool; Chondrocytes; Collagen Type II; Female; Gene Expression Profiling; Humans; Hyperostosis; Male; Osteogenesis; Phenotype; Signal Transduction; Synovial Fluid; Synovial Membrane; Transforming Growth Factor beta

In several murine models of autoimmune arthritis, Th17 cells are the dominant initiators of inflammation. In human arthritis the majority of IL-17-secreting cells within the joint express a cytokine phenotype intermediate between Th17 and Th1. Here we show that Th17/1 cells from the joints of children with inflammatory arthritis express high levels of both Th17 and Th1 lineage-specific transcription factors, RORC2 and T-bet. Modeling the generation of Th17/1 in vitro, we show that Th17 cells "convert" to Th17/1 under conditions that mimic the disease site, namely low TGFbeta and high IL-12 levels, whereas Th1 cells cannot convert to Th17. Th17/1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin between Th17 and Th17/1 cells in arthritis. Using CD161, a lectin-like receptor that is a marker of human Th17, we show synovial Th17 and Th17/1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. We provide evidence to support a Th17 origin for Th1 cells expressing CD161. In vitro, Th17 cells that convert to a Th1 phenotype maintain CD161 expression. In the joint CD161+ Th1 cells share features with Th17 cells, with shared TCR clonality, expression of RORC2 and CCR6 and response to IL-23, although they are IL-17 negative. We propose that the Th17 phenotype may be unstable and that Th17 cells may convert to Th17/1 and Th1 cells in human arthritis. Therefore therapies targeting the induction of Th17 cells could also attenuate Th17/1 and Th1 effector populations within the inflamed joint.

Topics: Amino Acid Sequence; Arthritis, Juvenile; Base Sequence; Cell Lineage; Child; Flow Cytometry; Gene Expression; Humans; Interferon-gamma; Interleukin-12; Interleukin-17; Molecular Sequence Data; NK Cell Lectin-Like Receptor Subfamily B; Nuclear Receptor Subfamily 1, Group F, Member 3; Receptors, CCR6; Reverse Transcriptase Polymerase Chain Reaction; T-Box Domain Proteins; T-Lymphocytes, Helper-Inducer; Th1 Cells; Transforming Growth Factor beta

A Th1 biased immune response in synovial fluid has been reported in children with polyarticular and extended oligoarticular-type juvenile idiopathic arthritis (JIA). We investigated T cell phenotypes including Th1, Th2, Th17, and Treg with emphasis on Th17 and Treg, in order to differentiate cytokines in the enthesitis-related arthritis (ERA) form of JIA.. The frequencies of Th1, Th2, Th17, and Treg cells were determined by flow cytometry in peripheral blood (PB) and synovial fluid from patients with ERA and healthy subjects. Levels of interleukin 1ss (IL-1ss), IL-6, IL-21, IL-23, and transforming growth factor ss (TGF-ss), cytokines that influence Th17 lineage cells, were measured in paired plasma and synovial fluid (SF) samples by ELISA. Frequencies are expressed as percentages and cytokine levels as pg/ml.. There were no differences in blood samples in the frequency of Th1, Th2, Th17, and Treg cells between patients and controls. In paired samples, the median frequency of CD4+IFN-gamma+ (20.49 vs 4.03; p < 0.005) and CD4+IL-17+ (2.27 vs 0.57; p < 0.01) cells was significantly higher in SF compared to PB, respectively; whereas the frequency of CD4+IL-4+ (1.79 vs 2.29; p < 0.04) cells was significantly reduced in the SF compared to PB. There was no difference in the frequency of regulatory T cells. Patients receiving methotrexate had fewer Th2 cells, whereas the Childhood Health Assessment Questionnaire score had a negative association with the frequency of Treg. Median levels of IL-1ss (p < 0.008), IL-6 (p < 0.0001), and IL-17 (p < 0.0001) were higher in SF than in plasma and levels of TGF-ss were lower (p < 0.001). Levels of IL-21 were similar in SF and plasma, whereas IL-23 was undetectable.. In patients with ERA, peripheral blood Th1, Th2, Th17, and Treg cells were unchanged, but Th1 and Th17 cells were increased and Th2 cells were reduced in the SF compared to blood. Elevated IL-1ss and IL-6 in SF may be responsible for increased Th17 cells.

Topics: Adolescent; Adult; Arthritis, Juvenile; Cell Lineage; Child; Flow Cytometry; Humans; Immunophenotyping; Interleukin-17; Interleukin-1beta; Interleukin-23; Interleukin-6; Interleukins; Male; Synovitis; T-Lymphocytes, Regulatory; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Young Adult

To determine whether peripheral blood mononuclear cells (PBMCs) from children with recent-onset polyarticular juvenile idiopathic arthritis (JIA) exhibit biologically or clinically informative gene expression signatures.. Peripheral blood samples were obtained from 59 healthy children and 61 children with polyarticular JIA prior to treatment with second-line medications, such as methotrexate or biologic agents. RNA was extracted from isolated mononuclear cells, fluorescence labeled, and hybridized to commercial gene expression microarrays (Affymetrix HG-U133 Plus 2.0). Data were analyzed using analysis of variance at a 5% false discovery rate threshold after robust multichip analysis preprocessing and distance-weighted discrimination normalization.. Initial analysis revealed 873 probe sets for genes that were differentially expressed between polyarticular JIA patients and healthy controls. Hierarchical clustering of these probe sets distinguished 3 subgroups within the polyarticular JIA group. Prototypical patients within each subgroup were identified and used to define subgroup-specific gene expression signatures. One of these signatures was associated with monocyte markers, another with transforming growth factor beta-inducible genes, and a third with immediate early genes. Correlation of gene expression signatures with clinical and biologic features of JIA subgroups suggested relevance to aspects of disease activity and supported the division of polyarticular JIA into distinct subsets.. Gene expression signatures in PBMCs from patients with recent-onset polyarticular JIA reflect discrete disease processes and offer a molecular classification of disease.

Topics: Adolescent; Antibodies, Antinuclear; Antirheumatic Agents; Arthritis; Arthritis, Juvenile; Case-Control Studies; Child; Child, Preschool; Female; Gene Expression Profiling; Genes, Immediate-Early; Humans; Leukocytes, Mononuclear; Male; Methotrexate; Multigene Family; Rheumatoid Factor; Transforming Growth Factor beta

The transcription factor forkhead box P3 (FOXP3 in humans; Foxp3 in mice) controls the development and function of regulatory T cells (Treg). In mice, CD4(+)CD25(-) T cells do not express Foxp3 following TCR activation. Whether FOXP3 is a common activation-induced molecule in human T cells--hence not Treg restricted--is currently a controversial issue. As FOXP3 can significantly modulate the function of T cells, understanding the mode (and regulation) of FOXP3 expression in human T cells is vital. Here we show that in conventional CD4(+)CD25(-) T cells, the induction of FOXP3 expression following TCR activation is both restricted to a fraction of the progeny and transient. Moreover, FOXP3 expression in vivo is particularly infrequent in activated effector CD4(+) T cells that accumulate within inflamed joints. We next demonstrate that the repression of FOXP3 transcription in resting conventional human CD25(-) T cells is linked to complete methylation of an evolutionarily conserved intronic CpG island. The dense methylation pattern is furthermore inherited after activation by progeny. This intronic CpG island, on the other hand, is frequently unmethylated in CD4(+)CD25(+) T cells. Importantly, blocking maintenance DNA methylation, by pharmacological inhibition of DNA methyltransferase-1, induced significant and stable activation-dependent FOXP3 expression in cycling conventional T cells, which was further amplified by co-treatment with transforming growth factor beta. In contrast to natural Treg, such induced CD4(+)FOXP3(+) T cells could produce pro-inflammatory cytokines upon activation. These results indicate that DNA methylation normally restricts FOXP3 transcription in conventional human T cells.

Topics: Arthritis, Juvenile; Azacitidine; CD4-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Decitabine; DNA Methylation; DNA Modification Methylases; Female; Forkhead Transcription Factors; Gene Expression Regulation; Humans; Interleukin-2 Receptor alpha Subunit; Lymphocyte Activation; Male; Synovial Fluid; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Single nucleotide polymorphisms (SNPs) in cytokine genes have been associated with risk of a number of autoimmune diseases. Moreover, some SNPs are associated with variations in rates of in vitro gene expression, and it is therefore possible that these functional polymorphisms may differentially affect inflammatory processes and disease outcome. This project's objective was to determine whether cytokine genotypes correlate with disease outcomes in patients with juvenile rheumatoid arthritis (JRA).. Genotypes of SNPs of pro-inflammatory cytokines, tumour necrosis factor-alpha -308G -->A, interleukin-6 (IL-6) -174G -->C and interferon-gamma +874G -->A, and anti-inflammatory, immunosuppressive cytokines, interleukin-10 -1082G -->A, -819C -->T and -592A -->C and transforming growth factor-beta1 (TGF-beta1) codon 10T -->C and codon 25G -->C, were determined for patients with JRA who previously participated in a long-term outcome study. Cytokine genotypes and clinical variables showing significant correlations with clinical outcomes at the alpha = 0.100 level in univariate analyses were entered in multivariate tests.. In multivariate tests, the IL-6 genotype -174G/G was positively correlated with pain [regression coefficient B = 0.899, 95% confidence intervals (CI) 0.185, 1.612, P = 0.014]. The homozygous TGF-beta1 codon 25G/G genotype showed a protective effect against joint space narrowing on radiographs taken within 2 yr of disease onset, but confidence intervals were wide [odds ratio (OR) 0.176, 95% CI 0.037, 0.837 P = 0.029].. The correlation of IL-6 genotype with pain and the possible association of the TGF-beta1 codon 25 genotype with short-term radiographic damage (G/C with greater risk and G/G with decreased risk) suggests that both these polymorphisms may be useful early prognostic indicators. Further studies of the relation between cytokine genotypes and outcomes in patients with all forms of juvenile idiopathic arthritis (JIA) are warranted.

Topics: Adolescent; Adult; Age of Onset; Arthritis, Juvenile; Child; Cytokines; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Interleukin-6; Male; Multivariate Analysis; Pain; Polymorphism, Single Nucleotide; Prognosis; Radiography; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study investigates the role of CD4(+)CD25(+) regulatory T cells during the clinical course of juvenile idiopathic arthritis (JIA). Persistent oligoarticular JIA (pers-OA JIA) is a subtype of JIA with a relatively benign, self-remitting course while extended oligoarticular JIA (ext-OA JIA) is a subtype with a much less favorable prognosis. Our data show that patients with pers-OA JIA display a significantly higher frequency of CD4(+)CD25(bright) T cells with concomitant higher levels of mRNA FoxP3 in the peripheral blood than ext-OA JIA patients. Furthermore, while numbers of synovial fluid (SF) CD4(+)CD25(bright) T cells were equal in both patient groups, pers-OA JIA patients displayed a higher frequency of CD4(+)CD25(int) T cells and therefore of CD4(+)CD25(total) in the SF than ext-OA JIA patients. Analysis of FoxP3 mRNA levels revealed a high expression in SF CD4(+)CD25(bright) T cells of both patient groups and also significant expression of FoxP3 mRNA in the CD4(+)CD25(int) T cell population. The CD4(+)CD25(bright) cells of both patient groups and the CD4(+)CD25(int) cells of pers-OA JIA patients were able to suppress responses of CD25(neg) cells in vitro. A markedly higher expression of CTLA-4, glucocorticoid-induced TNFR, and HLA-DR on SF CD4(+)CD25(bright) T regulatory (Treg) cells compared with their peripheral counterparts suggests that the CD4(+)CD25(+) Treg cells may undergo maturation in the joint. In correlation with this mature phenotype, the SF CD4(+)CD25(bright) T cells showed an increased regulatory capacity in vitro compared with peripheral blood CD4(+)CD25(bright) T cells. These data suggest that CD4(+)CD25(bright) Treg cells play a role in determining the patient's fate toward either a favorable or unfavorable clinical course of disease.

Topics: Adolescent; Adult; Arthritis, Juvenile; Biomarkers; Cartilage, Articular; CD4-Positive T-Lymphocytes; Cell Differentiation; Child; Child, Preschool; DNA-Binding Proteins; Female; Forkhead Transcription Factors; Humans; Interferon-gamma; Interleukin-10; Interleukin-2; Leukocytes, Mononuclear; Lymphocyte Count; Male; Receptors, Interleukin-2; Remission, Spontaneous; RNA, Messenger; Synovial Fluid; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

The aim of the present study was to investigate the patterns of cytokine production by T cell clones raised from in vivo activated synovial fluid (SF) mononuclear cells (MNC) of five patients with oligoarticular juvenile arthritis (JA). Freshly isolated SF T cells were cultured in vitro with low dose recombinant IL-2 and subsequently cloned by limiting dilution. Sixty-four clones were obtained from the five patients studied. Fifty-nine clones were TCR alpha/beta+, either CD4+ (n = 43) or CD8+ (n = 15). The remaining five clones were TCR gamma/delta+, CD4-, CD8-. Clone immunophenotypes differed in the individual patients. Forty-four T cell clones were stimulated with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) and supernatants tested for the presence of IL-2, IL-4, IL-5 and interferon-gamma (IFN-gamma) by ELISA or bioassays. Cytokine mRNA accumulation was tested by reverse transcriptase-polymerase chain reaction (RT-PCR). Most of 44 clones tested released large amounts of IFN-gamma irrespective of the immunophenotype. Of these, 27 were classified as Th1-type and 17 as Th0-type based upon the IFN-gamma/IL-4 ratio in culture supernatants. Finally, when 10 representative T cell clones were tested for pro- and anti-inflammatory cytokines, gene expression by RT-PCR, all of them were found to express the granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha), IL-10 and transforming growth factor-beta 1 (TGF-beta1) genes, and half of them IL-6 and IL-8 mRNA. In conclusion, T cell clones, that represent the progeny of in vivo activated SF T cells from oligoarticular JA patients, display heterogeneous immunophenotypes, but all share the ability to produce large amounts of IFN-gamma, with a predominant Th1/Th0 pattern. The expression of pro- and anti-inflammatory cytokine genes in these clones suggests that in vivo activated SF T cells modulate joint inflammation in a complex fashion.

Topics: Adolescent; Arthritis, Juvenile; CD4 Antigens; CD8 Antigens; Cells, Cultured; Child; Clone Cells; Cytokines; Female; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-gamma; Interleukin-2; Interleukins; Leukocytes, Mononuclear; Male; Phytohemagglutinins; Polymerase Chain Reaction; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; Recombinant Proteins; RNA, Messenger; Synovial Fluid; T-Lymphocytes; Tetradecanoylphorbol Acetate; Th1 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To investigate the effect of synovial fluid (SF) from patients with juvenile rheumatoid arthritis (JRA) on proliferation and induction of degradative and invasive phenotype in normal synovial fibroblasts, and to elucidate the contribution of SF cells to this activity.. SF and/or conditioned medium (CM) from SF cells were evaluated for their ability to (1) stimulate a proliferative response, (2) induce the "activated phenotype" capable of invading cartilage matrix, and (3) promote the release of key matrix metalloproteinases (MMP) in normal synovial fibroblasts.. Proliferation of normal synovial fibroblasts exposed to SF or CM from SF cells of patients with JRA was up to 3 times greater than untreated controls. Concomitant with induction of an activated phenotype in the treated synovial fibroblasts, the activated form exhibited up to 250% invasiveness of cartilage matrix compared to untreated synovial fibroblasts (100%), in addition to releasing increased MMP activity, not normally associated with these quiescent cells. This induction was not solely due to tumor necrosis factor-alpha, transforming growth factor-beta, interleukin 1beta (IL-1beta), and IL-6, as SF and/or CM depleted of these cytokines sustained about 40% of their invasive and inducing ability. We observed that the mononuclear cell (MNC) population that infiltrated into the joint cavity secretes this "inducing activity," which can be maintained in culture up to several weeks.. Our data suggest that the cellular component of SF releases soluble factor(s) that directly or indirectly contribute to (a) proliferation of synovial fibroblasts, and (b) production and release of extracellular MMP by synovial fibroblasts, thereby inducing a degradative and invasive phenotype culminating in cartilage and bone destruction.

Topics: Antibodies; Arthritis, Juvenile; Binding, Competitive; Cartilage; Cell Division; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Humans; Immunophenotyping; Interleukin-1; Interleukin-6; Male; Metalloendopeptidases; Monocytes; Neutralization Tests; Synovial Fluid; Synovial Membrane; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The synovial expression of the mucosal lymphocyte integrin alpha E beta 7 and its ligand E-cadherin was analysed in order to study the relationship between T lymphocytes of the gastrointestinal tract and the synovium in patients with rheumatoid arthritis (RA). Immunohistochemical evaluation of synovium revealed that the alpha E beta 7-expression was detectable in 16 of the 38 samples examined. A concomitant examination on circulating lymphocytes by flow cytometry showed that alpha E beta 7-expressing lymphocytes occur less frequently in peripheral blood (PB). In vitro culture of lymphocytes increased the alpha E beta 7-expression on synovial lymphocytes six-fold, whereas PB lymphocytes expressed a two-fold increase. The addition of PHA to the culture medium did not dramatically increase the alpha E beta 7-expression on synovial lymphocytes, in contrast to PB lymphocytes where a 24-fold increase was detected. The addition of TGF-beta 1 to the culture of PB lymphocytes increased the alpha E beta 7-expression three-fold. E-cadherin expression was found in all synovial tissues analysed by immunohistochemistry. These results demonstrate that synovial T lymphocytes have the capacity to express the 'mucosal-type' integrin alpha E beta 7, possibly due to high levels of intra-articular TGF-beta 1. This expression might be of physiological importance since E-cadherin, the ligand for alpha E beta 7, is richly expressed by synoviocytes. In addition, the results indicate that a high in vivo expression of alpha E beta 7 is suppressed in the synovial tissue by a hitherto unknown mechanism.

Topics: Adult; Arthritis, Juvenile; Arthritis, Rheumatoid; Cadherins; Cells, Cultured; Female; Humans; Integrins; Leukocytes, Mononuclear; Ligands; Male; Middle Aged; Synovial Membrane; T-Lymphocytes; Transforming Growth Factor beta