Synovial tissue heterogeneity in rheumatoid arthritis in relation to disease activity and biomarkers in peripheral blood.

OBJECTIVE: To investigate the clinical relevance of synovial tissue subtypes in rheumatoid arthritis (RA) and to search for peripheral blood (PB) markers that may serve as biomarkers for tissue subtypes. METHODS: Gene expression analysis using complementary DNA microarrays was applied on paired synovial tissue biopsy and PB samples obtained from 17 RA patients. Molecular tissue subtypes were correlated with histologic parameters (CD3, CD22, CD38, CD68, CD163, tumor necrosis factor alpha, intercellular adhesion molecule 1, vascular cell adhesion molecule, and E-selectin), disease characteristics, and PB markers. PANTHER classification was used for pathway analysis. RESULTS: Genomic subtyping of high- and low-inflammation rheumatoid synovial tissues based on gene expression profiles exactly matched immunohistochemical classification. The patients with the high-inflammation tissue type had higher Disease Activity Scores in 28 joints, higher C-reactive protein levels, higher erythrocyte sedimentation rates, increased numbers of platelets, and shorter disease durations. Comparative analysis of PB gene expression profiles yielded no statistically significant differences between the 2 tissue groups at the single-gene expression level. PANTHER pathway analysis revealed a significant association of increased protein biosynthesis with high-inflammation tissue. CONCLUSION: High-inflammation tissue is associated with more severe disease and shorter disease duration. While pathway-level analysis revealed that coordinate differential expression of genes involved in protein synthesis in PB is associated with high-inflammation tissue types, differential tissue pathology was not reflected in the PB by differential expression of single genes.

Loss of imprinting of IGF2 characterises high IGF2 mRNA-expressing type of fibroblast-like synoviocytes in rheumatoid arthritis.

OBJECTIVE: Increased expression of insulin-like growth factor 2 (IGF2) by fibroblast-like synoviocytes (FLS) was associated with low inflammatory synovium of patients with rheumatoid arthritis (RA). The aim of this study was to analyse whether the differential expression of IGF2, whose expression is normally restricted to one allele, is due to activation of the normally suppressed allele. METHODS: IGF2 gene expression of RA FLS was quantified by quantitative real-time PCR. FLS heterozygous for a 3'-untranslated region IGF2 polymorphism were selected to measure the relative contribution of the allelic transcripts by allele-specific transcript quantification assay. Proliferation was determined by [(3)H]thymidine incorporation. RESULTS: IGF2 was shown to contribute to RA FLS proliferation. FLS could be classified in IGF2 high and IGF2 low-expressing cell lines. Allelic IGF2 transcript quantification analysis revealed that in part of the RA FLS the normally suppressed allele was activated, resulting in biallelic expression of the IGF2 gene. Biallelic expression was associated with increased levels of IGF2 mRNA production. CONCLUSION: The findings indicate that the imprinting status of IGF2 might underlie the increased expression of IGF2, which may contribute to autonomous growth of RA FLS of low inflammatory synovial tissues.

B lymphocyte autoimmunity in rheumatoid synovitis is independent of ectopic lymphoid neogenesis.

B lymphocyte autoimmunity plays a crucial role in the pathogenesis of rheumatoid arthritis. The local production of autoantibodies and the presence of ectopic lymphoid neogenesis in the rheumatoid synovium suggest that these dedicated microenvironments resembling canonical lymphoid follicles may regulate the initiation and maturation of B cell autoimmunity. In this study, we assessed experimentally the relevance of ectopic lymphoid neogenesis for B cell autoimmunity by a detailed structural, molecular, and serological analysis of seropositive and seronegative human synovitis. We demonstrate that synovial lymphoid neogenesis is a reversible process associated with inflammation which is neither restricted to nor preferentially associated with autoantibody positive rheumatic conditions. Despite the abundant expression of key chemokines and cytokines required for full differentiation toward germinal center reactions, synovial lymphoid neogenesis in rheumatoid arthritis only occasionally progresses toward fully differentiated follicles. In agreement with that observation, we could not detect Ag-driven clonal expansion and affinity maturation of B lymphocytes. Furthermore, ectopic lymphoid neogenesis is not directly associated with local production of anti-citrullinated protein Abs and rheumatoid factor in the rheumatoid joint. Therefore, we conclude that synovial lymphoid neogenesis is not a major determinant of these rheumatoid arthritis-specific autoantibody responses.

Inflammation and ectopic lymphoid structures in rheumatoid arthritis synovial tissues dissected by genomics technology: identification of the interleukin-7 signaling pathway in tissues with lymphoid neogenesis.

OBJECTIVE: In approximately 25% of synovial tissues from rheumatoid arthritis (RA) patients, infiltrates of T cells, B cells, and follicular dendritic cells (FDCs) are spatially organized into structures resembling lymph nodes with germinal centers. The remainder of the tissues lack FDCs and show either a diffuse or an aggregated T cell and B cell infiltrate. To gain more insight into this specific disease process, we sought to identify the genes expressed in RA tissues with ectopic lymphoid structures. METHODS: Gene expression profiling of RA synovial tissues was determined by complementary DNA microarray analysis and quantitative real-time polymerase chain reaction. The presence of lymphoid follicles and localization of interleukin-7 (IL-7) in synovial tissue sections was determined by immunofluorescence staining using specific antibodies. RESULTS: Findings of gene expression analysis confirmed previous reports that tissues with lymphoid structures showed elevated expression of CXCL13, CCL21, CCR7, and lymphotoxin alpha and beta messenger RNA. In addition, the tissues also showed enhanced expression of the chemokines CXCL12 and CCL19 and the associated receptors CXCR4 and CXCR5, which are important for the attraction of T cells, B cells, and dendritic cells. Pathway analysis revealed increased expression of genes involved in JAK/STAT signaling, T cell- and B cell-specific pathways, Fcepsilon receptor type I signaling in mast cells, and IL-7 signal transduction in the tissues with ectopic lymphoid follicles, accompanied by increased expression of IL-7 receptor alpha (IL-7Ralpha)/IL-2Rgamma chains and IL-7. Protein expression of IL-7 in RA tissues was localized within fibroblast-like synoviocytes, macrophages, and blood vessels and was colocalized with extracellular matrix structures around the B cell follicles. CONCLUSION: Activation of the IL-7 pathway may play an important role in lymphoid neogenesis, analogous to its role in the development of normal lymphoid tissue.

Fibroblast-like synoviocytes derived from patients with rheumatoid arthritis show the imprint of synovial tissue heterogeneity: evidence of a link between an increased myofibroblast-like phenotype and high-inflammation synovitis.

OBJECTIVE: Given the heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues, we sought to determine whether this variability was also reflected at the level of the fibroblast-like synoviocyte (FLS) cultured from RA synovial tissues. METHODS: Gene expression profiles in FLS cultured from synovial tissues obtained from 19 RA patients were analyzed using complementary DNA microarrays and hierarchical cluster analysis. To validate the subclassification, we performed prediction analysis and principal components analysis. Genes that differed significantly in their expression between FLS cultures were selected using Statistical Analysis of Microarrays software. Real-time quantitative polymerase chain reaction was performed to validate the microarray data. Immunocytochemistry was applied to study the expression of the genes of interest in FLS and synovial tissues. RESULTS: Hierarchical clustering identified 2 main groups of FLS characterized by distinctive gene expression profiles. FLS from high-inflammation synovial tissues revealed increased expression of a transforming growth factor beta/activin A-inducible gene profile that is characteristic of myofibroblasts, a cell type considered to be involved in wound healing, whereas increased production of growth factor (insulin-like growth factor 2/insulin-like growth factor binding protein 5) appeared to constitute a characteristic feature of FLS derived from low-inflammation synovial tissues. The molecular feature that defines the myofibroblast-like phenotype was reflected as an increased proportion of myofibroblast-like cells in the heterogeneous FLS population. Myofibroblast-like cells were also found upon immunohistochemical analysis of synovial tissue. CONCLUSION: Our findings support the notion that heterogeneity between synovial tissues is reflected in FLS as a stable trait, and provide evidence of a possible link between the behavior of FLS and the inflammation status of RA synovium.