OBJECTIVETo explore the pathogenesis of rheumatoid arthritis (RA) by studying the expression of T cell receptors (TCRs).

METHODST cell receptor Vbeta (TCR Vbeta) gene usage and expression were analyzed from synovial membrane and peripheral blood of 8 RA patients, 2 osteoarthritis patients and 2 accident amputees. The complementary determining region 3 (CDR3) of 25 TCR Vbeta subfamily genes in unselected T cell populations were amplified semi-quantitatively by reverse transcription-polymerase chain reaction (RT-PCR). The products were further studied by genescan for frequency of Vbeta usage.

RESULTSThe numbers of Vbeta subfamilies expressed by T cells from RA peripheral blood and synovial membrane were not significantly restricted. More importantly, biased Vbeta gene expression in RA synovium was observed and Vbeta6, Vbeta17, and Vbeta22 genes were the predominant subfamilies. It was noteworthy that the expression of Vbeta17 in RA synovium was significantly increased.

CONCLUSIONOur data were consistent with the hypothesis that several antigen or superantigen-driven processes may be involved in the pathogenesis of RA.

Arthritis, Rheumatoid ; genetics ; immunology ; Genes, T-Cell Receptor beta ; Humans ; Synovial Membrane ; metabolism ; T-Lymphocytes ; immunology

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent joint swelling and progressive destruction of cartilage and bone. Current RA treatments are largely empirical in origin and their precise mechanism of action is uncertain. Increasing evidence shows that chronic inflammatory diseases such as RA are caused by prolonged production of proinflammatory cytokines including tumor necrosis factor (TNF) and interleukin 1 (IL-1). The nuclear factor kappaB (NF-kappaB) plays an essential role in transcriptional activation of TNF and IL-1. NF-kappaB is induced by many stimuli including TNF and IL-1, forming a positive regulatory cycle that may amplify and maintain RA disease process. NF-kappaB and enzymes involved in its activation can be a target for anti-inflammatory treatment. Aspirin and sodium salicylate inhibit activation of NF-KB by blocking IkappaB kinase, a key enzyme in NF-kappaB activation. Glucocorticoids suppress expression of inflammatory genes by binding glucocorticoid receptor with NF-kappaB, and increasing expression of inhibitory protein of NF-kappaB, IkappaBalpha. Sulfasalazine and gold compounds also inhibit NF-kappaB activation. Continuing advances in our understanding of action mechanism of antirheumatic agents will benefit the future development of RA regimens with greater efficacy and less toxicity.
Animal ; Antirheumatic Agents/therapeutic use* ; Arthritis, Rheumatoid/therapy* ; Arthritis, Rheumatoid/metabolism ; Arthritis, Rheumatoid/immunology ; Cytokines/immunology ; Cytokines/genetics ; Gene Expression Regulation ; Human ; Macrophages/immunology ; NF-kappa B/metabolism* ; NF-kappa B/immunology ; NF-kappa B/biosynthesis ; Tumor Necrosis Factor/genetics

We have used a surface plasmon resonance biosensor (SPR, BIACORE 2000) to detect antibodies against glucose 6-phosphate isomerase (GPI) in synovial fluids of rheumatoid arthritis (RA) and osteoarthritis (OA). Recombinant human GPI proteins fused with or without NusA were expressed in E. coli, purified to homogeneity and immobilized in flow cells of CM5 sensor chips. The flow cells immobilized with NusA protein or bovine serum albumin were used to monitor non-specific binding. Synovial fluid samples from RA patients showed a significantly higher level of binding to recombinant GPI proteins than samples from OA patients. Proteins which bound to the recombinant GPI proteins were confirmed to be immunoglobulin through the administration of anti-human immunoglobulin. NusA fusion protein was excellent for this assay because of a low background binding activity in the SPR analysis and its advantage of increased solubility in recombinant protein production. These results suggested a useful utilization of recombinant NusA-GPI fusion protein for the detection of autoantibodies against GPI in RA patients.
Aged ; Antibodies/*immunology ; Arthritis, Rheumatoid/*immunology ; Female ; Glucose-6-Phosphate Isomerase/genetics/*immunology/metabolism ; Human ; Male ; Middle Aged ; Osteoarthritis/immunology ; Peptide Elongation Factors/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Surface Plasmon Resonance ; Synovial Fluid/*immunology ; Transcription Factors/genetics/metabolism

Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2alpha causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2alpha on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2alpha-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-alpha treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2alpha-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2alpha-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-alpha-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-alpha neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2alpha-induced upregulation of specific receptors for TNF-alpha (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2alpha, and may suggest potential new anti-arthritis therapies.
Animals ; Arthritis/genetics/*immunology/pathology ; Arthritis, Rheumatoid/genetics/immunology/pathology ; Basic Helix-Loop-Helix Transcription Factors/genetics/*immunology ; Cells, Cultured ; Chondrocytes/immunology/metabolism/*pathology ; Coculture Techniques ; Fibroblasts/immunology/metabolism/*pathology ; Gene Expression Regulation ; Interleukin-6/genetics/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Osteoarthritis/genetics/immunology/pathology ; Synovial Membrane/immunology/metabolism/*pathology ; Tumor Necrosis Factor-alpha/genetics/*immunology ; Up-Regulation

UNLABELLEDTo investigate the effect of sinomenine on the expression of chemokines and chemokine receptors of dendritic cells in patients with rheumatoid arthritis in vitro.

METHODSThe peripheral blood mononuclear cells isolated from 8 patients with rheumatoid arthritis were induced to differentiate into dendritic cells with GM-CSF and IL-4. The dendritic cells were exposed to sinomenine at high (5 mmol/L), moderate (2 mmol/L), and low (1 mmol/L) concentrations or treated with the control medium. The expression of CCR5 and CCR7 on the surface of the dendritic cells were measured by flow cytometry, and the CCR5 and CCR7 mRNA expressions were detected by semi-quantitative PCR. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expressions of CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (ITAC).

RESULTSCompared with the control cells, the dendritic cells treated with sinomenine, especially at high and moderate concentrations, showed significantly lowered mRNA and protein expressions of CCR5 and CCR7. Similar results were observed in the expressions of CXCL9 (MIG) and CXCL10 (IP-10), but not in CXCL11 (ITAC).

CONCLUSIONSinomenine produces therapeutic effect on rheumatoid arthritis possibly by inhibiting the expression of chemokines and chemokine receptors in the dendritic cells to suppress the chemotactic migration of the dendritic cells.

Adult ; Arthritis, Rheumatoid ; drug therapy ; immunology ; metabolism ; Chemokines ; genetics ; metabolism ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Middle Aged ; Morphinans ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; Receptors, Chemokine ; metabolism

OBJECTIVETo compare the levels of STAT4 tyrosine phosphorylation in peripheral T-lymphocytes induced by IL-12 in rheumatoid arthritis (RA) and osteoarthritis (OA).

METHODSFrom May 2007 to August 2009, peripheral blood mononuclear cells (PBMCs) were isolated from RA patients [RA group, all the cases were female, the age was from 28 to 55 years with an average of (45.0 +/- 13.0) years] and OA patients [OA group, all the cases also were female; the age was from 55 to 75 years with an average of (67.0 +/- 9.6) years]. The purity of T-lymphocytes from PBMCs was accredited by flow cytometry. The IL-12 of 50 ng/ml added in T-lymphocytes, the levels of STAT4 tyrosine phosphorylation were detected by western blot after different time intervals (0, 10, 30, 60 min).

RESULTSThe purity of T-lymphocytes were above 91% through diremption and depuration for peripheral blood monouclear cells. The levels of STAT4 tyrosine phosphorylation in T-lymphocytes from RA induced by IL-12 were higher than that from OA in the different times (10, 30, 60 min); after 30 min, its levels from RA and OA achieved to crest value.

CONCLUSIONSTAT4 in peripheral T-lymphocytes of rheumatoid arthritis was more easily to be activated than osteoarthritis.

Adult ; Aged ; Arthritis, Rheumatoid ; immunology ; Female ; Humans ; Interleukin-12 ; pharmacology ; Middle Aged ; Osteoarthritis ; immunology ; Phosphorylation ; Polymorphism, Single Nucleotide ; STAT4 Transcription Factor ; genetics ; metabolism ; T-Lymphocytes ; drug effects ; metabolism ; Tyrosine ; metabolism

B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). High levels of B cell activating factor (BAFF) are detected in autoimmune diseases. BAFF and BAFF receptor (BAFF-R) are expressed in B and T cells of RA synovium. The study was undertaken to identify the NF-kappaB signal pathway involved in the induction of BAFF-R in human B cells. Immunohistochemical staining of NF-kappaB p65, NF-kappaB p50, BAFF, and BAFF-R was performed on sections of synovium from severe and mild RA and osteoarthritis (OA) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from control and RA patients and B cells were isolated from controls. BAFF-R was analyzed by flow cytometry, realtime PCR and confocal staining after treatment with NF-kappaB inhibitors. NF-kappaB p65, NF-kappaB p50, BAFF, and BAFF-R were highly expressed in severe RA synovium relative to mild RA synovium or OA synovium. BAFF-R expression was reduced by NF-kappaB inhibitors in PBMCs and B cells from normal controls. We also showed reduction in expression of BAFF-R via inhibition of the NF-kappaB pathway in PBMCs of RA patients. BAFF/BAFF-R signaling is an important mechanism of pathogenesis in RA and that BAFF-R reduction by NF-kappaB blocking therapy is another choice for controlling B cells in autoimmune diseases such as RA.
Arthritis, Rheumatoid/genetics/*metabolism/pathology/physiopathology ; B-Cell Activating Factor/genetics/metabolism ; B-Cell Activation Factor Receptor/genetics/*metabolism ; B-Lymphocytes/*drug effects/immunology/metabolism/pathology ; Cell Separation ; Cells, Cultured ; Disease Progression ; Enzyme Inhibitors/pharmacology ; Flow Cytometry ; Gene Expression Regulation/immunology ; Humans ; Immunohistochemistry ; NF-kappa B/*metabolism ; Signal Transduction/immunology ; Synovial Membrane/*pathology ; T-Lymphocytes/drug effects/immunology/metabolism/pathology ; Transcriptional Activation/drug effects

OBJECTIVETo construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries.

METHODSPeripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR. The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry.

RESULTSThe libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain (IgG1) were constructed. The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry. With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%, respectively, as shown by DNA sequence analysis of the randomly selected clones, the libraries showed an expressible combinatory diversity of 6.13×10(10).

CONCLUSIONThe constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.

Amino Acid Sequence ; Animals ; Antibodies ; genetics ; immunology ; Antibody Specificity ; Arthritis, Rheumatoid ; immunology ; Cell Surface Display Techniques ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; HEK293 Cells ; Humans ; Immunoglobulin G ; biosynthesis ; genetics ; Immunoglobulin Heavy Chains ; biosynthesis ; genetics ; Immunoglobulin kappa-Chains ; biosynthesis ; genetics ; Lymphocytes ; immunology ; metabolism ; Molecular Sequence Data ; Peptide Library ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Transfection

To select candidate genes, we attempted to comparative analysis of protein levels between rheumatoid arthritis (RA) patients and healthy controls by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). We identified 17 proteins that showed up- or down-regulated spots in RA patients. We found that coactosin-like1 (COTL1) were highly expressed in RA patients compared with healthy controls. We performed a case-control study to determine whether the COTL1 gene polymorphisms were associated with RA and systemic lupus erythematosus (SLE). The genotype frequency of c.-1124G>T and the allelic frequency of c.484G>A in RA patients, and the genotype frequency of c.484G>A in SLE patients were significantly different from healthy controls (P = 0.009, 0.027, and 0.025, respectively). We also investigated the correlation with the levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody in RA patients, and anti-nuclear antibodies (ANA) in SLE patients. The c.484G>A polymorphism in RA patients has significant association with the levels of anti-CCP antibody (P = 0.03). Our findings demonstrated that c.-1124G>T and c.484G>A polymorphisms of the COTL1 gene might be associated with the genetic susceptibility of autoimmune disorders.
Arthritis, Rheumatoid/*genetics/immunology/metabolism ; Autoimmune Diseases/genetics/immunology ; Case-Control Studies ; Electrophoresis, Gel, Two-Dimensional ; Genotype ; Humans ; Lupus Erythematosus, Systemic/genetics/immunology ; Microfilament Proteins/*genetics/metabolism ; Polymorphism, Genetic/*genetics ; Proteome/genetics ; Proteomics/*methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

To select candidate genes, we attempted to comparative analysis of protein levels between rheumatoid arthritis (RA) patients and healthy controls by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). We identified 17 proteins that showed up- or down-regulated spots in RA patients. We found that coactosin-like1 (COTL1) were highly expressed in RA patients compared with healthy controls. We performed a case-control study to determine whether the COTL1 gene polymorphisms were associated with RA and systemic lupus erythematosus (SLE). The genotype frequency of c.-1124G>T and the allelic frequency of c.484G>A in RA patients, and the genotype frequency of c.484G>A in SLE patients were significantly different from healthy controls (P = 0.009, 0.027, and 0.025, respectively). We also investigated the correlation with the levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody in RA patients, and anti-nuclear antibodies (ANA) in SLE patients. The c.484G>A polymorphism in RA patients has significant association with the levels of anti-CCP antibody (P = 0.03). Our findings demonstrated that c.-1124G>T and c.484G>A polymorphisms of the COTL1 gene might be associated with the genetic susceptibility of autoimmune disorders.
Arthritis, Rheumatoid/*genetics/immunology/metabolism ; Autoimmune Diseases/genetics/immunology ; Case-Control Studies ; Electrophoresis, Gel, Two-Dimensional ; Genotype ; Humans ; Lupus Erythematosus, Systemic/genetics/immunology ; Microfilament Proteins/*genetics/metabolism ; Polymorphism, Genetic/*genetics ; Proteome/genetics ; Proteomics/*methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization