Synovial Fluid Of Rheumatoid Arthritis Patients Research Articles

MicroRNA-146a expresses in interleukin-17 producing T cells in rheumatoid arthritis patients

BackgroundInterleukin (IL)-17 is an important factor in rheumatoid arthritis (RA) pathogenesis. MicroRNA (miRNA)s are a family of non coding RNAs and associated with human diseases including RA. The purpose of this study is to identify the miRNAs in the differentiation of IL-17 producing cells, and analyze their expression pattern in the peripheral blood mononuclear cells (PBMC) and synovium from RA patients.MethodsIL-17 producing cells were expanded from CD4+T cell. MiRNA microarray was performed to identify the miRNAs in the differentiation of IL-17 producing cells. Quantitative polymerase chain reaction was performed to examine the expression patterns of the identified miRNAs in the PBMC and synovium from RA and osteoarthritis (OA) patients. Double staining combining in situ hybridization and immunohistochemistry of IL-17 was performed to analyze the expression pattern of identified miRNA in the synovium.ResultsSix miRNAs, let-7a, miR-26, miR-146a/b, miR-150, and miR-155 were significantly up regulated in the IL-17 producing T cells. The expression of miR-146a and IL-17 was higher than in PBMC in the patients with low score of Larsen grade and short disease duration. MiR-146a intensely expressed in RA synovium in comparison to OA. MiR-146a expressed intensely in the synovium with hyperplasia and high expression of IL-17 from the patients with high disease activity. Double staining revealed that miR-146a expressed in IL-17 expressing cells.ConclusionThese results indicated that miR-146a was associated with IL-17 expression in the PBMC and synovium in RA patients. There is the possibility that miR-146a participates in the IL-17 expression.

Extracellular calreticulin is present in the joints of patients with rheumatoid arthritis and inhibits FasL (CD95L)–mediated apoptosis of T cells

The binding of FasL (CD95L) to its receptor, Fas (CD95), induces apoptosis. Studies have shown that in patients with rheumatoid arthritis (RA), T lymphocytes are resistant to FasL-induced apoptosis in vivo but are susceptible to FasL-induced apoptosis in vitro. Dysfunction in this mechanism may be an important contributor to the pathophysiology of RA. Thus, the present study was undertaken to determine which factors might inhibit FasL-Fas binding in vivo and those that would inhibit apoptosis of T lymphocytes in an in vitro model system. Human Jurkat T cells rendered apoptotic by FasL exposure were analyzed by flow cytometry. Necrosis was determined according to measurement of lactate dehydrogenase release. Quantification of calreticulin in plasma and synovial fluid and of calreticulin-FasL binding was performed by enzyme-linked immunosorbent assay. Measurement of nitrite/nitrate in the plasma and synovial fluid was carried out by chemiluminescence assay. Extracellular calreticulin was present at a significantly higher concentration in the plasma (median 10.3 ng/ml, interquartile range [IQR] 14.8 ng/ml) and synovial fluid (median 10.3 ng/ml, IQR 12.0 ng/ml) of RA patients (each P < 0.05) compared with the plasma (median 3.1 ng/ml, IQR 1.3 ng/ml) and synovial fluid (median 2.9 ng/ml, IQR 0.9 ng/ml) of patients with psoriatic arthritis and the plasma of healthy control subjects (median 2.9 ng/ml, IQR 0.9 ng/ml). Calreticulin concentrations in the synovial fluid correlated with the tender and swollen joint counts and the activity scores on the 28-joint Disease Activity Score assessment. Calreticulin also bound directly to FasL. In vitro, calreticulin (2-16 ng/ml) inhibited FasL-induced apoptosis of Jurkat T cells. Calreticulin was present at higher concentrations in the plasma and synovial fluid of RA patients. Calreticulin had the capacity to bind directly to FasL and to inhibit FasL-mediated apoptosis of Jurkat T cells, and thus might play a role in inhibiting apoptosis of inflammatory T cells in RA.

Synoviocyte-Derived Angiopoietin-Like Protein 2 Contributes to Synovial Chronic Inflammation in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by symmetrical polyarticular synovitis of the diarthrodial joints. Several proinflammatory cytokines derived from both infiltrating inflammatory cells and activated resident cells within the RA joint play a fundamental role in the processes that cause inflammation. However, anticytokine treatment is beneficial but not curative, the effects are only partial, and nonresponses are common. Therefore, an effort has been made to identify other key regulators of inflammation in articular structures to develop new therapies to suppress synovial inflammation and joint destruction in RA. Adipose tissue-derived angiopoietin-like protein 2 (Angptl2) activates an inflammatory cascade in endothelial cells and induces chemotaxis of monocytes/macrophages in obesity, resulting in initiation and propagation of inflammation within adipose tissues and obesity-related metabolic diseases. Angptl2 mRNA and protein are abundantly expressed in hyperplastic rheumatoid synovium of RA patients, especially in fibroblast-like and macrophage-like synoviocytes, but not in B and T lymphocytes. Angptl2 concentration in joints of RA patients was also significantly increased in comparison with patients with osteoarthritis, which in comparison with RA represents a significantly lower inflammatory grade form of arthritis. Notably, Angptl2 promoted increased chemotactic activities of CD14+CD16- monocytes from synovial fluid of RA patients. Therefore, Angptl2 acts as an important rheumatoid synovium-derived inflammatory mediator in RA pathogenesis.

Targeting the function of extracellular cyclophilins as a novel approach for the treatment of Rheumatoid Arthritis (RA). (35.13)

Abstract Elevated levels of extracellular cyclophilins A and B are present in the synovial fluid of RA patients and have been reported to correlate with disease severity. Based on the findings that cyclophilins have potent chemotactic properties for various subsets of pro-inflammatory leukocytes, we propose that extracellular sources of cyclophilins may contribute to the development of RA pathology by promoting leukocyte recruitment into synovial spaces and tissues. Using the collagen-induced arthritis (CIA) mouse model of RA, we tested the impact of treating CIA mice with non immunosuppressive analogs of cyclosporine A (CsA) to inhibit the function of cyclophilins. Our findings demonstrate that treatment with non-immunosuppressive CsA leads to a significant reduction in CIA clinical disease severity, with corresponding reductions in histopathology and levels of pro-inflammatory cytokines and enzymes within the joints. Inhibiting the function of cyclophilin A might provide a novel approach for reducing RA-mediated joint inflammation.

Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

IntroductionRheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients.MethodsIC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining.ResultsCirculating IC in the serum of RA patients and healthy controls contain fibrinogenβ and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogenγ and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogenβ were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients.ConclusionsCitrullinated fibrinogenβ and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA- RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA.

Rheumatoid arthritis and the role of oral bacteria

Rheumatoid arthritis (RA) and periodontal disease (PD) have shown similar physiopathologic mechanisms such as chronic inflammation with adjacent bone resorption in an immunogenetically susceptible host; however, PD has a well-recognized bacterial etiology while the cause of RA is unclear. Some reports have indicated that an infectious agent in a susceptible host could be one possible trigger factor for RA, and it has been suggested that oral microorganisms, specialty periodontal bacteria could be the infectious agent (mainly Porphyromonas gingivalis). It has been reported that PD is more frequent and more severe in patients with RA, suggesting a positive association between both diseases. There have been reports regarding the detection of antibodies against periodontal bacteria while other studies have identified periodontal bacterial DNA in serum and synovial fluid of RA patients and have explored the possible pathways of transport of periodontal bacterial DNA. In conclusion, there is no question that RA and PD have pathologic features in common and there is strong evidence of an association between both diseases, but further studies, including experimental models, are needed to demonstrate the arthritogenicity of oral microorganisms.

A Dual Action of Rheumatoid Arthritis Synovial Fibroblast IL-15 Expression on the Equilibrium between CD4+CD25+ Regulatory T Cells and CD4+CD25− Responder T Cells

We previously described that fibroblast-like cells from the synovium of rheumatoid arthritis patients (RASFib) constitutively express intracellular and surface IL-15, which induces activation of cocultured T cells. Our objective was to study the effect of RASFib IL-15 expression on the function of human CD4(+)CD25(+) regulatory T cells (Treg). RASFib, through their constitutive IL-15 expression, were able to induce the proliferation of human Tregs stimulated through their TCR, and at the same time potentiated their suppressive action on the cytokine secretion of CD4(+)CD25(-) responder T cells (Tresp). In parallel, constitutive RASFib IL-15 expression mediated an up-regulated response of Tresp. Subsequently, total CD4(+) T cells, containing natural proportions of Treg and Tresp, secreted an increased amount of pathogenic cytokines when cocultured with RASFib despite the presence of proliferating Treg with superior regulatory potency. In summary, RASFib IL-15 exerts a dual action on the equilibrium between Treg and Tresp by potentiating the suppressive effect of Treg while augmenting the proinflammatory action of Tresp; the result is a shift of the Treg/Tresp balance toward a proinflammatory state. This alteration of the Treg/Tresp equilibrium is not observed in the presence of osteoarthritis synovial fibroblasts or dermal fibroblasts, which do not constitutively express surface IL-15. Additionally, Treg with superior suppressive potency were present in the peripheral blood and the synovial fluid of RA patients, but this enhanced immunoregulatory activity was not able to overcome the increased secretion of pathogenic cytokines by RA-Tresp, indicating that rheumatoid arthritis patients demonstrate an altered Treg/Tresp equilibrium in vivo.

Vaspin and omentin: new adipokines differentially regulated at the site of inflammation in rheumatoid arthritis

Scientific interest in adipose tissue-derived peptides has increased dramatically in recent years (1). Several mediators known as adipo(cyto)kines were first associated with the pathophysiology of obesity-related complications; however a significant...

Terminal monosaccharide screening of synovial immunoglobulins G and A for the early detection of rheumatoid arthritis

The expressions of some terminal glycotopes of synovial immunoglobulins G, A, and M were analysed in relation to rheumatoid arthritis (RA) progression defined according to early and advanced radiological changes in patients' hands. The relative amounts of terminal monosaccharides were determined by lectin-immunoblotting of immunoglobulin preparations using appropriate lectins able to recognize alpha2,6-linked (Sambucus nigra agglutinin) and alpha2,3-linked (Maackia amurensis agglutinin) sialic acid, galactose (Ricinus communis agglutinin I), N-acetylglucosamine (Griffonia simplicifolia agglutinin II) as well as alpha1,6-linked (Aleuria aurantia lectin), alpha1,3-linked (Lotus tetragonolobus agglutinin), and alpha1,2-linked (Ulex europaeus agglutinin) fucose. The results indicate differences between early and advanced RA stages in the terminal sugar exposition of synovial IgG and IgA, but not IgM. The galactose-deficient glycotope with exposed N-acetylglucosamine of the synovial 33.1-kDa IgG fragment appeared exclusively in the early stage of RA. In contrast, this glycotope of intact synovial IgG and IgA was present in both groups, although with higher proportions in advanced RA. The proportions of the sialyl and fucosyl determinants of intact synovial A and G immunoglobulins were clearly lower in the early RA group than in the advanced. The analysis of terminal oligosaccharide exposition in IgG, IgG fragments, and IgA present in the synovial fluid of RA patients might be applicable as a stage-specific marker in the diagnosis and therapy of RA patients.

TREM-1 expression is increased in the synovium of rheumatoid arthritis patients and induces the expression of pro-inflammatory cytokines

To investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) in the synovium of human RA patients as well as the level of soluble TREM-1 in the plasma of RA patients. Twenty-four RA synovial samples were analysed by gene expression oligonucleotide microarrays. Expression levels of TREM-1 mRNA in murine CIA paws were determined by quantitative PCR (qPCR). TREM-1 protein expression was detected by immunohistochemistry in five RA synovial samples and two OA synovial samples. TREM-1-positive cells from five RA synovial tissues were analysed by FACS staining to determine the cell type. Activation of TREM-1 was tested in five RA synovial samples. Soluble TREM-1 was measured in serum from 32 RA patients. The expression of TREM-1 mRNA was found to increase 6.5-fold in RA synovial samples, whereas it was increased 132-fold in CIA paws. Increased numbers of TREM-1-positive cells were seen in RA synovium sections and these cells co-expressed CD14. Using a TREM-1-activating cross-linking antibody in RA synovial cultures, multiple pro-inflammatory cytokines were induced. The average amount of soluble TREM-1 in plasma from RA patients was found to be higher than that in plasma from healthy volunteers. These findings suggest that the presence of high levels of functionally active TREM-1 in RA synovium may contribute to the development or maintenance of RA, or both. Inhibiting TREM-1 activity may, therefore, have a therapeutic effect on RA. High levels of soluble TREM-1 in the plasma of RA patients compared with healthy volunteers may indicate disease activity.

Circulating CD21lowB cells in common variable immunodeficiency resemble tissue homing, innate-like B cells

The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. CD21(low) B cells are polyclonal, unmutated IgM(+)IgD(+) B cells but carry a highly distinct gene expression profile which differs from conventional naïve B cells. Interestingly, while clearly not representing a memory population, they do share several features with the recently defined memory-like tissue, Fc receptor-like 4 positive B cell population in the tonsils of healthy donors. CD21(low) B cells show signs of previous activation and proliferation in vivo, while exhibiting defective calcium signaling and poor proliferation in response to B cell receptor stimulation. CD21(low) B cells express decreased amounts of homeostatic but increased levels of inflammatory chemokine receptors. This might explain their preferential homing to peripheral tissues like the bronchoalveolar space of CVID or the synovium of rheumatoid arthritis patients. Therefore, as a result of the close resemblance to the gene expression profile, phenotype, function and preferential tissue homing of murine B1 B cells, we suggest that CD21(low) B cells represent a human innate-like B cell population.

Rheumatoid arthritis is linked to oral bacteria: etiological association

The purpose of this review is to evaluate the association between rheumatoid arthritis (RA) and periodontopathic bacteria. Clinical studies of RA and periodontal disease have provided evidence for a significant association between the two disorders. Patients with long-standing active RA have a substantially increased frequency of periodontal disease compared with that among healthy subjects. High levels of oral anaerobic bacterial antibodies have been found in the serum and synovial fluid of RA patients. Porphyromonas gingivalis, Tannerella forsythensis, and Prevotella intermedia have been identified in RA synovial fluid. Ornidazole, levofloxacin, and clarithromycin are used in the treatment of infections caused by anaerobic bacteria. These antibiotics have been shown to be effective against RA. The evidence in this review indicates that oral bacteria directly associate with etiopathogenesis of RA.

The Presumed Hyporesponsive Behavior of Rheumatoid Arthritis T Lymphocytes Can Be Attributed to Spontaneous Ex Vivo Apoptosis rather than Defects in T Cell Receptor Signaling

Genetic associations and the clinical success of compounds targeting TCR costimulatory proteins suggest an active role for TCR signaling in the initiation and perpetuation of rheumatoid arthritis (RA). Paradoxically, T cells isolated from affected joints in RA show impaired proliferative and cytokine responses following stimulation with mitogens and recall Ags attributed in part to chronic T cell exposure to oxidative stress and inflammatory cytokines. Therefore, it is uncertain how local autoreactive TCR signaling contributes to pathology in established RA. Using single-cell analysis, we show that in contrast to results obtained in bulk culture assays, T cells from the synovial fluid of RA patients proliferate and produce cytokines (IL-2, TNF-alpha, and IFN-gamma) as efficiently, if not more so, than T cells isolated from healthy donors and RA patient peripheral blood following TCR/CD28 stimulation. RA synovial fluid T cell hyporesponsiveness observed in bulk cultures can be attributed to spontaneous apoptosis ex vivo, which is associated with altered ratios of proapoptotic Noxa and anti-apoptotic Mcl-1 expression. The absence of RA synovial T cell proliferation and cytokine production in situ, despite the capacity of these cells to support productive TCR signaling, suggests that T cells contribute to local pathology in established RA by TCR-independent mechanisms.

Expression of CC Chemokine Ligand 5 in Patients with Rheumatoid Arthritis and Its Correlation with Disease Activity and Medication

To determine the levels of CC chemokine ligand 5 (CCL5) in serum and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and their relations with disease activity and medication. CCL5 in serum and SF was quantified by enzyme-linked immunosorbent assay (ELISA) in 28 RA patients and 21 osteoarthritis (OA) patients. In RA patients, the correlations of CCL5 levels in serum and SF with disease activity were analyzed. Meanwhile, the serum CCL5 levels among RA patients treated with disease-modifying antirheumatic drugs (DMARDs), Tripterygium Glucosides, and other Chinese herbs without disease-modifying effects were also compared. CCL5 levels in both serum and SF of RA patients were significantly higher than those of OA patients (P < 0.05). Moreover, the level of CCL5 was higher in SF than that in serum of RA patients (P < 0.01). Serum CCL5 level was correlated significantly with the number of swollen joints (r = 0.3329, P < 0.05), erythrocyte sedimentation rate (r = 0.4001, P < 0.05), and C reactive protein (r = 0.3735, P < 0.01). In addition, the level of CCL5 had a trend of lower in patients treated with DMARDs or Tripterygium Glucosides than those treated with other Chinese herbs, although the difference was not significant among those patients due to the small number of patients in each group. In RA patients, the expression of CCL5 increases and correlates with some clinical and laboratory parameters of RA, which indicate that CCL5 plays an important role in RA and may serve as a useful marker of disease activity. DMARDs and Tripterygium Glucosides might exert their clinical effects through reducing CCL5 production in RA.

Induction of CCL13 expression in synovial fibroblasts underlines a significant role of oncostatin M in rheumatoid arthritis

Rheumatoid arthritis (RA) is a severe, chronic disease characterized by a profound inflammatory response that leads to both joint destruction as well as extraarticular symptoms with a significant impact on both morbidity and mortality. The cause and pathologic process underlying RA have not yet been fully elucidated. However, it is clear that both the cellular immune system and the cytokine network are subject to profound dysregulations. The success of an anti-proinflammatory cytokine therapy directed against either tumor necrosis factor alpha (TNFα) or, to a lesser extent, interleukin-1 (IL-1), has highlighted the significance of these cytokines in the pathological progession of RA. Nevertheless, these therapeutical strategies are not beneficial to all patients, suggesting that other cytokines like IL-6, oncostatin M, IL-4, IL-13, IFNγ are also present in the synovial fluid of chronically inflamed joints. Here we demonstrate that among a number of cytokines present in the synovial fluid of RA patients, only OSM induces a prolonged expression of the chemokine CCL13. The expression of CCL13 appears to be tissue-specific since neither in dermal nor lung or cervix fibroblasts CCL13 expression could be observed. We identify human synovial fibroblasts (HSF) as well as synovial fibroblasts of RA patients (RASFs) as producers of CCL13. The expression of CCL13 is mediated through STAT5 activation and p38 MAPK-dependent pathways and results in increased migration of monocytic cells.

Expression, regulation, and signaling of the pattern‐recognition receptor nucleotide‐binding oligomerization domain 2 in rheumatoid arthritis synovial fibroblasts

Since pattern-recognition receptors (PRRs), in particular Toll-like receptors (TLRs), were found to be overexpressed in the synovium of rheumatoid arthritis (RA) patients and to play a role in the production of disease-relevant molecules, we sought to determine the expression, regulation, and function of the PRR nucleotide-binding oligomerization domain 2 (NOD-2) in RA. Expression of NOD-2 in synovial tissues was analyzed by immunohistochemistry. Expression and induction of NOD-2 in RA synovial fibroblasts (RASFs) were measured by conventional and real-time polymerase chain reaction (PCR) analyses. Levels of interleukin-6 (IL-6) and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) and expression of matrix metalloproteinases (MMPs) by ELISA and/or real-time PCR. NOD-2 expression was silenced with small interfering RNA. Western blotting with antibodies against phosphorylated and total p38, JNK, and ERK, as well as inhibitors of p38, JNK, and ERK was performed. Activation of NF-kappaB was measured by electrophoretic mobility shift assay. NOD-2 was expressed by fibroblasts and macrophages in the synovium of RA patients, predominantly at sites of invasion into articular cartilage. In cultured RASFs, no basal expression of messenger RNA for NOD-2 was detectable, but was induced by poly(I-C), lipopolysaccharide, and tumor necrosis factor alpha. After up-regulation of NOD-2 by TLR ligands, its ligand muramyl dipeptide (MDP) increased the expression of IL-6 and IL-8 via p38 and NF-kappaB. Stimulation with MDP further induced the expression of MMP-1, MMP-3, and MMP-13. Not only TLRs, but also the PRR NOD-2 is expressed in the synovium of RA patients, and activation of NOD-2 acts synergistically with TLRs in the production of proinflammatory and destructive mediators. Therefore, NOD-2 might contribute to the initiation and perpetuation of chronic, destructive inflammation in RA.

Role of placenta growth factor and its receptor flt‐1 in rheumatoid inflammation: A link between angiogenesis and inflammation

To investigate the direct effects of placenta growth factor (PlGF) and its specific receptor, flt-1, which are known to mediate angiogenesis, on the inflammatory process of rheumatoid arthritis (RA). Expression of PlGF and flt-1 in the synovial tissue of RA patients was examined using immunohistochemistry. Enzyme-linked immunosorbent assay was used to determine the concentrations of PlGF, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) in culture supernatants of either mononuclear cells or synoviocytes. The flt-1 expression level in mononuclear cells was analyzed by flow cytometry. Experimental arthritis was induced in mice either by immunization with type II collagen (CII) or by injection of anti-CII antibody. PlGF was highly expressed in the synovium of RA patients, and its primary source was fibroblast-like synoviocytes (FLS). When stimulated with IL-1beta, FLS from RA patients produced higher amounts of PlGF than did FLS from patients with osteoarthritis. Exogenous PlGF specifically increased the production of TNFalpha and IL-6 in mononuclear cells from RA patients (but not those from healthy controls) via a calcineurin-dependent pathway. The response to PlGF was associated with increased expression of flt-1 on RA monocytes, which could be induced by IL-1beta and TNFalpha. A novel anti-flt-1 hexapeptide, GNQWFI, abrogated the PlGF-induced increase in TNFalpha and IL-6 production, and also suppressed CII-induced arthritis and serum IL-6 concentrations in mice. Moreover, genetic ablation of PlGF prevented the development of anti-CII antibody-induced arthritis in mice. Our data suggest that enhanced expression of PlGF and flt-1 may contribute to rheumatoid inflammation by triggering production of proinflammatory cytokines. The use of the novel anti-flt-1 peptide, GNQWFI, may be an effective strategy for the treatment of RA.

Alterations in peripheral blood memory B cells in patients with active rheumatoid arthritis are dependent on the action of tumour necrosis factor

IntroductionDisturbances in peripheral blood memory B cell subpopulations have been observed in various autoimmune diseases, but have not been fully delineated in rheumatoid arthritis (RA). Additionally, the possible role of tumour necrosis factor (TNF) in regulating changes in specific peripheral blood memory B cell subsets in RA is still unclear.MethodsThe frequency and distribution of B cell subsets in the peripheral blood and synovial membrane of active RA patients with long-standing disease have been analysed. Additionally, the possible role of TNF in causing disturbances in memory B cell subsets in RA patients was assessed in a clinical trial with the specific TNF-neutralising antibody, infliximab.ResultsRA patients, independent of disease duration, have a significantly lower frequency of peripheral blood pre-switch IgD+CD27+ memory B cells than healthy individuals, whereas post-switch IgD-CD27+ accumulate with increased disease duration. Notably, both pre-switch IgD+CD27+ and post-switch IgD-CD27+ memory B cells accumulate in the synovial membrane of RA patients. Finally, anti-TNF therapy increased the frequency of pre-switch IgD+CD27 memory B cells in the peripheral blood.ConclusionsThe data suggest that decreases in peripheral blood IgD+CD27+ pre-switch memory B cells in RA reflect their accumulation in the synovial tissue. Moreover, the significant increase in the peripheral blood pre-switch memory B cells in patients who underwent specific TNF-blockade with infliximab indicates that trafficking of memory B cells into inflamed tissue in RA patients is regulated by TNF and can be corrected by neutralising TNF.

Chemokine receptor expression and functional effects of chemokines on B cells: implication in the pathogenesis of rheumatoid arthritis

IntroductionAccumulation of B cells in the rheumatoid arthritis (RA) synovium has been reported, and it has been thought that these cells might contribute to the pathogenesis of RA by antigen presentation, autoantibody production, and/or inflammatory cytokine production. Chemokines could enhance the accumulation of B cells in the synovium. The aims of this study were to determine chemokine receptor expression by B cells both in the peripheral blood of normal donors and subjects with RA, and at the inflammatory site in RA, and the effects of chemokines on B cell activation.MethodsCell surface molecule expression was analyzed by flow cytometry. Cellular migration was assessed using chemotaxis chambers. Cellular proliferation was examined by 3H-thymidine incorporation. Tumor necrosis factor (TNF) production was assayed by enzyme-linked immunosorbent assay.ResultsSignificant numbers of peripheral blood B cells of healthy donors and subjects with RA expressed CC chemokine receptor (CCR)5 and CXCR3, and most B cells expressed CCR6, CCR7, CXCR4 and CXCR5. CCR5 expression was more frequent on CD27+ than CD27- peripheral blood B cells of healthy donors and RA. Synovial B cells more frequently expressed CCR5, but less often expressed CCR6, CCR7 and CXCR5 compared to peripheral blood in RA. Further functional analyses were performed on peripheral blood B cells from healthy donors. Migration of peripheral blood B cells, especially CD27+ B cells, was enhanced by CC chemokine ligand (CCL)20, CCL19, CCL21 and CXCL12. All four chemokines alone induced B cell proliferation; with CCL21 being the most effective. CCL21 also enhanced the proliferation of anti-immunoglobulin (Ig)M-stimulated B cells and blockade of CCR7 inhibited this effect. CCL20, CCL21 and CXCL12 enhanced TNF production by anti-IgM mAb-stimulated B cells. Finally, stimulation with CXCL12, but not CCL20, CCL19 and CCL21, enhanced inducible costimulator-ligand (ICOSL) expression by peripheral blood B cells of healthy donors and RA, but did not increase B cell-activating factor receptor or transmembrane activator and CAML-interactor.ConclusionsThe data suggest that CCR5, CCR6, CCR7, CXCR3, CXCR4 and CXCR5 may be important for the B cell migration into the synovium of RA patients, and also their local proliferation, cytokine production and ICOSL expression in the synovium.

Anti-Inflammatory Effects of Sphingosine Kinase Modulation in Inflammatory Arthritis

Sphingosine kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P). SphK/S1P play a critical role in angiogenesis, inflammation, and various pathologic conditions. Recently, S1P(1) receptor was found to be expressed in rheumatoid arthritis (RA) synovium, and S1P signaling via S1P(1) enhances synoviocyte proliferation, COX-2 expression, and prostaglandin E(2) production. Here, we examined the role of SphK/S1P in RA using a potent SphK inhibitor, N,N-dimethylsphingosine (DMS), and a molecular approach against one of its isoenzymes, SphK1. We observed that levels of S1P in the synovial fluid of RA patients were significantly higher than those of osteoarthritis patients. Additionally, DMS significantly reduced the levels of TNF-alpha, IL-6, IL-1beta, MCP-1, and MMP-9 in cell-contact assays using both Jurkat-U937 cells and RA PBMCs. In a murine collagen-induced arthritis model, i.p. administration of DMS significantly inhibited disease severity and reduced articular inflammation and joint destruction. Treatment of DMS also down-regulated serum levels IL-6, TNF-alpha, IFN-gamma, S1P, and IgG1 and IgG2a anti-collagen Ab. Furthermore, DMS-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Moreover, similar reduction in incidence and disease activity was observed in mice treated with SphK1 knock-down via small interfering RNA approach. Together, these results demonstrate SphK modulation may provide a novel approach in treating chronic autoimmune conditions such as RA by inhibiting the release of pro-inflammatory cytokines.