Synovial Fluid T-cell Research Articles

Expression and activity of NOD1 and NOD2/RIPK2 signalling in mononuclear cells from patients with rheumatoid arthritis

Objectives: The aim of this study was to analyse the expression and function of nucleotide-binding oligomerization domain (NOD)1 and NOD2 in isolated cells of patients with rheumatoid arthritis (RA).Method: mRNA expression levels of NOD1, NOD2, and receptor-interacting serine/threonine kinase 2 (RIPK2) genes were determined by quantitative polymerase chain reaction (qPCR) in peripheral blood mononuclear cells (PBMCs) and synovial fluid T cells (SFTCs) isolated from RA and osteoarthritis (OA) patients. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA) in plasma and cell culture supernatants. The stimulatory effect of RA SF was assessed by an in-vitro NOD2 activation assay using nuclear factor kappa B (NF-κB) luciferase-transfected cells.Results: A significantly higher level of NOD2 and RIPK2 mRNA expression, but not NOD1, was observed on PBMCs and SFTCs isolated from RA patients compared to the OA control group. In addition, the NOD2 pathway up-regulation was functional, as stimulation of PBMCs with muramyl dipeptide (MDP) induced the production of higher amounts of tumour necrosis factor (TNF)-α, interleukin (IL)-8, and IL-1β compared with OA PBMCs. Incubation of PBMCs from healthy donors with recombinant TNF-α or RA serum induced the expression of NOD2 mRNA. Finally, SF isolated from RA patients is able to activate the NF-κB signalling pathway in HEK293T-transfected cells in a NOD2-dependent manner.Conclusions: Our findings suggest that NOD2/RIPK2 signalling is up-regulated in immune cells of RA patients. Moreover, it seems that there is a NOD2 agonist in the SF of RA patients. Therefore, NOD2/RIPK2 activation can modulate the innate immune response and may play a role in the perpetuation of the inflammatory response in RA.

AB0021 In Psoriatic Arthritis Synovial Tissue Harbors Expanded T-Cell Clones Which Are not Fully Represented in Synovial Fluid or Blood Samples

BackgroundT-cells play a key role in psoriatic arthritis (PsA). Nevertheless, the distribution and exact role of these cells remains unclear. Here, we study whether expanded T-cell clones are present in...

Selective elimination of pathogenic synovial fluid T-cells from Rheumatoid Arthritis and Juvenile Idiopathic Arthritis by targeted activation of Fas-apoptotic signaling

In Rheumatoid Arthritis (RA) and Juvenile Idiopathic Arthritis (JIA) elimination of autoreactive T-cells by FasL/Fas-mediated Activation-Induced Cell Death (AICD) appears to be inhibited resulting in the perpetuation of the inflammatory response and concomitant progressive tissue destruction. Here, we report on a novel strategy that aims to overcome the local inhibition of AICD by using rationally designed recombinant fusion proteins in which sFasL is genetically fused to a T-cell selective targeting domain. The series included sFasL fusion proteins with engineered binding specificity for various T-cell surface-expressed proteins including CD7, CD28, RANKL and CD40L. The proposed mode of action is that selective binding of a given sFasL fusion protein results in its accretion at the cell surface of T-cells only, displaying a surplus of sFasL that is available to reactivate AICD in pathogenic synovial T-cells. Of the series of T-cell targeting FasL fusion proteins a CD7-targeted fusion protein, designated scFvCD7:sFasL, proved to be the most potent, with significant pro-apoptotic activity towards synovial fluid T-cells in all patient samples tested (RA; n=22: JIA; n=6). Treatment with scFvCD7:sFasL induced up to 80% apoptosis in CD3-positive synovial T-cells. Importantly, scFvCD7:sFasL potently activated Fas-signaling in synovial TH1-cells as well as synovial Treg cells, but not in synovial TH2 cells. These findings indicate that scFvCD7:sFasL may be of therapeutic value for the selective elimination of pathogenic synovial T-cells of the TH1 subtype in both RA and JIA.

Successful induction of severe destructive arthritis by the transfer of in vitro-activated synovial fluid T cells from patients with rheumatoid arthritis (RA) in severe combined immunodeficient (SCID) mice.

In order to investigate the role of pathogenic T cells in RA, the establishment of an RA model using patients' T cells is thought to be essential. In this study, multiple and severe destructive arthritis was established by transferring in vitro-stimulated synovial fluid T (SFT) cells from patients with RA through simultaneous injection into knee joint and peritoneal cavity of SCID mice without causing xenogeneic graft-versus-host disease (GVHD). Neither the transfer of unstimulated SFT cells nor sole i.p. injection was sufficient to induce severe arthritis. Interestingly, in contrast with SFT cells, in vitro-activated peripheral blood lymphocytes from RA patients failed to trigger such arthritis, suggesting that pathogenic T cells might be concentrated in synovial fluid of RA patients. This, the first severe arthritis model mimicking RA induced by RA patients' T cells, is expected to provide important information about RA pathogenesis and a possible therapeutic approach.

The cellular immune reaction in synovial fluid lymphocytes to Ureaplasma antigens in patients with Reiter's syndrome

Reiter's syndrome (RS) is an seronegative arthritis that occurs after urogenital or enteric infection which in addition with occular and/or mucocutaneous manifestations presents complete form of disease. According to previous understanding arthritis in the RS is the reactive one, which means that it is impossible to isolate its causative agent. However, there are the more and more authors suggesting that arthritis in the urogenital form of disease is caused by the infective agent in the affected joint. This suggestion is based on numerous studies on the presence of Chlamydia trachomatis and Ureaplasma urealyticum in the inflamed joint by using new diagnostic methods in molecular biology published in the recent literature [1-3]. Besides, numerous studies of the humoral and cell-mediated immune response to "triggering" bacteria in the affected joint have supported previous suggestions [4-7]. Aim of the study was to determine whether synovial fluid T-cells specifically recognize the "triggering" bacteria presumably responsible for the Reiter's syndrome. The 3H-thymidine uptake procedure for measuring lymphocyte responses was applied to lymphocytes derived concurrently from synovial fluid (SF) and from peripheral blood (PB) [8]. Ureaplasma antigen and mitogen PHA stimulated lymphocytes in 24 RS patients (24 PB samples, 9 SF samples) and the results were compared with those found in 10 patients with rheumatoid arthritis (RA) (10 PB samples, 5 SF samples). Preparation of ureaplasma antigen. Ureaplasma was cultured on cell-free liquid medium [9]. Sample of 8 ml was heat-inactivated for 15 minutes at 601C and permanently stirred with magnetic mixer. The sample was centrifuged at 2000 x g for 40 minutes and than deposits carefully carried to other sterile glass tubes (Corex) and recentrifuged at 9000 x g for 30 minutes. The deposit was washed 3 times in sterile 0.9% NaCl, and final sediment was resuspended in 1.2 ml sterile 0.9% NaCl. BACTERIOLOGY: Chlamydia trachomatis was isolated by cell culture using cycloheximide-treated McCoy cells [10], while Ureaplasma urealyticum was identified according to its biochemical properties grown on cell-free liquid medium [9]. Proliferative response of the PB lymphocytes to stimulation by mitogen and ureaplasma antigen did not differ between RS and RA patients. Also, there was no difference in proliferative response of SF lymphocytes to mitogen stimulation between RS and RA patients (Figure 1). However, proliferation of SF lymphocytes stimulated by ureaplasma antigen was significantly elevated in RS patients compared with the control group. This difference is statistically significant (p < 0.05) (Figure 2). Difference in proliferative response of the PB and SF lymphocytes stimulated by the ureaplasma antigen was not found in RS patients. It was found that SF lymphocytes of RS patients showed significantly elevated proliferative response to stimulation by the ureaplasma antigen compared with SF lymphocytes of the control group. There was no difference when the lymphocytes were stimulated by the mitogen. Our findings suggest that elevated proliferative response of lymphocytes is the sign of stimulation cell-mediated immunity to antigen present in inflamed joint. Hence, the main immune response to Ureaplasma is on the cell-mediated level in the affected joint. This confirms the earlier finding reported by Ford et al. who concluded that synovial rather than peripheral blood lymphocytes indicate the microbiological cause of arthritis [11, 12]. Horowitz et al. demonstrated the correlation between clinical remission after antibiotic therapy and eradication of Ureaplasma, together with a decrease in cellular immune response synovial fluid lymphocytes to ureaplasma antigen stimulation [13]. In that study Horowitz did not find statistically significant difference of ureaplasma proliferative response between PB and SF lymphocytes in patients with RS. We obtained the same results. Than we concluded that sensibilization of immune system exist in the presence of foreign antigen in RS patients. The other authors demonstrated higher stimulation indices than the ones we found in our patients [11-15]. This difference may be the result of different preparation of antigens, in other words selection of serotype of Ureaplasma for antigen preparation different conditions of lymphocyte cultivation. We concluded that the presence of antigen, antigen-specific T cells and efficient antigen-presenting cells (CD4+ T cells) in the joint of RS patients strongly suggests that a T-cell-mediated response to bacteria has the central role in the pathogenesis of Reiter's syndrome.

T-cell activation without proliferation in juvenile idiopathic arthritis.

A study was done to determine if the differentiation and activation phenotype of T cells in synovial fluid (SF) from patients with juvenile idiopathic arthritis (JIA) is associated with T-cell proliferation in situ. Mononuclear cells were isolated from 44 paired samples of peripheral blood and SF. Differentiation and activation markers were determined on CD4 and CD8 T cells by flow cytometry. Cell-cycle analysis was performed by propidium iodide staining, and surface-marker expression was also assessed after culture of the T cells under conditions similar to those found in the synovial compartment. The majority of the T cells in the SF were CD45RO+CD45RBdull. There was greater expression of the activation markers CD69, HLA-DR, CD25 and CD71 on T cells from SF than on those from peripheral blood. Actively dividing cells accounted for less than 1% of the total T-cell population in SF. The presence or absence of IL-16 in T-cell cultures with SF or in a hypoxic environment did not affect the expression of markers of T-cell activation. T cells from the SF of patients with JIA were highly differentiated and expressed early and late markers of activation with little evidence of in situ proliferation. This observation refines and extends previous reports of the SF T-cell phenotype in JIA and may have important implications for our understanding of chronic inflammation.

Oligoclonal T-receptor (V beta) use in the response to connective tissue antigens by synovial fluid T-cells from rheumatoid arthritis patients.

Oligoclonal T-receptor (V beta) use in the response to connective tissue antigens by synovial fluid T-cells from rheumatoid arthritis patients.

Analysis of synovial fluid T-cell repertoires by CDR3 size spectratyping reveals possible antigen and superantigen stimulation.

Analysis of synovial fluid T-cell repertoires by CDR3 size spectratyping reveals possible antigen and superantigen stimulation.

Patterns of T-Cell Receptor Variable β Gene Expression by Synovial Fluid and Peripheral Blood T-Cells in Rheumatoid Arthritis

Conflicting data have been reported regarding the presence or absence of a predominant variable (V) region T-cell receptor (TCR) gene in the peripheral blood or synovial fluid of patients with rheumatoid arthritis (RA). In this study we have used the polymerase chain reaction (PCR) to compare the level of TCR Vβ gene expression by peripheral blood mononuclear cells (PBMC) and by synovial fluid cells obtained from HLA DRB1 *0401 and *0404 RA patients. PCR was performed using cDNA synthesized from freshly obtained cells (not stimulated in vitro). The pattern of expression observed for most of the Vβ genes studied showed either preferential expression by PBMC or similar levels of expression between PBMC and synovial fluid T-cells. However, among individual patients ( N = 5), several Vβ genes were identified that were expressed to a significantly greater degree by synovial fluid cells. Vβ14 expression was detected in PBMC of all patients and the level of transcripts encoding Vβ14 increased following stimulation with immobilized anti-CD3 and IL-2. In vitro manipulation of populations of T-cells was found to alter the level of expressed Vβ gene products. A Vβ gene common to all patients that was consistently deleted from PBMC or expressed to a greater degree by resident, unsorted synovial fluid cells compared to PBMC was not identified.

Salmonella-reactive Synovial Fluid T-cell Clones in a Patient with Post-infectious Salmonella Arthritis

From a patient with reactive arthritis following Salmonella typhimurium enteritis, synovial fluid T-lymphocytes were cloned and expanded in vitro. Seven out of 74 clones showed a marked proliferative response to antigens of heat-killed Salmonella typhimurium with autologous T-cell-depleted peripheral blood mononuclear cells as antigen-presenting cells. The Salmonella-reactive clones were of the CD4+ phenotype, antigen-induced proliferation could be inhibited by a monoclonal antibody to HLA class II. One clone recognized both Salmonella and Campylobacter jejuni antigens in the proliferation assay. The multiclonality of Salmonella-reactive synovial fluid T-cells indicates that the microorganisms have been present, at least transiently, within the affected joint and thus recruited specific T-lymphocytes that might initiate synovitis.

Clonal heterogeneity of synovial fluid T lymphocytes from patients with rheumatoid arthritis.

Although substantial evidence suggests that synovial T lymphocytes are critical in the pathogenesis of rheumatoid arthritis (RA), little is known regarding their antigenic specificities, antigen receptor gene rearrangements, and mechanisms of activation. To assess the extent of expansion of specific clones among RA synovial fluid T cells, Southern blot analyses of T-cell receptor (TCR) gene rearrangements were performed on 40 RA synovial fluid T-cell clones, as well as on both fresh and polyclonally activated T cells from RA synovial fluid, RA peripheral blood, and normal peripheral blood. Two of the clones had identical TCR rearrangement patterns, but the remainder were unique. The nonclonal RA T-cell samples showed the same pattern of TCR beta-chain rearrangement that was observed among normal peripheral blood T cells, indicating no dominant clonal T-cell population in these samples. It was noted that with sufficient exposure of autoradiograms of the Southern blots, discrete TCR gene rearrangements, representing in some cases common D beta J beta (D, diversity; J, joining) rearrangements, were evident in T cells from peripheral blood of normal individuals and patients with RA, as well as T cells from RA synovial fluid. Taken together, the findings indicate that only a minor degree of oligoclonality can be demonstrated among T lymphocytes from RA synovial fluid.

A minority of patients with rheumatoid arthritis show a dominant rearrangement of T-cell receptor beta chain genes in synovial lymphocytes.

The clonality of T lymphocytes isolated from the synovial fluid and peripheral blood of patients with rheumatoid arthritis was investigated by restriction enzyme fragment mapping of the rearrangements of the beta chain gene of the T-cell antigen receptor. Three patients showed a dominant rearrangement amongst their synovial fluid T cells which was not seen in their peripheral blood T-cell population, suggesting the presence of a predominating T-cell clone. However, most of the patients examined (8 out of 11) demonstrated polyclonal T-cell populations in both their synovial fluid and peripheral blood. Of four synovial fluid T-cell lines investigated, one showed evidence of a dominant T-cell clone.

Generation of long-term T-cell lines from synovial fluid

Investigations of human autoimmune diseases have largely involved study of circulating T cells. The development of T-cell cloning technology has made possible the study of the small numbers of T cells found at sites of pathological involvement in autoimmune diseases. In this initial communication, the feasibility of in vitro propagation of synovial fluid T cells from patients with arthritis is demonstrated. The generation of long-term, phenotypic helper/inducer, as well as phenotypic suppressor/cytotoxic, interleukin 2-dependent synovial fluid T-cell lines from patients with arthritis is reported. The ability to generate such synovial fluid T-cell lines should now allow for new investigative approaches to human autoimmune arthritic diseases.