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Objective: Oxidative stress contributes to the inflammatory properties of rheumatoid arthritis (RA) synovial T lymphocytes. This study was undertaken to investigate the mechanisms leading to production of reactive oxygen species (ROS) and oxidative stress in RA synovial T lymphocytes., Methods: ROS production in T lymphocytes from the peripheral blood (PB) of healthy donors and from the PB and synovial fluid (SF) of RA patients was measured by ROS-dependent fluorescence of 6-carboxy-2',7'-dichlorofluorescein. Rap1 GTPase activation was assessed by activation-specific probe precipitation. Proliferation of RA PB and SF T lymphocytes was assayed by 3H-thymidine incorporation. In some experiments, RA PB T cells were preincubated with autologous SF or with PB or SF adherent cells. Experiments were performed in the absence or presence of transwell membranes or CTLA-4Ig fusion proteins. Short- and long-term stimulations of healthy donor PB T lymphocytes were performed with inflammatory cytokines, in the absence or presence of activating anti-CD28 antibodies., Results: T lymphocyte ROS production and Rap1 inactivation were mediated by cell-cell contact with RA synovial adherent cells, and this correlated with T cell mitogenic hyporesponsiveness. CTLA4-Ig blockade of synovial adherent cell signaling to CD28 T cells reversed the inhibition of Rap1 activity and prevented induction of ROS. Introduction of active RapV12 into T cells also prevented induction of ROS production. Coincubation of T cells with stimulating anti-CD28 antibodies and inflammatory cytokines synergistically increased T cell ROS production., Conclusion: Cell-cell contact between T cells and RA synovial adherent cells mediates Rap1 inactivation and subsequent ROS production in T lymphocytes following exposure to inflammatory cytokines. This process can be blocked by CTLA4-Ig fusion protein.

Objective: To investigate the cellular and molecular sources of oxidative stress in patients with rheumatoid arthritis (RA) through analysis of the production of reactive oxygen species (ROS) in synovium., Methods: Cytochemical procedures based on the 3,3'-diaminobenzidine (DAB)-Mn2+ deposition technique were used on unfixed cryostat sections of synovium from RA patients and rheumatic disease controls. For immunophenotyping, sections were incubated, fixed, and stained with fluorescein isothiocyanate-labeled antibodies. Fluorescence-activated cell sorter analysis of the ROS-reactive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate-di(acetoxymethyl ester) was used to measure intracellular ROS in T lymphocytes from peripheral blood and synovial fluid. To determine which enzymes produced ROS, different inhibitors were tested., Results: Large quantities of DAB precipitated in the majority of RA synovial T lymphocytes, indicative of intracellular ROS production. These ROS-producing T lymphocytes were observed throughout the synovium. Polymerization of DAB was observed to a lesser extent in other forms of chronic arthritis, but was absent in osteoarthritis. DAB staining of cytospin preparations of purified RA synovial fluid T cells confirmed the presence of ROS-producing cells. One of the ROS involved appeared to be H2O2, since catalase suppressed intracellular ROS production. Superoxide dismutase, which uses superoxide as a substrate to form H2O2, diphenyleneiodonium (an inhibitor of NADPH oxidase), N(G)-monomethyl-L-arginine (an inhibitor of nitric oxide synthesis), nordihydroguaiaretic acid (an inhibitor of lipoxygenase), and rotenone (an inhibitor of mitochondrial ROS production) failed to suppress ROS production., Conclusion: Our findings show that chronic oxidative stress observed in synovial T lymphocytes is not secondary to exposure to environmental free radicals, but originates from intracellularly produced ROS. Additionally, our data suggest that one of the intracellularly generated ROS is H2O2, although the oxidase(s) involved in its generation remains to be determined.

Objective: Rheumatoid arthritis (RA) is characterized by inflammation and destruction of synovial joints. Fibroblast-like synoviocytes (FLS) harvested from synovial tissue of patients with RA can invade normal human cartilage in severe combined immunodeficient (SCID) mice and Matrigel basement membrane matrix in vitro. This study was undertaken to investigate the association of these in vitro characteristics with disease characteristics in patients with RA., Methods: Synovial tissue samples from 72 RA and 49 osteoarthritis (OA) patients were obtained. Samples of different joints were collected from 7 patients with RA. The FLS invasiveness in Matrigel was studied, and the intraindividual and interindividual differences were compared. From the patients with FLS who exhibited the most extreme differences in in vitro ingrowth (most and least invasive FLS), radiographs of the hands and feet were collected and scored according to the Sharp/van der Heijde method to determine the relationship between in vitro invasion data and estimated yearly joint damage progression., Results: FLS from patients with RA were more invasive than FLS from patients with OA (P < 0.001). The mean intraindividual variation in FLS invasion was much less than the mean interindividual variation (mean +/- SD 1,067 +/- 926 and 3,845 +/- 2,367 for intraindividual and interindividual variation, respectively; P = 0.035), which shows that the level of FLS invasion is a patient characteristic. The mean +/- SEM Sharp score on radiographs of the hands or feet divided by the disease duration was 4.4 +/- 1.1 units per year of disease duration in patients with the least invasive FLS (n = 9), which was much lower compared with the 21.8 +/- 3.1 units per year of disease duration in patients with the most invasive FLS (n = 9) (P < 0.001)., Conclusion: The ex vivo invasive behavior of FLS from RA patients is associated with the rate of joint destruction and is a patient characteristic, given the much smaller intraindividual than interindividual FLS variation.

Objective: To determine immunohistological markers in synovial tissue of patients with early rheumatoid arthritis (RA) which are associated with unfavourable disease outcome., Methods: Synovial tissue was obtained from 36 patients with RA within 1 year after the initial symptoms and before starting disease modifying antirheumatic drug treatment. Clinical, laboratory, and radiological assessments (Larsen score) were performed at the time of the biopsy and at the end of follow up (mean 58 months, range 38-72). Immunohistological analysis was performed to detect T cells, B cells, plasma cells, fibroblast-like synoviocytes (FLS), macrophages, and granzyme B+ cytotoxic cells. The sections were evaluated by digital image analysis., Results: Patients were divided into two groups based upon the radiological progression per year of follow up: group I with mild progression (n = 20; Larsen <2 points/year); group II with more severe progression (n = 16; Larsen > or =2 points/year). Regression analysis with a univariate model showed that the numbers of granzyme B+ cytotoxic cells (relative risk (RR) = 12, p = 0.003), T cells (RR = 11, p = 0.013), and FLS (RR = 10, p = 0.020) discriminated between groups I and II. A multivariate model demonstrated that the numbers of T cells (RR = 1.2, p = 0.015) and FLS (RR = 1.4, p = 0.013) were independent discriminators between groups I and II., Conclusion: The numbers of granzyme B+ cytotoxic cells, T cells, and FLS in synovial tissue of patients with RA are related to the severity of joint damage. The data suggest a pathogenetic role for these cells in the process of joint damage.

Background: Synovial tissue (ST) from end stage destructive rheumatoid arthritis (RA) and arthroscopic biopsies obtained during active inflammation might exhibit different characteristics., Objective: To define the cell infiltrate and the expression of proinflammatory cytokines, angiogenic factors, and matrix metalloproteinases (MMPs) in ST selected at arthroscopy compared with that from end stage RA., Methods: Synovial biopsy specimens were obtained from the actively inflamed knee joints of 13 patients with chronic RA by arthroscopy and compared with ST from 10 patients with end stage, destructive RA. Immunohistological analysis was performed to detect T cells, plasma cells, macrophages, fibroblast-like synoviocytes (FLS), and the expression of interleukin (IL)1beta, IL6, tumour necrosis factor alpha (TNFalpha), MMP-1, MMP-3, MMP-13, TIMP-1, and VEGF., Results: The expression of CD68+ macrophages was significantly higher in ST selected at arthroscopy than in samples obtained at surgery, both in the intimal lining layer and in the synovial sublining. The expression of CD3+ T cells also tended to be higher in arthroscopic samples. The expression of TNFalpha, IL6, MMP-1, MMP-3, MMP-13, TIMP-1, and VEGF was on average higher in ST obtained at arthroscopy. In contrast, the expression of IL1beta was on average higher in surgical samples., Conclusion: Active arthritis activity is associated with increased cell infiltration, expression of proinflammatory cytokines, MMPs, and angiogenic growth factors in synovial biopsy samples selected at arthroscopy. Increased expression of IL1beta in the synovium of patients with destructive RA requiring joint replacement may well reflect the important role of IL1beta in cartilage and bone destruction.

Rheumatoid arthritis (RA) is a heterogeneous disease. We used cDNA microarray technology to subclassify RA patients and disclose disease pathways in rheumatoid synovium. Hierarchical clustering of gene expression data identified two main groups of tissues (RA-I and RA-II). A total of 121 genes were significantly higher expressed in the RA-I tissues, whereas 39 genes were overexpressed in the RA-II tissues. Among the 121 genes overexpressed in RA-I tissues, a relative majority of nine genes are located on chromosome 6p21.3. An interpretation of biological processes that take place revealed that the gene expression profile in RA-I tissues is indicative for an adaptive immune response. The RA-II group showed expression of genes suggestive for fibroblast dedifferentiation. Within the RA-I group, two subgroups could be distinguished; the RA-Ia group showed predominantly immune-related gene activity, while the RA-Ib group showed an additional higher activity of genes indicative for the classical pathway of complement activation. All tissues except the RA-Ia subgroup showed elevated expression of genes involved in tissue remodeling. These results confirm the heterogeneous nature of RA and suggest the existence of distinct pathogenic mechanisms that contribute to RA. The differences in expression profiles provide opportunities to stratify patients based on molecular criteria.

Background: Matrix metalloproteinases (MMPs) have a pivotal role in the destruction of cartilage in rheumatoid arthritis (RA), which is mediated by the fibroblast-like synoviocytes (FLS)., Objective: To examine the in vitro invasiveness of synoviocytes obtained from inflamed joints of patients with arthritis in relation to the expression of MMP 1-14, 17, 19, cathepsin-K, the tissue inhibitors of matrix metalloproteinases TIMP-1 and TIMP-2 by FLS., Methods: FLS were derived from 56 patients (30 with RA, 17 with osteoarthritis (OA), and nine with avascular necrosis (AVN)). Invasive growth of FLS through an artificial matrix (Matrigel) was measured in a transwell system. The number of cells that migrated through the matrix were counted. Proliferation rate was determined by counting the FLS after seven days of culturing. Expression of MMPs, cathepsin-K and TIMPs was investigated with reverse transcriptase-polymerase chain reaction and related to the expression of a household gene, beta-actin., Results: FLS from RA showed greater invasive growth than FLS from OA and AVN. The median number of cells that grew through the matrix membrane was 4788 for RA, significantly higher than the number for OA, 1875 (p<0.001) and for AVN, 1530 (p=0.014). The median rate of proliferation of RA FLS was 0.27 per day compared with OA 0.22 per day (p= 0.012) and AVN 0.25 per day, but there was no correlation between the rate of proliferation and invasive growth in vitro. FLS from RA and OA that expressed MMP-1, MMP-3, or MMP-10 were significantly more invasive (median number of invasive cells: 3835, 4248, 4990, respectively) than cells that did not express these MMPs (1605, p=0.03; 1970, p=0.004; 2360, p=0.012, respectively). There was also a significant relationship between the expression of MMP-1 and MMP-9 and the diagnosis RA (both p=0.013). The expression levels of mRNA for MMP-1 and MMP-2 correlated with the protein levels produced by the synoviocytes as measured by an enzyme linked immunosorbent assay (ELISA)., Conclusion: FLS of RA invade more aggressively in a Matrigel matrix than OA and AVN FLS; this is not because of a higher rate of proliferation of RA FLS. The significant correlation between the expression of MMP-1, MMP-3, and MMP-10 and invasive growth in a Matrigel transwell system suggests that these MMPs play a part in the invasive growth of FLS obtained from patients with RA.

Objective: To provide a comprehensive understanding of the humoral immune response that takes place at the site of inflammation in rheumatoid arthritis (RA), we studied the functional properties of synovial B cells. In particular, the response to various modes of mitogen stimulation was investigated., Methods: Purified synovial fluid (SF) B cells were cultured in the presence of CD40 ligand (CD40L)-expressing fibroblasts and cytokines, activated T cells, or phorbol myristate acetate (PMA)/ionomycin. Proliferation was determined by 3H-thymidine incorporation. Release of intracellular calcium was studied by flow cytometry., Results: The inflamed joints of RA patients contained a population of CD20+,CD38- B cells with dramatically impaired mitogen responsiveness. Although the Ig-producing capacity was intact, these cells failed to proliferate in response to (a) CD40 in the presence of interleukin-2 (IL-2) and IL-10, (b) activated T cells, or (c) stimulation via the B cell receptor. Moreover, SF CD20+,CD38- B cells revealed a defective B cell receptor-induced Ca2+ influx, reminiscent of anergic B cells. Release of intracellular Ca2+ by ionomycin in the presence of the protein kinase C activator PMA did not restore the proliferative capacity. These findings indicate blockades in the proximal and distal intermediates involved in mitogen signaling., Conclusion: SF CD20+,CD38- B cells have functionally impaired proliferative responsiveness. The capacity of these cells to respond to activation by the production of Ig supports the notion that these cells might serve as Ig-producing effector cells and, as such, play a role in the pathophysiology of RA.

Objective: Digital image analysis (DIA) offers the opportunity to quantify the stained area and staining intensity when synovial tissue (ST) is investigated by immunohistochemical analysis. This study aimed at determining the sensitivity of DIA compared with semiquantitative analysis (SQA)., Methods: Paired ST samples were obtained from the knee joint of 10 patients with rheumatoid arthritis (RA) with active disease and after follow up when complete clinical remission was achieved. ST samples of 10 subjects with non-inflammatory knee pain served as controls. Immunohistochemistry with antibodies against interleukin 1beta (IL1beta) and vascular cell adhesion molecule 1 (VCAM-1) was applied using two staining protocols with 3-amino-9-ethylcarbazole (AEC) or p-diethylaminobenzaldehyde (DAB) as dye. All sections were analysed semiquantitatively (0-4) and DIA of up to a maximum of 60 high power fields (HPF). The average integrated optical density was calculated as the product of the stained area (corrected for total tissue area) and the optical density., Results: Both SQA and DIA enabled the assessment of differences in IL1beta and VCAM-1 expression between ST from active RA, RA in remission, and controls. SQA and DIA showed excellent correlations (IL1beta rs=0.867; p<0.0001: VCAM-1 rs=0.828; p<0.0001). A limited analysis of one region with six HPF still allowed adequate discrimination compared with an extended analysis of three regions with a total of 60 HPF. In general, the red dye (AEC) resulted in better discrimination than the brown (DAB) staining., Conclusion: DIA offers a reliable, reproducible, and sensitive analysis of ST sections stained for cytokines and adhesion molecules.

Objective: To obtain an adenoviral vector with increased infection efficiency in the synovial tissue compared with conventional vectors based on adenovirus serotype 5 (Ad5), without compromising the specificity of infection., Methods: Coxsackie adenovirus receptor (CAR) expression was assessed in cultured synoviocytes. Chimeric adenoviruses based on Ad5 but carrying the DNA encoding the fiber of adenovirus from subgroup B (Adll, 16, 35) or D (Ad24, 28, 33, 45, or 47) were constructed and produced on PER.C6 cells. The gene transfer efficiency of these chimera was tested on cultured synoviocytes and peripheral blood mononuclear cells (PBMC)., Results: No surface expression of CAR protein was observed on synoviocytes. CAR messenger RNA expression of synoviocytes was found to be low. Of all fiber chimeric vectors tested, vectors carrying the fiber of Ad16 (Ad5.fib16) were most potent, yielding approximately150 times increased transgene expression in cultured synoviocytes compared with those of Ad5. Flow cytometry showed that the increase in transgene expression was caused by the transduction of higher percentages of synoviocytes and higher gene expression per synoviocyte. Experiments with 500 virus particles/cell of Ad5.GFP or Ad5.fib16.GFP resulted in an infection efficiency of 0.6% and 1% in PBMC and 43% and 76% in synoviocytes, respectively., Conclusion: Synoviocytes hardly express CAR, which hampers Ad5-mediated gene transfer. Ad5.fib16 is superior to Ad5 vectors for transducing synoviocytes, without compromising the specificity of infection. Our data suggest that Ad5.fib16-mediated gene transfer to synovial tissue improves the therapeutic window.

Objective: To investigate the cartilage-degrading capacity of granzyme B and the presence of granzyme B-positive cells at sites of erosion in the rheumatoid synovium., Methods: Granzyme B was added to [(3)H]proline/[(35)S]sulphate-labelled cartilage matrices and to cartilage explants. Proteoglycan degradation was assessed by the release of (35)S and glycosaminoglycans into the medium and collagen degradation was assessed by the release of (3)H and hydroxyproline and by measuring the fraction of denatured collagen. Granzyme B expression was studied at the invasive front of the synovium by immunohistochemistry., Results: Granzyme B induced loss of both newly synthesized, radiolabelled proteoglycans in cartilage matrices and resident proteoglycans of the cartilage explants. No effect on collagen degradation was found. Granzyme B-positive cells were present throughout the synovium and at the invasive front., Conclusion: The presence of granzyme B-positive cells at the invasive front of the synovium together with its ability to degrade articular proteoglycans supports the view that granzyme B may contribute to joint destruction in rheumatoid arthritis.

Paired synovial tissue samples were obtained from both clinically uninvolved (CU) and clinically involved (CI) knee joints of eight rheumatoid arthritis (RA) patients. In addition, biopsies were taken from five control subjects. We observed the expression of the chemokines CXCL8, CXCL9, CXCL10, CCL2 and CCL4 in CI and CU joints of RA patients. In particular, CXCL8 protein levels were specifically increased in CI joints compared with CU joints, which was confirmed by immunohistochemistry and in situ hybridization.

Objective: To determine the effect of synovial fluid (SF) from rheumatoid arthritis (RA) patients on adenovirus type 5 (Ad5)-mediated gene transfer to synoviocytes, and to explore new strategies for vector development based on the neutralization data obtained., Methods: SF was derived from 63 randomly selected R4 patients. Ten samples were used to study the effect of SF on Ad5-mediated gene transfer in synoviocytes. IgG and <100-kd fractions were purified from these 10 SF, and their effect on gene transfer was determined. Neutralizing activity against wild-type Ad5 (wt-Ad5), wt-Ad26, wt-Ad34, wt-Ad35, and wt-Ad48 was tested in the SF from the remaining 53 patients., Results: Seven of 10 SF samples inhibited Ad5-mediated gene transfer. Purified antibodies exhibited inhibition patterns similar to those seen with unfractionated SF. In 5 of 10 SF samples, low molecular weight fractions inhibited gene transfer at low dilutions. Neutralization of wt-Ad35 by SF from RA patients was less frequent than neutralization of other wt-Ad tested (4% versus 42-72%; n = 53)., Conclusion: SF from 70% of the RA patients contained neutralizing antibodies that hamper Ad5-mediated gene transfer to synoviocytes. The activity of neutralizing antibodies may be circumvented in the majority of RA patients when vectors based on an Ad35 backbone are used.

Objective: Constitutive differences between individuals in cytokine production may determine the variation in the course of inflammatory arthritis., Methods: The association between interleukin 10 (IL-10) production and joint destruction was studied by comparing IL-10 mRNA content in synovial biopsies from seven patients with destructive joint disease and six patients with non-destructive joint disease. The IL-10 mRNA content was 0.4 +/- 0.6 arbitrary units in erosive joints compared with 2.3 +/- 1.2 arbitrary units in non-erosive joints (P: < 0.03, Mann-Whitney U:-test). As this difference suggested that IL-10 production was associated with joint destruction, we tested whether the IL-10 locus determined the extent of joint damage., Results: Innate differences in IL-10 production are locus-dependent. In line with these data, we showed that innate differences in IL-10 protein production were also present as differences in IL-10 mRNA levels. We tested if polymorphisms in the promoter of IL-10 were associated with the extent of joint damage., Discussion: In a cohort study of female rheumatoid arthritis patients followed for 12 yr, the extent of joint destruction differed significantly between patients with different IL-10 genotypes. In patients with the -1082AA genotype who were studied prospectively, the mean increase in radiographic damage score (modified Sharp score of X-rays of hands and feet) during the first 6 yr was 9 +/- 9 per yr vs 19 +/- 16 per yr for patients with the genotype -1082GG (P: < 0.02). In line with these data, cultures of endotoxin-stimulated whole blood from 158 donors showed that the presence of the allele associated with less joint destruction correlated with slightly higher IL-10 production., Conclusions: Both the immunogenetic and the synovial biopsies suggest that a variation in IL-10 production is associated with joint destruction.

Objectives: To compare the activity of the CytoMegaloVirus promoter (CMV) and the Major Late promoter (MLP) in synoviocytes in vitro and in vivo. To determine the phenotype of infected cells and the induction of inflammation. To investigate the effects of the cytomegalovirus (CMV) or major late (MLP) promoter on adenovirus-mediated reporter gene transduction of synoviocytes in vitro and in vivo., Methods: After infection with adenoviral vectors harboring CMV- and MLP-driven luciferase and lacZ genes, gene expression was examined in cultured synoviocytes and in the synovium of rhesus monkeys with collagen-induced arthritis. Immunohistochemical staining for the macrophage-marker CD68 and lacZ expression was performed. Inflammation was scored in the synovial membrane of injected and non-injected joints., Results: CMV-driven reporter gene expression was found to be 6 to 10 times higher than MLP-driven gene expression in both cultured synoviocytes and monkey synovium. Both CD68 positive and CD68 negative cells were lacZ positive. Inflammation in joints injected with CMV-driven adenoviral vectors was not higher than that in MLP-driven adenoviral vectors- or non-injected joints., Conclusion: These experiments show that the CMV promoter induces higher gene expression in synoviocytes than the MLP promoter. Both fibroblast-like and macrophage-like synoviocytes can be infected with adenoviral vectors. No deleterious effects of the CMV-promoter driven adenoviral vectors were observed.

Objective: Leflunomide and methotrexate have proven to be efficacious in reducing joint inflammation and slowing destruction in clinical trials of patients with rheumatoid arthritis (RA). This study was conducted to provide more insight into the mechanism of action of these agents in synovial tissue., Methods: In a 2-center, prospective, randomized, double-blind clinical trial, we compared leflunomide (20 mg/day, after a 3-day 100 mg/day loading dose) and methotrexate (increased stepwise to 15 mg/week) treatment in patients with active RA. Paired synovial tissue biopsy samples were obtained by knee arthroscopy at baseline and after 4 months of treatment. Frozen synovial tissue sections were stained for macrophages (CD68), T cells (CD3), adhesion molecules (intercellular adhesion molecule 1 [ICAM-1], vascular cell adhesion molecule 1 [VCAM-1]), cytokines (tumor necrosis factor alpha, interleukin-1beta [IL-1beta]), matrix metalloproteinase 1 (MMP-1), and tissue inhibitor of metalloproteinases 1 (TIMP-1)., Results: Paired synovial tissue sections were available in 35 patients (16 taking leflunomide, 19 taking methotrexate). Both drugs displayed equal clinical efficacy, with 8 leflunomide-treated patients (50%) and 10 methotrexate-treated patients (53%) fulfilling the American College of Rheumatology 20% response criteria. Both compounds showed similar effects on synovial tissue: reduced numbers of macrophages and reduced ICAM-1 and VCAM-1 expression were noted after 4 months of treatment. Both leflunomide- and methotrexate-treated patients exhibited a decreased MMP-1:TIMP-1 ratio in the synovial tissue. In the subset of patients fulfilling the 20% response criteria of the American College of Rheumatology, a more pronounced reduction in the expression of ICAM-1, VCAM-1, IL-1beta, and MMP-1 was found compared with the nonresponders., Conclusion: Leflunomide and methotrexate are clinically efficacious drugs that interfere with mechanisms involved in joint inflammation and destruction of joint integrity.

Objective: B lymphocytes accumulate in the inflamed joints of patients with rheumatoid arthritis (RA) and are responsible for production of high amounts of (auto)-antibodies. The aim of this study was to determine the capacity of fibroblast-like synoviocytes (FLS) to contribute to the accumulation of synovial fluid (SF) B cells by extending their life span., Methods: Highly purified SF B cells were cultured with FLS in the presence or absence of blocking antibodies directed against cell adhesion molecules, and cell viability was determined after various time intervals by trypan blue, annexin V, propidium iodide, or Hoechst staining. Phenotypic characterization of peripheral blood and SF B cells and FLS was carried out by flow cytometry., Results: Synovial B cells, which consist predominantly of memory B cells and plasma cells (PC), undergo spontaneous cell death by apoptosis upon removal from their in vivo environment, despite expression of Bcl-2. Coculture with FLS rescued synovial B cells from apoptosis in a cell contact-dependent manner. Blocking studies using monoclonal antibodies demonstrated a role for the molecular interaction of SF B cells with vascular cell adhesion molecule 1 (VCAM-1; CD106) in FLS-induced survival. The ability of FLS to induce SF B cell survival was not related to the rheumatoid origin since FLS from non-RA patients had similar properties., Conclusion: These findings indicate a crucial role for FLS in the survival of synovial B cells at the site of inflammation in RA through the interaction with VCAM-1 expressed on FLS. Consequently, memory B cells and PC accumulation arise and persist not only as a result of maturation and recruitment of these cells, but also by active prevention from cell death by the microenvironment.

Objective: The experience with digital image analysis (DIA) in the assessment of synovial inflammation is limited. In this study we compared DIA with two currently applied methods for the evaluation of the synovium., Methods: Synovial tissue (ST) specimens were obtained by arthroscopy from knee joints of rheumatoid arthritis (RA) patients with variable disease expression and in control subjects. CD68+ macrophages and CD3+ T-cells were detected by immunohistochemical staining using two different labelling techniques. The labelled ST sections were quantified using three different analysis techniques: manual cell counting (MC), semi-quantitative analysis (SQA) and DIA., Results: We observed strongly positive correlations between the three methods when examining T-cell infiltration: between MC and SQA: sigma=0.91 (P<0. 0001), between MC and DIA: sigma=0.95 (P<0.0001), and between SQA and DIA: sigma=0.83 (P<0.0001). Similarly, for the analysis of synovial intimal macrophages, positive correlations were noted between MC and SQA (sigma=0.62; P=0.002), MC and DIA (sigma=0.56; P<0.01), and SQA and DIA (sigma=0.79; P<0.0001). Finally, the analysis of synovial sublining macrophages revealed positive correlations between MC and SQA (sigma=0.95; P<0.0001), MC and DIA (sigma=0.64; P=0.001) and SQA and DIA (sigma=0.69; P<0.0001)., Conclusion: These three different methods generated similar results. DIA offers the opportunity of a reliable and time-efficient analysis of the synovial infiltrate.

Objective: An early diagnosis in patients presenting with arthritis is important to provide information about prognosis and to initiate treatment. The objective of this study was to determine which markers applied in immunohistological analysis of synovial tissue (ST) specimens could be used to differentiate rheumatoid arthritis (RA) from other forms of arthritis., Methods: Synovial biopsies were obtained by blind needle techniques from 95 patients with early arthritis. After follow-up of at least 2 yr to verify the diagnosis, the patients could be classified as follows: RA (n=36), undifferentiated arthritis (UA; n=21), osteoarthritis (OA; n=17), reactive arthritis (ReA; n=10), ankylosing spondylitis (AS; n=3), psoriatic arthritis (PsA; n=2) and crystal-induced arthritis (CA; n=6). ST sections were analysed by immunohistochemistry using monoclonal antibodies against CD3, CD4, CD8, CD22 (B cells), CD38 (plasma cells), CD68 (macrophages) and CD55 (fibroblast-like synoviocytes)., Results: Logistic regression analysis revealed that the higher scores for the numbers of CD38+ plasma cells and CD22+ B cells in RA were the best discriminating markers comparing RA to non-RA patients (CD38: P=0.0001; CD22: P<0.05). Polychotomous regression analysis comparing three diagnostic categories (1: RA; 2: UA, ReA, AS and PsA; 3: OA and CA) also identified the score for the number of CD38+ plasma cells (P<0.0001) as well as the numbers of CD68+ macrophages in the synovial sublining (P=0.05) as discriminating markers., Conclusion: The results suggest that immunohistochemical analysis of ST specimens from early arthritis patients can be used to differentiate RA from non-RA patients. The numbers of plasma cells, B cells and macrophages are especially increased in ST of patients with RA. Future studies in early arthritis patients with clinical features which do not allow an immediate confident diagnosis may clarify the role of this test system in differential diagnosis.

Objective: Analysis of serial synovial biopsy specimens is increasingly used as an outcome measure for the evaluation of therapeutic interventions. However, observations in placebo treated groups are scarce. We describe the immunohistologic features of the synovium in placebo treated patients with RA and in those who received interleukin 10 (IL-10)., Methods: Ten patients with active RA received dosages of either placebo (n = 7) or 5 microg/kg (n = 1) or 10 microg/kg (n = 2) of recombinant human IL-10 (rhIL-10; SCH 52000, Schering-Plough, Kenilworth, NJ, USA) daily for 28 consecutive days. Synovial biopsy specimens from the knee joint were obtained by needle arthroscopy before and 4 weeks after initiation of treatment. Immunohistochemistry was performed using monoclonal antibodies specific for the following surface markers and cytokines: CD3, CD4, CD8, CD38, CD68, CD55, IL-1beta, IL-6, and tumor necrosis factor-alpha., Results: No patient exhibited clinical improvement after treatment with placebo or any rhIL-10 dosage. Microscopic analysis of synovial tissue revealed no significant change in the scores for infiltration by inflammatory cells or in the scores for the expression of cytokines after treatment., Conclusion: Studies of serial synovial biopsies from patients treated with placebo or IL-10 revealed no changes in immunohistologic scores. This suggests that the biopsy procedure itself has no effect on the features of the synovium.

Gene transfer to synovial tissue by adenoviral vectors (Ad) was studied in vitro in cultured human synoviocytes and in vivo in seven primates with arthritis. Hyperplastic synovium was efficiently transduced with Ad.lacZ in vitro and in vivo in rhesus monkeys with collagen-induced arthritis, whereas chondrocytes were not transduced. Intraarticular injection of recombinant Ad harboring the luciferase gene showed the presence of reporter gene products only in Ad-injected joints. In addition, the feasibility of synovectomy by Ad harboring the herpes simplex virus thymidine kinase gene (tk) was studied. In vitro infection of synovium from rheumatoid arthritis patients with Ad.TK, followed by administration of ganciclovir, resulted in death of >90% of the synoviocytes. By mixing Ad.TK-infected with noninfected cells, it appeared that the presence of 10% infected synoviocytes resulted in the killing of more than 85% of the synoviocytes, demonstrating a substantial bystander effect. Intraarticular injection of Ad.TK in the knees of rhesus monkeys with arthritis, followed by treatment with ganciclovir for 14 days, resulted in increased apoptotic cell death in the synovium of Ad.TK-injected as compared with noninjected joints and ablation of the synovial lining layer. The procedure revealed no toxic side effects. These data suggest that nonsurgical synovectomy by tK gene therapy is feasible.

Objective: The p53 tumor suppressor gene is overexpressed in synovial tissue (ST) from patients with longstanding rheumatoid arthritis (RA), and may contain somatic mutations. The aim of this study was to determine p53 expression in ST from RA patients in different stages of the disease, compared with disease controls., Methods: ST biopsy specimens were obtained from the knee joints of 31 RA patients in varying disease phases, 8 patients with reactive arthritis (ReA), 10 patients with inflammatory osteoarthritis (OA), and 6 control patients (4 with meniscus pathology, 2 with vascular insufficiency). ST was also obtained from the clinically uninvolved knee joints of 9 RA patients. Expression of p53 was determined by immunohistology with DO1 monoclonal antibody (mAb) in all patients and by Western blot analysis with DO7 mAb in a subgroup of the patients., Results: The p53 protein was detected by immunohistology in 10 of the 13 patients with early RA (duration <6 months) and in 12 of the 14 patients with longstanding RA (duration >5 years). The p53 protein was also demonstrated in clinically uninvolved knee joints. Western blots revealed immunoreactive p53 in ST extracts from all RA patients. Expression of p53 was about twice as high in ST from patients with longstanding RA as in early RA samples, but the difference did not reach statistical significance. Small amounts of p53 were also detected in ST from ReA and OA patients, although the expression in RA synovium was significantly higher. Immunohistologic analysis of normal ST gave negative results for p53., Conclusion: This study shows that p53 overexpression is specific for RA, compared with OA and ReA. This phenomenon is probably secondary to increased production of wild-type p53 protein in response to DNA damage and secondary to somatic mutations caused by the genotoxic local environment in inflamed ST. Of interest, p53 overexpression can also be found in the earliest stages of RA and in clinically uninvolved joints.

Objective: Fibroblast-like synoviocytes (FLS) express decay-accelerating factor (CD55) at high levels. Recently, it was found that CD55 is a specific cellular ligand for the 7-span transmembrane receptor CD97. The objective of this study was to define the expression of this receptor-ligand pair in synovial tissue (ST) to provide more insight into the interaction between FLS and surrounding cells., Methods: Antibodies against CD97 and CD55 were used for immunohistologic analysis of synovial biopsy specimens from 16 patients with rheumatoid arthritis (RA) and 15 patients with osteoarthritis (OA). In addition, an enzyme-linked immunosorbent assay system was used to determine the expression of soluble CD97 (sCD97) in synovial fluid (SF) from 30 patients with RA, 13 with OA, and 10 with reactive arthritis (ReA)., Results: In both RA and OA ST sections, strong expression of CD55 was confirmed on FLS in the intimal lining layer, where it was also found that all macrophages expressed CD97. The percentage of macrophages that expressed CD97 was lower in the synovial sublining (P = 0.005). The mean levels of sCD97 in SF were significantly higher in RA patients than in patients with OA or ReA (P < 0.0001)., Conclusion: These results suggest that FLS are able to interact with macrophages via the CD97/CD55 receptor-ligand system. In this respect, the CD97/CD55 pair may account for the specific architecture of the intimal lining layer and may be of primary importance in maintaining and amplifying synovial inflammation. The specific increase in sCD97 levels in RA SF might be related to the presence of activated proteolytic systems or to the increase in synovial mass, rather than a consequence of local receptor-ligand interaction.

The cellular infiltrates and cytokine patterns in synovial tissue (ST) from patients with rheumatoid arthritis (RA) and reactive arthritis (ReA) were compared in order to determine the mechanisms responsible for the chronic and destructive course of RA. Since the results could be influenced by differences in disease duration, ST was studied from patients in both early and late stages of the disease. Ten patients had early RA (< 1 year), ten long-standing RA (> 1 year), six early ReA (< 1 year), and five long-standing ReA (> 1 year). Histological analysis demonstrated that the scores for infiltration by lymphocytes and plasma cells, and the scores for inflammation, were significantly higher in RA than in ReA. Immunolabelling studies showed that in particular, the scores for infiltration by CD38+ plasma cells, granzyme B+ cells, and interferon-gamma (IFN gamma)+ cells were significantly higher in RA than in ReA. The results were independent of the disease duration. The increased number of lymphocytes, plasma cells, and granzyme B+ cells in rheumatoid synovial tissue supports the paradigm that RA is the result of specific immune recognition in the joint and that granzyme B+ cells play an important role in joint destruction.

We compared the state of activation and proliferation of T cells in synovial tissue (ST) from rheumatoid arthritis (RA) patients in early and late stages of the disease to find out whether T-cell-driven immune responses vary during the course of the disease. ST was obtained from 12 patients with early RA (< 1 year) and 12 patients with longstanding RA (> 5 years). T cells and interferon-gamma (IFN-gamma)-positive cells were detected in ST using immunohistologic methods. To determine the percentage of T cells expressing the interleukin-2 receptor, IFN-gamma, or the proliferation associated antigen Ki-67, immunofluorescence double-staining techniques were used. The scores for the number of T cells and for the expression of IFN-gamma as well as the percentages of T cells expressing CD25, IFN-gamma, or Ki-67 in rheumatoid synovium were not dependent on disease duration. These results do not support the assumption that the responsiveness of T cells in ST of RA patients differs between early and late stages of the disease. The data indicate that at present no arguments exist that the effect of T-cell-directed interventions on synovial inflammation might vary in different stages of the disease., (Copyright 1998 Academic Press.)

Methotrexate (MTX) is one of the most widely prescribed drugs in the treatment of rheumatoid arthritis (RA). The mechanism by which MTX exerts its anti-rheumatic effect has not yet been defined. The aim of the present study was to investigate the effect of MTX treatment (7.5-15 mg/week) on synovial tissue in RA. For this purpose, synovial biopsies were taken from 11 RA patients before and 16 weeks after initiation of MTX therapy. Immunohistochemistry was performed using monoclonal antibodies (MAb) specific for CD3, CD4, CD8, CD22, CD25, CD38, CD68, MAb67, Ki67, interferon gamma (IFN-gamma), interleukin (IL)-1alpha, IL-1beta, tumour necrosis factor alpha (TNF-alpha), E-selectin, ICAM-1 and VCAM-1. All parameters for disease activity improved during the period of treatment. Immunohistochemical analysis revealed a statistically significant decrease in scores for CD3, CD8, CD38, CD68, Ki67, IL-1beta, TNF-alpha and the adhesion molecules E-selectin and VCAM-1. The observed decrease in synovial scores for inflammatory cells, monokines and adhesion molecules suggests that the anti-inflammatory effect of MTX is, in part, dependent on a reduction in monokine-inducible vascular adhesion molecules and subsequent reduction of cell traffic into joints.

In rheumatoid arthritis (RA), the functional status of T cells is incompletely understood. Synovial T cells display phenotypic evidence of former activation, but there is poor production of T cell-derived cytokines in the synovium. In addition, synovial T cell proliferation upon mitogenic and antigenic stimulation was decreased compared with that in peripheral blood T cells. Moreover, previous reports revealed that early Ca2+ rises induced by TCR/CD3 stimulation were decreased in RA T cells compared with those in healthy controls. To investigate the molecular mechanisms of RA synovial T cell hyporesponsiveness, we analyzed the TCR/CD3-mediated protein tyrosine phosphorylation in RA peripheral blood and synovial fluid (SF) T cells. SF T cells exhibited a decreased overall tyrosine phosphorylation pattern upon stimulation. Most notably, the induction of phosphorylation of p38 was virtually absent. Moreover, we found that tyrosine phosphorylation of the TCR zeta-chain, one of the most proximal events in TCR signaling, is clearly diminished in RA SF T cells. The decrease in tyrosine phosphorylation was accompanied by a decrease in detectable levels of zeta-protein within synovial T cells. These results suggest that a defective TCR signaling underlies the hyporesponsiveness of synovial T cells in RA.

Recently, a new player in the cytokine network has been described that is produced by monocytes and can be detected in the rheumatoid synovium: interleukin-15 (IL-15). Since this cytokine may play a role in the accumulation and activation of T-cells, B-cells, and natural killer (NK) cells characteristic of synovial tissue (ST) from patients with rheumatoid arthritis (RA), the expression of IL-15 was studied in ST from RA patients in comparison with ST from patients with reactive arthritis (ReA) and osteoarthritis (OA) and the phenotype of IL-15-positive cells was determined. IL-15 expression was investigated by immunohistochemical analysis of ST from ten patients with RA, ten patients with Yersinia enterocolitica-induced ReA, and nine patients with OA. The immunohistological findings were quantified and the results obtained in the different patient groups were compared. To determine the phenotype of IL-15-expressing cells, double-labelling immunofluorescence was performed. The expression of IL-15 was significantly higher in ST from patients with RA than in ST from patients with ReA or OA. In double-label experiments, co-expression was observed with markers for macrophages, T-cells, and NK cells. The composition of the cellular infiltrate in the synovium of patients with RA might be partly explained by the specific increase in expression of IL-15 in rheumatoid ST. It can be speculated that IL-15 production by inflammatory cells other than macrophages may occur in the rheumatoid synovium.

Objective: To define variations in the cellular infiltrate and in the expression of monokines in synovial tissue (ST) from rheumatoid arthritis (RA) patients with different durations of disease and different levels of disease activity., Methods: The immunohistologic features of synovial biopsy specimens from 31 patients with early RA (< 1 year) and 35 patients with longstanding RA (> 5 years) were compared. The possible associations between these features and local disease activity, as measured by the score for pain in the biopsied knee joint were also evaluated., Results: The immunohistologic features were not dependent on disease duration. We found a positive correlation between the scores for knee pain and the semiquantitative scores for the number of macrophages, as well as the expression of interleukin-6 and tumor necrosis factor alpha, whereas the correlation with the scores for CD4+ T cells was negative. Multivariate analysis showed that these correlations were highly statistically significant (P < 0.003)., Conclusion: The results do not support the view that inflammatory mechanisms in the synovial tissues of RA patients differ between early and late stages of the disease. The findings presented here are consistent with the concept that early RA is the result of a synovitis process of longer duration and that macrophage-derived cytokines play an important role in maintaining the clinical signs of inflammation.

To gain insight into the nature of the immune response with respect to accumulation and composition of the T-cell receptor (TCR) repertoire in synovial tissue in rheumatoid arthritis (RA), we have determined the nucleotide sequence of TCRBV regions transcribed by T lymphocytes derived from synovial tissue. Synovial tissue was obtained by needle biopsies from three different sites of the same joint in two early RA patients. We found that the TCRBV region repertoire among synovial tissue-infiltrating mononuclear cells was heterogeneous when the different biopsies taken from each patient were compared. However, DNA sequence analysis of TCRBV rearrangements of synovial T lymphocytes showed conserved amino acid usage profiles in the CDR3 domains of different TCRBV regions, which exhibited an individual specific character. These CDR3 motifs were not present in paired samples of peripheral blood. The existence of homologous CDR3 amino acid profiles within the TCRBV regions derived from synovial tissue is indicative of an antigen-driven immune response.

Objective: The effect of chimeric anti-tumor necrosis factor alpha (TNF alpha) monoclonal antibody (MAb) therapy on synovial inflammation was studied in order to address the hypothesis that anti-TNF alpha therapy leads to down-regulation of adhesion molecules and a decrease in inflammatory cell influx in synovial tissue (ST)., Methods: The immunohistologic features of synovial biopsy specimens, both before and 4 weeks after anti-TNF alpha MAb (cA2) therapy, were studied in 14 patients with rheumatoid arthritis (RA). The patients either received a placebo (n = 2), or were given intravenous doses of cA2 at 10 mg/kg (n = 5) or 20 mg/kg (n = 7)., Results: A significant (P < 0.03) reduction in the mean scores for T cells and for the adhesion molecules, vascular cell adhesion molecule 1 and E-selectin, was observed after therapy with 10 mg/kg or 20 mg/kg of cA2 in RA patients., Conclusion: The reduced expression of adhesion molecules, and the decrease in cellularity of rheumatoid ST after cA2 administration support the hypothesis that the antiinflammatory effect of anti-TNF alpha therapy might be partly explained by down-regulation of cytokine-inducible vascular adhesion molecules in ST, with a consequent reduction of cell traffic into joints.

Proteolytic joint destruction in inflammatory and non-inflammatory arthropathy is believed to be mediated, at least in part, by the plasminogen activation (PA) system. To further investigate possible involvement of the PA system, we quantified immunoreactive urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), both plasminogen activator inhibitors (PAI-1 and PAI-2) and u-PA-receptor (u-PAR) in synovial tissue extracts of 14 patients with rheumatoid arthritis (RA) and 12 with osteoarthritis (OA). u-PA, PAI-1, PAI-2 and u-PAR concentrations were significantly higher in RA than in OA patients. t-PA antigen levels were significantly lower in RA than in OA synovial tissue extracts. Immunohistochemistry was performed to compare the distribution and staining intensity of these components in samples of RA and OA synovial tissue. Intense immunostaining of u-PA, u-PAR, PAI-1 and, to a lesser degree, PAI-2 was observed predominantly in the synovial lining of RA patients. In OA patients, u-PA, PAI-1, PAI-2 and u-PAR were barely detectable. t-PA immunostaining was restricted to the endothelial side of vascular walls in both groups. We conclude that the observed increase of u-PA, u-PAR and PAI expression, distributed mainly in the synovial lining area of proliferative and invasively growing synovial tissue in RA patients, supports a pathogenic role for the PA system in destructive arthritis. Depressed t-PA-mediated plasminogen activation might contribute to delayed intra-articular fibrin removal.

Data concerning the presence of T-cell-derived cytokines in the rheumatic joint are conflicting, challenging the hypothesis that rheumatoid arthritis (RA) is a T-cell-mediated disease. In this study synovial tissue specimens of 11 patients with RA and eight patients with osteoarthritis (OA) were stained for interferon-gamma (IFN-gamma) and its receptor. The level of expression of IFN-gamma was compared with that in tissue specimens of delayed-type hypersensitivity (DTH) reactions of the skin and of chronic tonsillitis. Furthermore, the percentage of T-lymphocytes which stained positive for IFN-gamma was determined using double staining techniques. IFN-gamma and its receptor were detected in all patients with RA and in 7/8 and 3/8, respectively, of patients with OA. Expression of IFN-gamma (P<0.02) and IFN-gamma receptor (P<0.01) in synovial tissue of patients with RA was more abundant compared with that in patients with OA. Although IFN-gamma could be detected in RA synovial tissue, the level of expression was less when compared with DTH reactions of the skin and tonsillitis. The percentage of CD3+ cells being positive for IFN-gamma was approximately 1% in RA, whereas in DTH reactions of the skin it was >90% and in tonsillitis approximately 30%. We conclude that the presence of IFN-gamma and its receptor in RA synovial tissue suggests a role for this cytokine in the ongoing immunological reaction of the inflamed joint.

Our objective was to investigate the mechanism of accumulation of 99Tcm-labelled non-specific polyclonal human immunoglobulin (99Tcm-HIG) in inflamed synovial tissue (ST) in an experimental animal model of arthritis. Following 99Tcm-HIG scintigraphy, the in vivo localization of 99Tcm-HIG in the ST of knee joints of rats with adjuvant arthritis was studied using immunohistochemical techniques. In addition, the in vitro binding of 99Tcm-HIG to extracellular matrix proteins was analysed by means of immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). After 99Tcm-HIG scintigraphy, 99Tcm-HI was detected in the ST of rats with adjuvant arthritis. 99Tcm-HIG was diffusely distributed and not bound to cells. In vitro incubation of 99Tcm-HIG on the ST of rats with adjuvant arthritis revealed binding of 99Tcm-HIG to inflamed, but not to non-inflamed, ST. In addition, specific binding of 99Tcm-HIG to fibronectin, fibrin, collagen type I and III was demonstrated by ELISA. We conclude that the accumulation of 99Tcm-HIG in inflamed ST can be explained by the binding of 99Tcm-HIG to extracellular matrix proteins.

The objective of this paper is to define the expression of adhesion molecules in synovial tissue (ST) from patients with rheumatoid arthritis (RA) with respect to disease duration. Antibodies against adhesion molecules were used for immunohistochemistry to examine ST sections from 11 patients with early RA ( < 1 year), 14 patients with long-standing RA ( > 5 years), and 15 patients with osteoarthritis (OA). Increased cellular infiltration and increased expression of E-selectin, ICAM-1, VCAM-1, PECAM-1, VLA-4, and Mac-1 were found in ST from patients with RA compared to ST from patients with OA. The immunohistological findings were similar for the different stages of RA. The upregulation of adhesion molecules in ST of patients with RA of < 1 year's duration suggests the activation of chronic inflammatory processes in these patients. Therefore, the mechanisms by which therapies directed toward these adhesion molecules exert their effects are likely to be similar for patients with so-called early RA and patients with long-standing RA.

Objective: To define the expression and the phenotype of granzyme (Gran) A and B positive cytotoxic cells in synovial tissue (ST) from patients with rheumatoid arthritis (RA) with respect to disease duration and activity., Methods: Using antibodies against GranA and GranB, which serve as markers of activated natural killer (NK) cells and cytotoxic T lymphocytes, ST sections from 10 patients with early RA, 10 patients with longstanding RA, and 10 patients with osteoarthritis were examined. The phenotype of Gran+ cells was determined with double-labeling techniques., Results: Gran+ cells, the majority of which were NK cells, were found in ST from patients in all groups. Several of these cells did not express the surface markers CD16, CD56, and CD57. The highest ST expression of GranB was found in patients with early RA. In RA patients, there was a positive correlation of GranB expression with serum levels of acute-phase reactants, but not with histologic scores for inflammation., Conclusion: Gran+ cells are mainly NK cells, a substantial proportion of which do not express conventional NK cell surface markers. GranB expression is specifically increased in the synovial tissues of patients with RA of short duration.

In this study we have analyzed the TCR V alpha and V beta regions at the DNA level in the CD4+CD45RO+ memory T cell population of synovial tissue infiltrating T lymphocytes of three rheumatoid arthritis (RA) patients and one patient with chronic arthritis. Cell lines of CD4+CD45RO+, CD4+CD45RO-, CD8+CD45RO+ and CD8+CD45RO- T lymphocyte populations were generated following FACS cell sorting of freshly isolated synovial tissue mononuclear cell infiltrates (STMC) and of freshly isolated peripheral blood mononuclear cells (PBMC) of these patients. The phenotypic and molecular analyses have revealed the following. (i) The TCR repertoires of tissue infiltrating T lymphocytes in the various subsets were extensive on the basis of TCR V gene family usage. (ii) Furthermore, each patient displayed individual specific TCR V gene expression patterns in the various STMC and PBMC derived T cell subsets. However, the majority of these arthritis patients manifested increased expression of multiple TCR V gene families in the synovial tissue derived CD4+CD45RO+ T cell population when compared with the peripheral blood derived CD4+CD45RO+ subset. Of these gene families, we found enhanced expression of the TCR V alpha 7 and V beta 11 gene segments in synovial tissue to be shared by all four patients analyzed. (iii) Nucleotide sequence analysis of the CDR3 regions of a number of TCR V regions in the CD4+CD45RO+ T cell subsets has revealed that the CDR3 regions comprised within synovial tissue derived TCR V regions differed from those found in peripheral blood derived TCR V regions. These differences in CDR3 diversity might be the consequence of a specific interaction with particular MHC-peptide complexes expressed at the site of inflammation. (iv) The CDR3 region analysis also showed individual specific amino acid motifs within the N-D-N regions of all analyzed TCR V beta genes derived from PBMC as well as STMC.

Objective: To investigate synergistic interaction between cyclosporine (Cy) and chloroquine (Chl) in an in vitro system, with regard to interferon-gamma (IFN) production by OKT3 activated T cell clones., Methods: CD4+ and CD8+ T cell clones, derived from synovial tissue of a patient with rheumatoid arthritis (RA) were activated with plastic coated OKT3 monoclonal antibody in the presence or absence of various concentrations of Cy, Chl and their combinations. After 24 h of incubation the supernatants were assayed for IFN by ELISA:, Results: Cy as well as Chl were able to completely inhibit in a concentration dependent fashion the IFN production by CD4+ and CD8+ T cell clones. Combinations of Cy and Chl, which in themselves give minor inhibition of IFN production, were able to inhibit in a synergistically enhanced fashion the production of IFN by these clones. The synergy was formally proven by the construction of isoboles. This synergy was most pronounced when drug concentrations were used which individually gave minor inhibition of IFN production., Conclusion: We conclude that the results of our in vitro experiments may give rise to further investigation of the promising combination of Cy and Chl in the treatment of RA.

The presence of activated T cells in the synovial membrane of patients with rheumatoid arthritis (RA) suggests a role for these cells in the pathogenesis of the disease. Recent evidence indicates that human T cells may fall into functional categories dependent on their cytokine profile and cytotoxic capacity. The human Th1 subset is cytolytic and produces high levels of IFN-gamma whereas the Th2 type of T cell produces IL-4. In order to investigate whether Th1 or Th2 type cells are present in the inflammatory synovial membrane in RA, a panel of synovial membrane derived T-cell clones (n = 19) was generated and studied functionally. Anti-CD3-induced cytotoxicity assays were performed to demonstrate the cytotoxic potential of clones. Except for two, all clones were cytolytic in this test. Clone cells were activated to initiate cytokine production and assessment of the cytokine levels showed that all clones produced large amounts of IFN-gamma (18 out of 19 clones: over 50,000 pg/ml) whereas IL-4 was absent or present in minimal amounts (17 out of 19 clones: less than 1000 pg/ml). The production of IL-1, IL-2 and IL-6 was variable. The functional characteristics of the clones studied indicate that they may resemble the Th1 subtype of T cells. Our data suggest a relation between Th1-type functions the chronic inflammation characteristic of RA.

In this study T-cell receptor (TcR) beta-chain gene rearrangements of T-cell lines prepared from multiple sites (n = 92) of synovial tissue derived from both knees of a patient with rheumatoid arthritis were analysed. In the majority of T-cell lines, dominant TcR beta-chain gene rearrangements were detected, involving C beta 1 as well as C beta 2. The dominant rearrangement patterns of T-cell lines from different tissue fragments showed significant variability, but some of the DNA restriction fragments were shared by T-cell lines from multiple sites in both knees. The latter observation suggests that identical T-cell clones may be present at different sites in the synovial tissue and in different joints. However, since many T-cell lines yielded different rearrangement patterns, these data also indicate considerable heterogeneity of T cells in the joints. Apart from theoretical implications, this TcR heterogeneity of T cells within an individual patient also has practical consequences for studies on synovial T cells obtained by biopsy.

It has been suggested that the reduced resistance of patients with rheumatoid arthritis (RA) to bacterial joint infections may be due in part to polymorphonuclear cell (PMN) function. To obtain further insight into the mechanism that contribute to the increased susceptibility of RA patients to such infections we investigated the influence of different solid surfaces on the ingestion of various bacterial strains by PMN. Both in the presence and absence of serum, phagocytosis of bacteria by PMN was significantly lower on monolayers of synovial fibroblasts as compared to monolayers of endothelial cells and embryonic fibroblasts. It could be shown that the relative influence of the solid surface on the results of the phagocytosis assay increased when decreasing concentrations of purified IgG were used. The results of this study suggested that the effect of synovial fibroblasts on PMN may lead to reduced clearance of bacteria from the joint.

The dominant presence of specific T-cell populations in the rheumatoid joint as detected by Southern blot analysis of T cell receptor (TCR) gene rearrangements would indicate local antigen recognition and T cell proliferation. We therefore studied TCR beta chain gene rearrangements using a C beta 2 probe in paired samples of T cell populations from synovial tissue and peripheral blood (n = 6) as well as synovial fluid (n = 16) and peripheral blood (n = 18) of patients with rheumatoid arthritis (RA). Peripheral blood mononuclear cells from healthy donors (n = 7) served as a control. T cells were studied directly after isolation or after non-specific expansion with OKT3 monoclonal antibody (MoAb) and T cell growth factor (TCGF). DNA samples were digested with EcoRI and HindIII to detect rearrangements to C beta 1 and C beta 2, respectively. Extra bands were detected in all EcoRI-digested DNA samples prepared from both freshly isolated and non-specifically expanded T cell populations of patients and healthy donors, possibly representing 'common' (V-) D-J rearrangements. Dominant rearrangements were found in only two out of 16 synovial fluid T cell populations (one freshly isolated and one expanded) and not in peripheral blood or synovial tissue derived T cell populations. No extra bands were detected in HindIII-digested DNA samples. To investigate the effect of in vitro culture techniques on rearrangement patterns we studied DNA samples prepared from synovial tissue T cells obtained both by outgrowth from tissue with TCGF or by enzyme digestion and subsequent expansion either with TCGF or with OKT3 MoAb and TCGF. Whereas the latter T cell population yielded 'common' rearrangements, the former T cell populations yielded different dominant rearrangements. These data indicate that oligoclonality of the T cell populations in synovial tissue and synovial fluid of patients with RA is a rare event. The data also show the influence of in vitro culture techniques on the result of TCR gene rearrangement analysis.

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function.

T-cell receptor (TcR) beta and delta gene rearrangements were studied in anti-CD3 expanded T-cell populations cultured from the synovial membrane (SM) (n = 5) or synovial fluid (SF) (n = 2) of rheumatoid arthritis (RA) patients. Dominant TcR beta-chain gene rearrangements to C beta 1 were demonstrated in all the patients tested and 1-3 expanded clones per patient were found. Clonal rearrangements to C beta 2 were detected in one SM sample (two clones) and one SF sample (one clone). The TcR delta gene was deleted in all the samples tested. We conclude that clonal dominance may be found in expanded T-cell populations from SM and SF of RA patients. Multiple clones may be present, either using the C beta 1 or C beta 2 gene segment.