Immunological characteristics of lymphocytes in synovial fluid and peripheral blood in patients with rheumatoid arthritis.

The investigation has been carried out on 30 patients with rheumatoid arthritis. Rosettes (E, EA, EAC) technique and immunofluorescence test were used to detect the surface markers of lymphocytes. Cytotoxic activity of T and K cells in vitro was established. It has been proved that cells occurred to a larger extend in synovial fluid than in the peripheral blood. B cells and lymphocytes with Fc receptors were only a few in synovial fluid. The percentage ofFc receptors bearing cells was higher in the peripheral blood of patients with rheumatoid arthritis than that in peripheral blood of the control group. Cytotoxic activity of K-cell was low in synovial fluid. PHA-cell mediated cytotoxicity was low in peripheral blood as well as in synovial fluid of patients with rheumatoid arthritis.     

Analysis of function-associated receptor molecules on peripheral blood and synovial fluid granulocytes from patients with rheumatoid and reactive arthritis

In this study we report the expression pattern of 13 different function-associated surface molecules on synovial fluid and peripheral blood granulocytes from rheumatoid and reactive arthritis patients. We found increased expression of the complement receptors 1 (CD35) and 3 (CD11b) and of the activation-associated antigens CD67, CD24, and M5 on synovial fluid granulocytes from rheumatoid and/or reactive arthritis patients compared to autologous peripheral blood granulocytes. In addition, synovial fluid granulocytes expressed IgG Fc receptor 1 (CD64) and complement receptor 4 (CD11c), neither of which can be found on peripheral blood granulocytes. Peripheral blood granulocytes from rheumatoid and reactive arthritis patients expressed higher levels of leucocyte function-associated antigen 1 (CD11a) and of the membrane proteins CD31, CD24, M5, and M6 compared to peripheral blood granulocytes from healthy controls and patients with degenerative joint disease. No significant differences in the expression of any of the molecules studied could be observed between cells from rheumatoid and cells from reactive arthritis patients, suggesting a similar activation process for granulocytes in these two diseases.     

Clonality of T lymphocytes expanded with IL-2 from rheumatoid arthritis peripheral blood,synovial fluid and synovial membrane

The association of rheumatoid arthritis (RA) with particular MHC class II genes suggests that autoantigen-spccific T cell clones present in joints could be central to the pathogenesis of the disease. Previous investigations on the clonal diversity of T cells infiltrating the rheumatoid synovial membrane have yielded conflicting results. With the use of Southern blot analysis, we investigated the clonalily of rheumatoid T cell lines expanded from peripheral blood, synovial fluid and synovial tissue. From peripheral blood lymphocyte (PBL) of RA patients and healthy normal controls, we also checked the consequences of two different culture conditions on the clonality of these cell lines. From control PBL, we found that in vitro non-specific expansion of non-clonal T cell populations does not create artefactual clonal selection. However, growing T cells in ritro with IL-2 seems to be able to lead to preferential expansion of cells bearing IL-2 receptor (IL-2R). We identified such in vivo activated IL-2-sensitive T cell clones frequently in RA synovial tissue (8/13) and more rarely in synovial fluid and peripheral blood (3/12). One patient presents the same T cell receptor gene rearrangements in synovial membrane of two affected joints. In RA synovial tissue, the frequency of these IL-2-responsive T cells is most prevalent among actively inflamed membranes removed early in the disease process. The role and the relevance to the disease of these I L-2-responsivc T cells remain to be elucidated.     

Antibody-dependent direct cytotoxicity of human lymphocytes. II. Studies on peripheral blood lymphocytes, synovial fluid cells and sera from patients with rheumatoid arthritis.

Antibody-dependent direct cytotoxicity (ADDC) by human lymphocytes was evaluated in patients with rheumatoid arthritis and normal controls. Purified peripheral blood and synovial fluid lymphocytes mediated normal ADDC when compared to control subjects. No correlation could be obtained between the percentages of T, B and null cells in effector cell populations and the degree of 51Cr released from target cells. Sera from 50% of patients with rheumatoid arthritis inhibited ADDC by normal lymphocytes; the degree of inhibition did not correlate with the titre of IgM rheumatoid factor. The pathogenic implications of these findings are discussed.     

Electrophoretic behaviour of blood and synovial fluid lymphocytes in rheumatoid arthritis.

Probit analysis of the electrophoretic mobilities of human blood lymphocytes identifies at least three main subpopulations. According to their rate of movement in an electrical field, the subpopulations are referred to as the fast, intermediate and slow cell distributions. Lymphocytes of the fast and intermediate populations appear to be T cells, while the slow cell population includes cells with B cell characteristics. Compared with normal subjects, lymphocytes of intermediate mobility are significantly increased in the blood of rheumatoid patients and comprise a major fraction of the lymphocyte exudate in rheumatoid synovial fluid.     

The influence of synovial fluid exudates from patients with rheumatoid arthritis on the proliferation of peripheral blood lymphocytes and the prostanoid release from monocytes

The present study was designed to characterize leukocytes of patients with rheumatoid arthritis (RA) with regard to proliferation of peripheral blood lymphocytes (PBL) and prostanoid release from circulating monocytes (M. Compared to cells of healthy individuals, PBL from RA patients exhibited a reduced mitogenic response to concanavalin A (Con A) which was associated with an increased capacity of circulating M to synthesize PGE and thromboxane B₂ (TXB₂). Addition of synovial fluid exudates of RA patients (RA-SFE) to peripheral blood leucocyte cultures produced three effects: A spontaneous proliferation of normal and RA-PBL, a reduction of the Con A response of normal and RA-PBL, and an enhanced release of PGE and TXB₂ from RA-M only. To elucidate the cellular origin of these activities, normal and RA-PBL were incubated with supernatants (SNT) of synovial cell cultures from RA patients and patients with non-RA joint diseases. SNT from Con A-stimulated synovial lymphocytes of both RA and control patients induced a spontaneous proliferation of normal and RA-PBL. In contrast, SNT from non-lymphoid adherent synovial cells of RA and control patients reduced the Con A response of normal and RA-PBL but a striking difference was noted in that an enhanced PGE and TXB₂ release occurred only from M of RA patients.     

Migration of blood and synovial fluid neutrophils obtained from patients with rheumatoid arthritis.

The unstimulated random migration and the serum-induced chemokinesis of neutrophils obtained from the peripheral blood of patients with rheumatoid arthritis (n = 19) was not different from those of controls (n = 20). However, neutrophils obtained from the joint fluid of rheumatoid patients (n = 10) demonstrated a reduced serum-induced chemokinesis which was correlated with the amount of immune complexes present in the synovial fluid. The chemotactic response of peripheral blood neutrophils from subjects with rheumatoid arthritis taking aspirin (n =11) was increased while that of those rheumatoid subjects not taking aspirin (n = 8) was the same as controls. It is concluded that although there is no impairment of the in vitro migratory capacities of peripheral blood neutrophils obtained from patients with rheumatoid arthritis, neutrophils obtained from synovial fluids exhibit a marked defect in chemokinesis which may be related to the ingestion of immune complexes within the joint space.     

Characterization of synovial T lymphocytes in rheumatoid arthritis. I. Production of IL-2 dependent T cell clones from synovial fluid and peripheral blood.

Lymphocytes obtained from the peripheral blood (PBL) or synovial fluids (SFL) of patients with rheumatoid arthritis (RA) or other inflammatory joint diseases were compared with the PBL from normal individuals, by cloning under limiting dilution conditions in the presence of interleukin 2 (IL-2). The precursor frequency estimates of IL-2 responsive cells from these sources did not differ appreciably. However there were marked differences in the surface marker phenotypes of the clones derived from the PBL as compared to SFL. There was a predominance of OKT4-8+ cells in SFL from RA and non RA donors with inflammatory joint disease while PBL from all sources showed a marked prevalence of OKT4+8- cells. Comparison of precursor frequencies in the presence of PBL and SFL indicated that there were variations in the capacities of the SFL and PBL IL-2 dependent cells to grow on these fillers. SFL derived cells grew equally well on PBL or SFL filler, while PBL clones grew efficiently only on PBL fillers. Collectively these results indicate that there are marked differences in the surface phenotypes and growth requirements of IL-2 responsive SFL as compared to PBL.     

Allotype-dependent stimulation of peripheral blood and synovial lymphocytes by IgG3 in rheumatoid arthritis.

The immunopathologic process of rheumatoid arthritis (RA) is primarily expressed in the synovium where rheumatoid factor (RF) synthesis is concentrated. We hypothesized that RF synthesized by rheumatoid synovial cells (RSC) may be driven via a T cell-mediated immune response developed against IgG3 epitopes. To identify and characterize specific RSC RF epitopes and T cell antigens, two 28 amino acid peptides homologous with the C-terminus of IgG1 (P1) and IgG3 [G3m(5)] (P3) were synthesized and used in RF-binding studies and lymphocyte proliferation assays. Our results indicate that (i) the C-terminus of the CH3 domain contains epitopes for IgG3-reactive RSC RF; (ii) IgG3-reactive RSC RF binds primarily to IgG3 [G3m(5)]; (iii) P3 stimulated proliferation of T lymphocytes from both RA peripheral blood and RSC; and (iv) RF production was enhanced by P3 in selected RA cell cultures. These observations suggest that the C-terminus of IgG3 allotype G3m(5) may be important in T cell activation and RF production in RA.     

Tg cells in peripheral blood lymphocytes from patients with rheumatoid arthritis

Using combinations of methods for detecting Fc receptors and B lymphocytes, non-B, non-T lymphocytes and T lymphocytes we have shown T_G cells to be significantly increased in rheumatoid arthritis (RA) patients' peripheral blood lymphocytes (PBL). The possible significance of this finding to the disease process is discussed.     

Expression of cytolytic mediators by synovial fluid lymphocytes in rheumatoid arthritis.

To understand the role of cytolytic lymphocytes in the pathogenesis of rheumatoid arthritis, we investigated the expression of lymphocyte cytotoxicity mediators, perforin, and serine esterases, in lymphocytes derived from the synovial fluid of 15 patients with rheumatoid arthritis. Previous work has shown that CD8+ lymphocytes that possess markers of activation appear to be present in rheumatoid arthritis (RA). By means of in situ hybridization techniques and immunohistochemical analysis, the authors show that perforin and two serine esterases (serine esterase 1/Hanukah factor/granzyme A, and serine esterase 2/granzyme B) are expressed by subpopulations of CD8+ and CD56+ lymphocytes obtained from synovial fluid. The presence of these cytotoxic mediators suggests a possible mechanism for tissue damage, and provides evidence implicating cytolytic lymphocytes in the pathogenesis of RA.     

Elevation of a gamma delta T cell subset in peripheral blood and synovial fluid of patients with rheumatoid arthritis.

We examined the levels of TcR delta 1+ T cells (total gamma delta T cell) and delta TCS1+ (gamma delta T cell subset) T cells in the peripheral blood (PB) and synovial fluid (SF) of 16 patients with rheumatoid arthritis (RA) and compared them to the levels in PB of patients with Felty's syndrome (FS) and 21 healthy control subjects (NML). Synovial fluid from eight patients with seronegative spondyloarthropathies (SSA) was also examined. The results demonstrated elevated levels of the delta TCS1+ subset in the PB of RA and FS patients relative to NML (P less than 0.05). No such differences were observed in the levels of PB TcR delta 1+ T cells. The results did not appear to reflect a non-specific inflammatory response since delta TCS1 T cells were elevated in the SF of RA patients relative to SSA SF and NML PB. delta TCS1 T cells in SSA PB and SSA SF were comparable to NML PB. TcR delta 1+ T cells levels in RA SF were higher than SSA SF levels but were comparable to those of NML PB. Taken together, the results support a pathogenic role for delta TCS1+ T cells in RA.     

Inflammatory cytokines and enzymes in synovial fluid of patients with rheumatoid arthritis and other arthritides.

Cytokines and lysosomal enzymes, which are produced by inflammatory cells, play a role in inflammation. We have found that synovial fluid (SF) in rheumatoid and septic arthritis contained a large number of white blood cells (WBCs) and high levels of cytokines and enzymes, while in contrast the SF of osteoarthritis and traumatic arthritis did not contain significant amounts. Measurements of WBCs, cytokines and enzymes in SF are useful for evaluating clinical disease activity. Assays for WBCs and enzymes are simple and rapid when compared to those for cytokines.     

Mucin from rheumatoid arthritis synovial fluid enhances interleukin-6 production by human peripheral blood mononuclear cells

The carbohydrate chains represented by mucins (MUCs) are expressed by a variety of normal and malignant secretory epithelial cells and induce a variety of immunoreactions. To find new mucins related to the pathogenesis of rheumatoid arthritis (RA), we examined high-molecular-weight molecules inducing cytokines on human peripheral blood mononuclear cells (PBMCs) in synovial fluid from affected joints. We found a high-molecular-weight substance that induces interleukin 6 production on PBMCs in RA synovial fluid on gel filtration. MUC-1 was present in the resulting fractions, although they had been purified by CsCl density gradient centrifugation. We also found that MUC-1 was expressed on synovial cells and infiltrating inflammatory mononuclear cells on the sublining layer and lymphoid follicles in RA synovial tissues. CD68-positive superficial synovial cells colocalized with MUC-1 and CD68-positive macrophages were in contact with MUC-1-positive mononuclear cells. These findings imply that mucins, including MUC-1, may be related to immunoinflammatory reactions in the pathogenesis of RA.     

Leflunomide,a novel immunomodulating drug,inhibits homotypic adhesion of peripheral blood and synovial fluid mononuclear cells in rheumatoid arthritis

Objective and Design: A novel immunomodulating drug, leflunomide has been shown recently to be effective and well tolerated in patients suffering from rheumatoid arthritis (RA). The present study evaluated the effect of the drug on cell adhesion in RA. Material and Treatment: Peripheral blood and synovial fluid mononuclear cells were obtained from a clinical trial, undertaken primarily to evaluate the efficacy and pharmacokinetic profile of multiple-dose pulsing leflunomide therapy in RA patients. PB MNC and corresponding synovial fluid (SF) MNC for in vitro homotypic aggregation (HA) assay were obtained from healthy volunteers and RA patients with active disease not treated with leflunomide in vivo. Methods: Expression of activation antigens (CD25, CD54, CD69, CD71, HLA-DR) on peripheral blood mononuclear cells (PB MNC), as well as ex vivo ability of cells to aggregate spontaneously were determined in patients before entering into the clinical trial and at the end of 6 months treatment. HA was measured by aggregation in vitro. Data were compared by Student's t-test. Results: There was a decreased expression of activation antigens and decreased spontaneous MNC clustering after leflunomide therapy. We found in the in vitro study that HA of PB and SF MNC was mainly mediated through β2-integrin molecules. The active metabolite of leflunomide, A77 1726, effectively suppressed both spontaneous and phorbol-ester (PMA)-induced HA. Disruption of cell aggregates by A77 1726 was dose-dependent and, most likely, unrelated to the quantitative modulation of integrin receptors. Conclusions: Results from this study support the idea that leflunomide elicits its immunomodulatory action, at least partially, by modulating the adhesion process. accepted by M. J. Parnham     

Interleukin-2 in rheumatoid arthritis: production of and response to interleukin-2 in rheumatoid synovial fluid, synovial tissue and peripheral blood.

Several aspects of interleukin-2 (IL-2) generation and function were studied employing mononuclear cells from synovial fluid (SF), synovial tissue (ST) and peripheral blood (PB) of patients with rheumatoid arthritis (RA). Decreased PHA stimulated IL-2 production by lymphocytes from rheumatoid ST, SF (P less than 0.02), and PB (P less than 0.01) was observed when compared to normal blood and SF of patients with gout. The proliferative response of rheumatoid lymphocyte blasts exposed to exogenous IL-2 was also defective (P less than 0.05-0.001). This defect was greater in SF than in rheumatoid PB (P less than 0.05-0.001). In addition to the proliferative response, the effect of IL-2 on interferon-gamma (IFN-gamma) production was also examined. Rheumatoid lymphocytes from both PB and SF produced less IFN-gamma after overnight treatment with IL-2 than did normal PB lymphocytes. This decreased IFN-gamma induction was discordant with the excellent enhancement by IL-2 of natural killer activity. Removal of adherent cells in synovial fluid did not correct this deficit. Abnormalities in the biology of IL-2 and IFN-gamma suggest that impaired T cell function could contribute to the immunopathogenesis of RA.     

Effect of piroxicam therapy on granulocyte function and granulocyte elastase concentration in peripheral blood and synovial fluid of rheumatoid arthritis patients

The effect of piroxicam therapy (20 mg/day for 15 days) on various polymorphonuclear granulocyte (PMN) responses and on PMN elastase concentration was investigated in nine patients with active rheumatoid arthritis. Peripheral biood and synovial fluid samples were collected before starting therapy and 12 h after the last dose of the drug. All patients were evaluable for peripheral blood analysis and six for synovial fluid analysis. Piroxicam therapy had no effect on PMN random migration and phagocytosis, while it significantly reduced both FMLP-induced aggregation and FMLP-induced chemotaxis. This seems mainly due to an effect on FMLP binding, as no differences were observed after therapy in PMA- and PHA-induced aggregation as well as in serum-induced chemotaxis. In contrast, a marked impairment of NBT test and PMA- and FMLP-induced superoxide anion (O₂ ^–) production was found after piroxicam therapy. This effect was as evident in peripheral blood as in synovial fluid PMN. Also, a significant reduction in synovial fluid PMN number and synovial fluid PMN elastase concentration (elastase- ₁-proteinase complex) was found after treatment. It is concluded that piroxicam may act at different sites on various PMN responses-Its effect on O₂ ^– generation and PMN elastase concentration in synovial fluid may have an important role in reducing destruction of arthritic joint tissue.     

类风湿关节炎患者外周血淋巴细胞的凋亡及其意义

目的研究类风湿关节炎(RA)患者外周血淋巴细胞的凋亡及其意义。方法采用流式细胞术和激光共聚焦显微镜,检测35例RA患者和30例健康志愿者的外周血淋巴细胞抵抗凋亡的能力,包括分析新鲜分离和培养的淋巴细胞凋亡率及其与临床病情严重度的相关性。结果两组间新鲜分离的淋巴细胞凋亡率差异无统计学意义[(2.63±1.47)%比(3.53±2.04)%,P>0.05]。活化淋巴细胞检测的结果显示,RA组的凋亡率明显低于对照组[(12.08±6.06)%比(18.66±7.27)%,P<0.05],并与DAS28评分呈负相关(r=-0.617,P=0.008)。结论RA患者活化的外周血淋巴细胞出现凋亡缺陷,并与RA病情活动度相关。提示这些细胞募集到组织损害和自身免疫炎性反应进程异常的部位并对炎性反应进程的发展和维持起重要作用。