Role of opioid peptide in rheumatoid arthritis--detection of beta-endorphin in synovial tissue]

The presence of beta-endorphin (beta-end) was immunohistologically identified in synovial tissue samples biopsied from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The amount of beta-end in culture supernatants of synovial tissue explants was also determined by RIA. beta-end was strongly detected in mainly superficial synovial cells, vascular endothelial cells and a few synovial interstitial cells in RA patients, but not in OA patients. In RA patients the beta-end concentration was significantly higher in the supernatants of tissue explants (26.4 +/- 8.3 pg/ml) than in the plasma of the same patients (15.3 +/- 2.5 pg/ml) (p < 0.01). Using isolated synovial cells, the beta-end concentration in the culture supernatants of non-adherent cells (19.4 pg/ml) was higher than that of adherent cells (4.0 pg/ml). It is suggested that beta-end is produced by non-adherent cells such as lymphocytes and neutrophils in addition to synovial lining cells and endothelial cells and may play some role in the pathology of RA synovial inflammation.     

The levels of soluble granzyme A and B are elevated in plasma and synovial fluid of patients with rheumatoid arthritis (RA).

Cytotoxic cells possess specialized granules which contain perforin and a group of serine proteinases termed granzymes. Granzyme-positive cells have been identified in synovial fluid and tissue of patients with RA, where they may play an important role as mediators of granule-mediated apoptosis, extracellular proteolysis, and cytokine induction. The aim here was to define further the involvement of cytotoxic cells in RA. Plasma and synovial fluid samples from the knee joint were obtained from 31 RA patients. The disease controls included 20 osteoarthritis (OA) patients and 10 reactive arthritis (ReA) patients. A recently developed capture ELISA was used to detect soluble granzymes A and B in all patients. Compared with OA and ReA disease controls, markedly increased levels of soluble granzymes A and B were detected in both plasma and synovial fluid of RA patients (P < 0.00001). When values for soluble granzymes A and B in plasma and synovial fluid were used simultaneously as independent variables, logistic regression analysis indicated that a diagnosis of RA could be predicted correctly in 84% of the RA patients and a diagnosis of non-RA in 90% of the controls. The markedly elevated levels of soluble granzymes A and B in plasma and synovial fluid of RA patients strongly suggest that cytotoxic cells are active participants in the pathogenesis of RA. Moreover, the results suggest that measurement of granzymes may assist the laboratory evaluation of patients with arthritis. Larger studies in patients with early disease may clarify the role of this test system in differential diagnosis.     

Elevated expression of TAM receptor tyrosine kinase in synovial fluid and synovial tissue of rheumatoid arthritis

To investigate the expression and roles of TAM (Tyro3/Axl/Mer) receptor tyrosine kinases (TK) in synovial fluid and synovial tissue of patients with rheumatoid arthritis (RA). The expression of TAM TKs in the synovial fluid and synovial tissues of RA and osteoarthritis (OA) patients was measured by ELISA and immunohistochemistry. The relationships between soluble TAM TKs (sTAM TKs) levels and the clinical features, laboratory parameters and disease activity were analyzed in RA. The concentrations of sTAM TK in the synovial fluids of RA patients were increased in comparison to those of OA patients. Compared with OA patients, the expression of membrane Tyro3 TK (mTyro3 TK) and mMer TK in RA patient synovial tissue were significantly increased, which may partly explain the possible mechanism of elevated levels of sTAM TK in RA patient synovial fluid. sAxl TK levels were decreased in RA patients under sulfasalazine treatment and elevated in patients under Iguratimod treatment. Furthermore, sTyro3 TK levels were positively correlated with erythrocyte sedimentation rate (ESR) and negatively correlated with white blood cells (WBCs), red blood cells (RBCs), and hemoglobin (HB) in RA patients. The levels of sMer TK were positively associated with disease duration and rheumatoid factor (RF) and negatively correlated with HB, complement 3 (C3), and C4. Taken together, TAM TKs might be involved in RA synovial tissue inflammation.     

Lack of T cell oligoclonality in enzyme-digested synovial tissue and in synovial fluid in most patients with rheumatoid arthritis.

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.     

Elevated angiogenin levels in synovial fluid from patients with inflammatory arthritis and secretion of angiogenin by cultured synovial fibroblasts

Angiogenesis is a key process in the pathogenesis of inflammatory arthritis. Angiogenin is one of the most potent inducers of neovascularization in experimental models in vivo. To look for evidence that angiogenin is involved in inflammatory joint disease, we examined plasma and synovial fluid (SF) samples from rheumatology patients and synovial fibroblast cell culture supernatants. Angiogenin levels were determined by radioimmunoassay and ELISA. Plasma angiogenin concentrations ranged from 96 to 478 ng/ml, with no significant difference between patients and normal controls. In SF, angiogenin concentrations were significantly higher in patients with acute or chronic synovitis (rheumatoid arthritis (RA): median, 104 ng/ml; range 13-748, n = 14; crystal-induced arthritis (CIA): median, 149 ng/ml; range, 37-616, n = 14, and other chronic inflammatory arthritis: median, 42 ng/ml; range, 15-205; n = 9) than in the 18 patients with osteoarthritis (OA) (median, 20 ng/ml; range 8-116) (P < 0.0001, anova). Angiogenin levels in SF from RA patients in remission with secondary OA were similar to those achieved in primary OA, and decreased in parallel with the resolution of acute gout. Angiogenin protein was released by cultured synovial fibroblasts from OA and RA patients, and reached 1.18 ng/106 cells/day. These data suggest that angiogenin may mediate local inflammation in arthritis via effects on angiogenesis and leucocyte regulation.     

Increase in Human Endogenous Retrovirus HERV-K (HML-2) Viral Load in Active Rheumatoid Arthritis

To study the viral loads of human endogenous retrovirus HERV-K (HML-2) type 1 and type 2 in rheumatoid arthritis (RA), we measured the viral loads of HERV-K (HML-2) type 1 and type 2 using nucleic acid sequence-based amplification (NASBA) technology. We analyzed plasma samples from RA patients ( n  = 79) and healthy volunteers (HV, n  = 46) and synovial fluid samples from RA ( n  = 10) and osteoarthritis (OA, n  = 10) patients. HERV-K type 1 and type 2 viruses were detected and quantified for the majority of plasma and synovial fluid samples from RA patients. HERV-K type 1 and type 2 viral loads were significantly elevated in RA patients compared with HV in plasma ( P  < 0.0001) and from RA patients compared with OA patients in synovial fluid (type 1: P  = 0.0007; type 2: P  = 0.023). Moreover, an association was observed between the HERV-K type 1 viral load in plasma and the disease activity in RA patients (RA patients with low activity versus high activity P  = 0.0129; RA patients with intermediate activity versus high activity P  = 0.037). Our findings showed that HERV-K (HML-2) viral load can be detected in plasma samples from RA patients, with higher levels observed for those with active disease. There was an association of HERV-K type 1 levels with the disease activity.     

Relationship between leukotriene B4 and immunological parameters in rheumatoid synovial fluids

Leukotriene B₄ (LTB₄) was measured in synovial fluid from 20 patients with rheumatoid arthritis and 15 patients with osteoarthritis. The level of LTB₄ was significantly higher in synovial fluid from rheumatoid arthritis patients as compared with synovial fluid from osteoarthritis patients. LTB₄ levels also significantly correlated with cell numbers, rheumatoid factor, and immune complexes in synovial fluid from rheumatoid arthritis patients. There was an inverse correlation between LTB₄ levels and complement components. The high-pressure liquid chromatography peak of immunoreactivity extracted from the synovial fluid occurred at a retention volume identical to that of authentic LTB₄. These results suggest that the increased level of this mediator in synovial fluid may contribute to perpetuation of inflammation and tissue destruction in rheumatoid arthritis.     

Terminal complement complexes and C1/C1 inhibitor complexes in rheumatoid arthritis and other arthritic conditions.

Terminal complement complex (TCC) and C1r-C1s-C1 inhibitor complex (C1/C1 INH) concentrations were measured in plasma and synovial fluid from patients with arthritis and related to other measures of disease activity. Both TCC and C1/C1 INH concentrations were significantly increased in patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis (plasma and synovial fluid, P less than 0.05) and normal subjects (plasma only, P less than 0.001). In the patients with RA, there was no correlation between plasma or synovial fluid TCC concentrations and IgM rheumatoid factor, immune complex or C1/C1 INH levels. However, in 10 patients with seronegative RA, C1/C1 INH and immune complex levels correlated significantly in synovial fluid (r = 0.69, P less than 0.05) although not in plasma (r = 0.52). Plasma and synovial fluid TCC and C1/C1 INH concentrations did not differ in rheumatoid patients with severe compared with mild joint disease (categorized by the Ritchie score). These results confirm a role for complement activation in RA but suggest that several mechanisms are involved in its pathogenesis.     

Different synovial fluid fibronectin levels in rheumatoid variants

Summary A circulating high-molecular-weight glycoprotein called fibronectin plays a part in cell adhesion and migration before phagocytosis and in morphology, differentiation, and metabolism in inflammatory synovial effusions of patients with rheumatic diseases. A technique of nephelometric immunoassay, based on the measurement of an antigen-antibody reaction, was applied to the analysis of fibronectin concentrations in synovial fluids from 20 patients with rheumatoid arthritis (RA) and other diseases (non-RA). RA synovial fluids have a significantly higher concentration than the specimens obtained from Yersinia arthritis patients (n=12). The mean concentration of other synovial fluids, from 12 patients with osteoarthritis of the knees, did not significantly differ from the synovial fluids of control values obtained from patients who underwent meniscectomy. There was a considerably negative correlation between fibronectin levels and overall indices of inflammatory activity, such as Ritchie articular indices or a whole number of painful rheumatoid arthritis joints. However, a particularly distinct correlation was obtained when raised fibronectin levels were compared with the inflammatory activity of the knee joint, from which the specimen was aspirated. Thus, these findings suggest that the measurements of fibronectin in synovial fluid may be of some differential-diagnostic value in rheumatoid variants, but may only serve as an indicator of inflammatory activity if the joint, from which the specimen is obtained, is taken into account.Dedicated to Prof. Dr. N. Zöllner on his 60th birthdaySupported in part by the Deutsche Forschungsgemeinschaft, Go 236/8–9     

类风湿性关节炎患者滑膜液中IL-10、IL-12和sFasL含量的检测

为了研究细胞因子和凋亡分子在类风湿性关节炎(RA)发病中的作用,用ELISA法分析了26例RA患者滑膜液和血清中IL-12、IL-10和可溶性FasL(sFasL)的含量。结果表明RA患者滑膜液中IL-12、IL-10含量分别为(419.9±89.2)pg/ml和(187.7±34.5)pg/ml,外周血中这两种细胞因子的含量均较低,分别为(65.3±34.2)pg/ml(IL-12)和(85.0±12.7)pg/ml(IL-10)。滑膜液中sFasL的含量为266pg/ml,明显高于血清含量(36pg/ml)。这一结果提示,RA患者滑膜液中IL-12含量和sFasL增高,这些炎性细胞因子增高可能参与了关节滑膜中的自身反应性T细胞的活化,继而造成免疫损伤。     

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.     

Changes in lipoxygenase products from synovial fluid in carrageenan induced arthritis in dogs

A non-suppurative chronic arthritis was induced in the juvenile dog knee by intra-articular instillations with Carrageenan. Lipoxygenase products of arachidonic acid were separated from synovial fluid by reversed-phase high-performance liquid chromatography (RP-HPLC). After ten weeks we observed an accumulation of leukotriene B4 (LTB4) in synovial fluid in five out of six experimental knees (0.94 to 5.5 ng/ml), as judged by integrated optical density. Biological activity of LTB4 was confirmed by chemokinesis. LTB4 was not detected in control knees. The 15-lipoxygenase products, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxy-9,11-octadecadienoic acid (13-HODD), being inhibitors of 5-lipoxygenase, were found in relatively high levels in the control knees compared to the arthritic knees. The results denote LTB4 as a pro-inflammatory local mediator during carrageenan-induced arthritis; possibly, the decreased levels of 15-HETE and 13-HODD in the arthritic knees may have a regulatory function, thus facilitating LTB4 generation.     

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

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.     

Excitatory amino acids, TNF-alpha, and chemokine levels in synovial fluids of patients with active arthropathies

The aim of this study was to assess the synovial fluid (SF) neurotransmitter excitatory amino acid (EAA) levels, including glutamate (Glu) and aspartate (Asp), in the context of SF levels of other amino acids, TNF-alpha and chemokines from patients with active arthropathies. The SF was collected from patients with active rheumatoid arthritis (RA), gout, or osteoarthritis (OA). The SF samples were analysed for levels of neurotransmitters glutamate and aspartate, tumour necrosis factor-alpha (TNF-alpha), Regulated upon Activation Normally T-cell Expressed and Secreted (RANTES), macrophage inhibitory factor-1 alpha (MIP-1alpha) and interleukin 8 (IL-8). SF WBC counts were also determined. Correlations between SF EAA, TNF-alpha and chemokines were determined by the Pearson product-moment correlation. Primary cultures derived from SF from active RA and gout patients were incubated with added l-glutamate, to assess if exposure to Glu could increase TNF-alpha levels. There were significant elevations in SF EAA, SF TNF-alpha and SF RANTES in RA patients compared to gout or OA patients. Significant correlations between SF EAA and SF RANTES, MIP-1alpha and IL-8 levels were seen, and SF EAA and SF TNF-alpha or SF WBC levels approached significance. Addition of exogenous neurotransmitter glutamate significantly increased TNF-alpha levels in primary cell cultures derived from RA and gout patients. The SF neurotransmitter EAA levels significantly correlated to selected SF chemokine levels, in clinically active RA, gout and OA patients, independent of disease. Added Glu resulted in significantly increased TNF-alpha levels in primary synovial cell cultures. These data expand the relationship of SF neurotransmitter EAA levels to SF cytokines and chemokines in patients with clinically active arthritis, and suggest that neurotransmitters Glu and Asp contribute to peripheral inflammatory processes.     

Immunoglobulin G and A antibody responses to Bacteroides forsythus and Prevotella intermedia in sera and synovial fluids of arthritis patients

The objective of the present study was to investigate immunoglobulin G (IgG) and IgA antibody immune responses to Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, and Candida albicans in the sera of patients with rheumatoid arthritis (RA), the synovial fluid (SF) of patients with RA (RA-SF samples), and the SF of patients without RA (non-RA-SF samples). An enzyme-linked immunosorbent assay was used to determine IgG and IgA antibody levels in 116 serum samples from patients with RA, 52 RA-SF samples, and 43 non-RA-SF samples; and these were compared with those in SF samples from 9 patients with osteoarthritis (OA-SF samples) and the blood from 100 donors (the control [CTR] group). Higher levels of IgG antibodies against B. forsythus (P < 0.0001) and P. intermedia (P < 0.0001) were found in non-RA-SF samples than in OA-SF samples, and higher levels of IgG antibodies against B. forsythus (P = 0.003) and P. intermedia (P = 0.024) were found in RA-SF samples than in OA-SF samples. Significantly higher levels of IgA antibodies against B. forsythus were demonstrated in both RA-SF and non-RA-SF samples than in OA-SF samples. When corrected for total Ig levels, levels of IgG antibody against B. forsythus were elevated in RA-SF and non-RA-SF samples compared to those in OA-SF samples. Lower levels of Ig antibodies against B. forsythus were found in the sera of patients with RA than in the plasma of the CTR group for both IgG (P = 0.003) and IgA (P < 0.0001). When corrected for total Ig levels, the levels of IgG and IgA antibodies against B. forsythus were still found to be lower in the sera from patients with RA than in the plasma of the CTR group (P < 0.0001). The levels of antibodies against P. gingivalis and C. albicans in the sera and SF of RA and non-RA patients were comparable to those found in the respective controls. The levels of IgG and IgA antibodies against B. forsythus were elevated in SF from patients with RA and non-RA-SF samples compared to those in OA-SF samples. Significantly lower levels of IgG and IgA antibodies against B. forsythus were found in the sera of patients with RA than in the plasma of the CTR group. This indicates the presence of an active antibody response in synovial tissue and illustrates a potential connection between periodontal and joint diseases.     

Levels of the soluble forms of CD80, CD86, and CD83 are elevated in the synovial fluid of rheumatoid arthritis patients

The release of soluble forms of CD80 (sCD80), CD86 (sCD86), and CD83 (sCD83) provide a potentially powerful immunoregulatory mechanism. We therefore investigated the potential presence and relative levels of these molecules in the synovial fluid (SF) and serum of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Serum and SF levels were measured by enzyme-linked immunosorbent assay. Serum levels of sCD80, sCD86, and sCD83 in RA and OA patients were similar to those present in normal donor serum (NDS) and the SF of OA patients. In contrast, when compared with NDS and OA SF levels, almost all RA SF samples had elevated sCD83 levels (32/35, >0.63 ng/ml) and a substantial proportion had elevated sCD80 (13/29, >0.22 ng/ml) or sCD86 (16/33, >2.31 ng/ml) levels. Analysis of matched pairs of serum and SF from RA patients demonstrated that the SF/serum ratio for sCD80 (95% CI = 1.7-3), sCD86 (95% CI = 1.5-3.1), and sCD83 (95% CI = 3.6-7.8) levels was >1 in almost all patients. In conclusion, this study shows that the SF from almost all RA patients contain elevated levels of sCD83 and the majority of these samples also contain elevated levels of sCD80 and/or sCD86. These molecules may play a role in modulating immune responses within the rheumatoid joint.     

Depressed degranulation response of synovial fluid polymorphonuclear leucocytes from patients with rheumatoid arthritis to IgG aggregates.

No difference was found between the degranulation responses to FMLP of synovial fluid (SF) polymorphonuclear leucocytes (PMNL), from patients with rheumatoid arthritis (RA), and either paired blood PMNL or blood PMNL from a healthy donor. In contrast, the response of SF PMNL to heat-aggregated IgG was often reduced compared with autologous blood PMNL. Similarly, SF from some (35%) RA patients stimulated degranulation of PMNL but the response of SF-derived PMNL to autologous stimulatory SF was reduced compared with the response of blood PMNL. The stimulatory activity of the SF was removed by sepharose-protein A. These results were taken to suggest that the activity is due to immunoglobulin aggregates and that SF PMNL (from some RA patients) are tachyphylactic to stimulation by immunoglobulin aggregates as measured by degranulation because they have been stimulated by immunoglobulin aggregates in vivo. In other studies the concentration of myeloperoxidase (MPO) was measured enzymically in RA SF and was found to be present in varying amounts. However, only a weak relationship was found between MPO levels and either PMNL numbers or levels of complement-bearing IgG aggregates in SF. It is considered that the relationship between MPO and immunoglobulin aggregates levels is obscured by the presence of a peroxidase inhibitor in the fluids and/or because only aggregates bound to tissue stimulate degranulation in vivo.