Interleukin-6 localisation in the synovial membrane in rheumatoid arthritis

Summary Polyclonal antibodies were raised in rabbits against Interleukin-6 (IL-6) by immunisation with a synthetic peptide of identical sequence to the amino terminal 12 amino acids of human IL-6. These antibodies reacted with recombinant IL-6 by ELISA and stained the cytoplasm of the IL-6 secreting bladder tumour cell line T24. Staining was abolished by prior incubation of the antibody with the IL-6 peptide. F(ab)₂ fragments made by pepsin digestion of the IgG were immunopurified, labelled with biotin and retained activity in the biochemical and histological assays. Sections of synovial membrane from patients with rheumatoid arthritis (RA) were stained with these antibodies, using an immunoperoxidase technique, and cells containing IL-6 were domonstrated in the thickened synovial lining layer and also in a perivascular distribution in the deeper synovium. In osteoarthritis there were fewer cells in the lining layer and hence localisation appeared similar in both the interstitial area and lining layer. Double-staining techniques with mouse monoclonal antibodies against cell subset markers in five RA synovial membranes showed that up to 13% of T-cells and 19% of antibody-producing cells stained or IL-6. However, up to 70% of the macrophages contained IL-6 and these were found in close proximity to lg-producing plasma cells. This study showed that Anacrophages were the major cells of the immune system in which IL-6 could be localised in RA, and suggests a cole for locally produced IL-6 in the stimulation of theumatoid factor production.     

Interleukin-6 levels in synovial fluids of patients with rheumatoid arthritis correlated with the infiltration of inflammatory cells in synovial membrane

To compare the histological appearance of synovial membrane and interleukin (IL)-6 levels in synovial fluids of patients with rheumatoid arthritis (RA). Synovial tissue and synovial fluids were obtained from 51 knee joints with RA undergoing synovectomy or joint replacements. A histological inflammation score was determined based on the hyperplasia of the synovial lining and infiltration of inflammatory cells. The concentrations of IL-6 in synovial fluids were measured by ELISA. The association between IL-6 levels and histological findings was evaluated. We found a positive correlation between the infiltration of inflammation cells in synovial tissues and the concentration of IL-6 in synovial fluids. The IL-6 level in synovial fluid partially reflects histological synovial inflammation.     

The thickening of basement membrane in synovial capillaries in rheumatoid arthritis

Summary Synovial tissues from seven rheumatoid arthritis (RA) patients were used for the ultrastructural investigation of capillary cellular components and basement membranes (BM). Attention has been specially paid to the mechanism of BM thickening of the capillaries in the inflammatory sites.The capillary BM were multilamellated in the inflammatory sites. The multilamellation was characteristic not only in the BM surrounding the endothelial cells and pericytes but also in the BM between these two types of cells. Cell debris was frequently encountered between the multilamellated BM. The hyperplasia and various stages of degeneration of the endothelial cells were observed in these regions. Some endothelial cells were activated and occasionally located in capillaries containing degenerated endothelial cells. The high incidence of these findings indicates the following hypotheses.The accelerated rate of death and replenishment of capillary cellular components may play a role in BM thickening in the inflammatory sites of RA synovium. These cells may not only produce one layer of BM in their life-time but may also be activated to produce excessive amounts of BM components to make several layers.     

Combined elevation of IgM and IgA rheumatoid factor has high diagnostic specificity for rheumatoid arthritis

The diagnostic value of measuring rheumatoid factor (RF) by agglutination or isotype-specific enzyme-linked immunosorbent assay (ELISA) was compared. The study included 70 patients with rheumatoid arthritis (RA) and 205 patients with various other rheumatic conditions. Of the RA patients, 74% were RF-positive by agglutination and 90% had one or more RF isotypes elevated by ELISA compared to 14% and 22%, respectively, of the other patients. Strikingly, 70% of the RF-positive RA patients had an elevation of two or more RF isotypes compared to only 16% of the other RF-positive patients (P<0.0001). Furthermore, a combined elevation of IgM and IgA RF was found in 52% of the RF-positive RA patients, but only in two (4%) of the other RF-positive patients (P<0.0001). It is concluded that a combined elevation of IgM and IgA RF is highly specific for RA and is very rarely found in rheumatic diseases other than RA. Isotype-specific RF assays are therefore diagnostically superior to agglutination tests. The detection of the RA-specific RF isotype pattern may be particularly helpful early in the course of RA even before the disease is fully differentiated. Received: 10 December 1997 / Accepted: 25 June 1998     

Two-dimensional electrophoresis of synovial tissue,synovial fluid and serum in patients with rheumatoid arthritis and related diseases

Summary Using the technique of two-dimensional (2D) electrophoresis with consecutive silver staining, we investigated samples of serum, synovial fluid and synovial tissue obtained from 19 patients suffering from rheumatoid arthritis (RA) or non-RA arthritis. From these experiments we have drawn the following conclusions. 2D electrophoresis of serum, synovial fluid and synovial tissue extracts taken from patients suffering from joint diseases is a reproducible method. Repeated runs of the same sample reveal an essentially constant protein spot pattern. The time period between surgery and tissue preparation did not influence the number of protein spots when less than 15 h was involved. The protein spot number is always lower in synovial fluid than in either synovial tissue or serum in RA and non-RA patients. The mean value for the number of spots is 68 for the inflamed tissue irrespective of the cause of arthritis (RA and non-RA group taken together) and 47 for the control group. This difference is significant. We were able to definitely identify 7 spots in the tissue extract. We did not find RA-specific protein spots in either serum, synovial fluid or tissue extracts from the synovial membrane. The only significant difference between RA patients and either non-RA or control group patients concerning the protein spot pattern is the increased size of the immunoglobulin spot (mainly IgG) in RA. In addition, we discuss possible reasons for failure of the 2D electrophoresis technique to detect disease-specific protein patterns.     

The neo-synovial membrane of patients with active rheumatoid arthritis at resynovectomy

Summary Neo-synovial membranes, which formed after primary synovectomy in 21 patients with rheumatoid arthritis (RA), were studied at resynovectomy. The clinical, histomorphological, and immunohistological data were compared with data derived from primary synovial membranes from RA and osteoarthritis (OA) patients. The clinical data suggest a less active rheumatoid inflammatory response after synovectomy. Histomorphologicaly, the synovitis in resynovectomized neosynovial membranes of RA revealed no qualitative differences when compared with synovitis in the primary synovium. However, the degree of the inflammatory rection evaluated by the different parameters was found to be distinctly lower. The immunohistological data correlated with these findings.     

Properties and reactivity of immune complexes in rheumatoid synovial fluid

Summary Fractionation of immune complexes (IC) from rheumatoid synovial fluid revealed the presence of three different fractions of IC. The largest molecular weight form, fraction I (above 1000 Kdaltons) was predominately composed of IgG and IgM and contained both IgM-RF and IgG-RF. The other IC, fraction II (480 Kdaltons) and fraction III (330 Kdaltons), contained predominately IgG with some IgA and only significant amounts of IgG-RF. All three fractions of IC can bind Clq and stimulate human monocyte prostaglandin E (PGE) production. Fraction I IC bound Clq most readily while fraction III IC were the most effective stimulators of monocyte PGE production. IC stimulation of monocyte PGE production was inhibited by staphylococcus protein A suggesting mediation via activation of Fc receptors. It remains to be determined whether this IC reactivity has any pathologic significance.     

Plasma and synovial fluid concentrations of sulphasalazine and two of its metabolites in rheumatoid arthritis

Summary A study was made of plasma and synovial fluid levels of sulphasalazine, one of its dissociation products — sulphapyridine and a metabolite of the latter — acetyl sulphapyridine in patients with rheumatoid arthritis (RA) who were in a steady state on sulphasalazine therapy. Combined sulphapyridine levels were significantly higher than those of sulphasalazine both in plasma and synovial fluid. Synovial fluid levels of both drugs correlated with their plasma levels and were generally slightly lower. Some patients accumulated sulphasalazine and sulphapyridine in the synovial fluid and the mean concentration of sulphasalazine was higher in the fluid than in the plasma. The explanation for this is uncertain. The concentration of combined sulphapyridine in synovial fluid was related to local joint inflammation and more active systemic disease. No consistent association was found between sulphasalazine levels and local or systemic activity. The higher sulphapyridine levels in synovial fluid found in this study suggest the possibility that this moiety could play a more active role in RA than it does in inflammatory bowel disease.     

Expression and citrullination of keratin in synovial tissue of rheumatoid arthritis

Keratin is the main component of cellular intermediate filaments, and its post-translational modification plays an important role in cell differentiation and apoptosis, as well as disease states. The conversion of peptidylarginine to citrulline, termed citrullination, is particularly involved in the pathogenesis of rheumatoid arthritis (RA). Immunohistochemistry using an antibody mixture that broadly recognized various keratin forms detected cytokeratin in many cells in the area lining the synovial membrane of RA. Furthermore, double immuno-florescent labeling showed that the cells expressing cytokeratin were also positive for citrulline when they were in the vicinity of extracellular deposits or approached the exterior of the synovial membrane. Western blot analysis demonstrated citrullination of keratin purified from RA synovial tissue by immuno-precipitation. The above results indicate the presence of citrullinated cytokeratin in synovial membranes in RA.     

Induction of a lymphotoxin-like mediator in peripheral blood and synovial fluid lymphocytes by incubation with synovial fluid from patients with rheumatoid arthritis

Summary A significantly increased spontaneous cell-mediated cytotoxicity (SCMC) has been reported in synovial fluid lymphocytes (SFL) as compared to peripheral blood lymphocytes (PBL) of patients with rheumatoid arthritis (RA) and that of normal controls [1–3]. To determine whether this increased SCMC activity is due to the production of a lymphokine and related to the production of a lymphotoxin (LT)-like mediator, PBL from normal controls and PBL and SFL from RA patients were incubated either with a human melanoma cell line (IGR 3) or with cell-free synovial fluid (SF) from RA patients. The SF and the cell-free supernatants of the different cultures were tested for LT activity by estimation of inhibition of DNA synthesis of HeLa cell monolayers and they were added to a SCMC assay system using normal PBL and IGR 3 as target.In the supernatants from cocultures of either PBL from controls or PBL and SFL from RA patients with IGR 3 cells, there was no significant difference in LT activity. An LT-like mediator was observed in the supernatants of all lymphocytes cocultured with SF, whereas SF alone and supernatants of lymphocytes alone exhibited little or no LT activity. In a control experiment, LT induction was not observed when normal lymphocytes were cultured with the serum of RA patients. Absorption of the culture supernatants with an insolubilised goat anti-human Ig did not remove LT activity. The demonstrated release of an LT-like mediator from lymphocytes incubated with SF might be one contributing mechanism to the inflammatory joint reaction in RA patients.     

Ultrasonographic evaluation of synovial effusion and synovial proliferation pattern in the knee joints of patients with rheumatoid arthritis

In the present study, 49 knee joints of 26 patients with rheumatoid arthritis and 17 knee joints of 17 healthy subjects were ultrasonographically examined. Lateral, superior, and medial aspects of the patella were scanned using an ultrasonograph with a 7.5-MHz annular array transducer to evaluate the thickness of synovial effusion and the synovial proliferation pattern. The overall mean thickness of synovial effusion (mean of all three sites) in the knee joints was 4.9 ± 3.4 mm for rheumatoid arthritis patients and 1.4 ± 0.5 mm for healthy subjects. In rheumatoid arthritis patients, the mean thickness of synovial effusion at the superior aspect of the patella (6.5 ± 4.1 mm) was significantly greater than that at the lateral aspect (4.5 ± 4.8 mm) (P < 0.05) and the medial aspect (4.0 ± 3.1 mm) (P < 0.01). Patients with the villonodular pattern of synovial proliferation had a shorter duration of disease than those with uniform thickening or an overlapping pattern. Received: May 1, 2001 / Accepted: August 6, 2001     

Immunohistochemical detection of factor XIIIa and factor XIIIs in synovial membranes of patients with rheumatoid arthritis or osteoarthritis

In spite of differences in etiology, RA and OA lead to astonishingly similar synovitic alterations. Fibroblastic transformation of the synovial membrane and an increase in monocytes constitute a rare but highly characteristic feature of RA. Monocytes synthesize factor (F) XIII, implying that FXIII (a and s) in synovial tissue might help to differentiate between RA and OA. Biopsies were obtained at open surgery from 98 unselected patients with the clinical diagnosis of RA (n=54) or OA (n=44). In a three-stage (ABC) immunoperoxidase technique, polyclonal antisera against factor XIIIa and factor XIIIs were investigated. Compared to OA sections, RA synovium showed more FXIIIa-positve cells - monocytes, fibrocytes, fibroblasts and synovial lining cells. In the subsynovial layer, band-like structure of FXIIIa-stained cells was observed in 27.8% of the RA patients, but in only one OA specimen. Higher proportions of FXIIIa-positive monocytes, macrophages, histiocytes and fibroblasts, as well as positive Langhans' giant cells and vascular wall regions (except endothelial cells), were observed in RA. OA specimens revealed more intense FXIIIa labeling of these cells with a lower percentage of stained cells. Overall, labeling with FXIIIs antibody resulted in less intense staining. In conclusion, distinction between synovitis caused by RA and synovitis due to OA is possible, as the former show higher numbers of FXIIIa-positive cells, including monocytes, fibroblasts, fibrocytes and synovial lining cells. Furthermore, RA tissue is stained less intensely than OA tissue. There is evidence for continuous excretion of FXIII in the synovial membrane by the above-mentioned cell systems.     

Phenotypic characterization of 3H-thymidine incorporating cells in rheumatoid arthritis synovial membrane

Summary Tritiated-thymidine incorporating cells in synovial tissue samples from ten patients with definite or classic rheumatoid arthritis (RA) were studied by combining two techniques. Tritiated-thymidine-labelled cells were seen in autoradiography and simultaneously the subtype of them was determined with immunoperoxidase staining using monoclonal antibodies. Tritiated-thymidine-labelled cells comprised 0.8±0.4% of all the inflammatory cells in RA synovial membrane. Of all ³H-thymidine-labelled cells 34±17% were positive for OKT8 and 19±8% for OKT4 monoclonal antibodies. OKM1-positive cells comprised 7±3% of all ³H-thymidine labelled cells, whereas only a few (3±4%) of them were positive for pan-B monoclonal antibody. This study emphasizes the importance of activated OKT8 lymphocytes in RA synovial membrane.     

Stimulation of rheumatoid synovial and blood T cells and lines by synovial fluid and interleukin-2: Characterization of clones and recognition of a co-stimulatory effect

Summary Rheumatoid arthritis (RA) is characterized by the presence of interleukin-2 (Il-2) receptor-positive T cells in the peripheral blood and synovial compartments. Utilizing the limiting dilution technique, the precursor frequencies of Il-2 responsive T cells were determined in peripheral blood and synovial sites from RA patients and in the blood of normal donors. The frequencies of Il-2 responsive T cells were significantly higher in RA patients (range from 1/180 to 1/7432) compared to normal donors (range from 1/400 to 1/8163). T-cell clones raised by the addition of Il-2 alone were predominantly of the CD4-positive phenotype. Peripheral blood T cells, synovial T-cell clones and lines derived from RA patients were co-stimulated with Il-2 and synovial fluid or supernatants from cultured synovial lining cells. This co-stimulation induced a strikingly enhanced proliferative T-cell response while synovial fluid alone was without effect. This stimulatory activity was found in the high molecular weight range (approximately 150 kDa) and could not be attributed to the action of immunoglobulins or known cytokines such as Il-2 or interleukin-1 (Il-1), suggesting the activity of a material that modulates the Il-2-dependent growth of T cells. The co-stimulatory capacity of synovial fluid with Il-2 may be relevant to the activated state, especially of synovial T cells.     

Pathways of mononuclear cell infiltration in rheumatoid synovitis

Summary The mononuclear cell infiltration which characterizes the chronic inflammatory reaction results from the migration of lymphocytes and monocytes through the endothelium of the postcapillary venule. The initial step in the emigration of these cells in their binding to the vascular endothelium. The binding capacity of the endothelial cell (EC) for lymphocytes and monocytes is increased by IFN-, IL-1, TNF, and TNF. Production of these cytokines by chronic inflammatory cells may be expected to amplify the chronic inflammatory reaction. Initiation of the chronic synovitis of rheumatoid and other chronic synovitides probably results from the interaction of antigen with sensitized T cells in the sublining region of the synovium. This interaction is facilitated by the presence of substantial numbers of DR+macrophage+accessory cells in the synovial interstitial space. It is likely that these accessory cells are bone marrow derived monocytes migrating to the synovial lining layer in response to chemotactic factors released by the hyperplastic synovial lining cells. Lymphocytes differ in their binding affinity for ECs, and more strongly binding lymphocytes may be preferentially bound. Since binding is the first step in lymphocyte emigration, this event may lead to the selection of more strongly binding lymphocytes in the perivascular infiltrate. The T cells present in the mononuclear cell infiltrates of rheumatoid arthritis, other chronic synovitides, and multiple sclerosis have been shown to be composed largely of the CDw29+CD4+, helper-inducer, memory cell subset. The predominance of this T-cell subset may result from its demonstrated greater binding affinity for ECs. The fact that this subset has been shown to react weakly with T-cell mitogens and strongly with soluble antigens, may explain two important properties of synovial fluid T cells in inflammatory disease: (1) their weak reactivity to T cells mitogens and (2) their strong reactivity to antigens. Thus, the hyperplasia of the synovial lining layer, which may be an important factor in the mobilization of accessory cells, and the increased EC binding of HI memory T cells may combine to facilitate the initiation and maintenance of chronic synovitis.     

Serum and synovial fluid leptin levels and markers of inflammation in rheumatoid arthritis

This study was designed to investigate the serum and synovial fluid leptin levels, and inflammatory markers in rheumatoid arthritis (RA) patients. Serum and synovial fluid leptin levels were significantly higher (P > 0.05) in RA patients than control group; RA patients with moderate disease activity (DAS < 2.7) having significantly higher leptin levels (P > 0.05) than those with low disease activity (DAS < 2.7). Leukocytes and erythrocyte sedimentation rate (ESR) were found to be significantly higher in moderate disease activity RA group compared to low activity group (P > 0.05, P < 0.001, respectively). Serum leptin level is found to be independent of age and inflammatory markers. ESR is positively correlated with DAS activity and CRP values. Our finding of no correlation between leptin and BMI shows that regulation of leptinemia is complex, and leptin levels cannot be used to assess RA activity.     

Endothelial cells are the major source of sICAM-1 in rheumatoid synovial tissue

The objective of this research was to investigate the cellular source of soluble ICAM-1 (siCAM-1) from rheumatoid synovial tissue (RS) and its relation to sICAM-1 in synovial fluid (SF) and serum, and to study the expression of ICAM-1 in isolated cells of RS. sICAM-1 was determined by using the enzyme-linked immunosorbent assay (ELISA) and Western blot analysis in supernatants from RS cultured for short periods (n = 19), in SF (n = 7) and in serum (n = 19). ICAM-1 expression, vascularization and inflammatory infiltration (CD3, CD68, CD22) were characterized immunohistochemically in cytospin preparations (n = 18), cryosections (n = 18) and in conventionally stained paraffin sections (n = 19) of RS. The degree of RS vascularization was analysed morphometrically in immunohistochemically stained cryosections (factor VIII related antigen). We found 90-kD sICAM-1 in supernatants of cultured cells, in SF and in sera. sICAM-1 in cellular supernatant correlated significantly (P < 0.01) with SF sICAM-1. The amount of sICAM in cellular supernatants showed no correlation to the score of inflammatory infiltration, but correlated significantly (P < 0.001) with the vascularization index of RS. The percentage of ICAM-1-expressing cells correlated significantly (P < 0.001) with the percentage of CD68-positive macrophages, but not with CD3- and CD22-positive lymphocytes. Macrophages, multinucleated giant cells and endothelial cells exhibited a higher expression of ICAM-1 as compared to lymphocytes and fibroblasts. The differential expression of ICAM-1 on infiltrating leucocytes and resident cells of RS indicates a functional role of ICAM-1 in the local inflammatory process. SF sICAM-1 originated in RS, but serum sICAM-1 did not. Shedding of sICAM-1 by RS was independent of inflammatory infiltration, but depended on the degree of vascularization, indicating that endothelial cells are the major source of sICAM-1 in RS. Received: 14 November 1996 / Accepted: 3 January 1997     

Early lymphocyte activation in the synovial microenvironment in patients with osteoarthritis: comparison with rheumatoid arthritis patients and healthy controls

Osteoarthritis (OA) is largely considered to be a non-inflammatory disease, although there is compelling evidence that subclinical inflammation is a common event, even in the absence of acute inflammatory flares. In this study we analyze, by means of CD5 and CD69 expression, the infiltration and early activation of CD5+cells, mostly lymphocytes, in both synovial membrane and synovial fluid from advanced OA patients and compare them with samples from patients with rheumatoid arthritis and healthy controls. The number of infiltrating CD5+ cells in both synovial membrane and synovial fluid from patients with advanced OA was significantly reduced as compared with rheumatoid arthritis patients. However, synovial membrane and synovial fluid CD5+ cells on OA exhibited a phenotype with evidence of recent activation comparable to that observed in RA.     

Characterization of IL-2 responsive synovial T lymphocytes in rheumatoid arthritis

Summary Lymphocytes from peripheral blood (PBL) and synovial fluid (SFL) were obtained from patients with rheumatoid arthritis (RA) and cloned under limiting-dilution conditions without prior activation but in the presence of exogenous interleukin (IL)-2. The precursor frequencies of such in vivo activated IL-2-responsive cells were higher in RA SFL (1/83) than in RA PBL (1/201) or normal PBL (1/377). These HLA-Dr/Ia-positive clones expressed T-cell markers CD3 and T101 and were either CD4- or CD8-positive but lacked NK markers CD11, CD16, and HNK-1. All such clones were cytotoxic for NK-sensitive K562 targets and NK-insensitive Raji cell targets. These cells, which most closely resemble non-major histocompatibility complex (MHC) restricted cytotoxic T (CTL) cells, are present with increased frequency in RA synovial fluids.     

Pokeweed mitogen-induced secretion of IgG- and IgM-rheumatoid factors by synovial fluid and blood B lymphocytes in rheumatoid arthritis

Summary Lymphocytes from patients with rheumatoid arthritis (RA) were assayed for their ability to secrete Ig and rheumatoid factors (RF) upon polyclonal activation in vitro. B lymphocytes secreting IgM-RF and IgG-RF were enumerated using hemolytic plaque forming cell (PFC) assays. The sensitivity of RF-PFC formation was similar to that of a reverse PFC assay detecting all cells secreting Ig. Blood mononuclear cells from RA patients generated a median of 12 IgM-RF and 15 IgG-RF-secreting cells/10⁶ after 6 days culture with pokeweed mitogen (PWM). Thus a median of approximately 0.5% of IgM- and IgG-secreting cells were identified as RF-PFC. Synovial fluid B lymphocytes co-cultured with autologous blood T lymphocytes in the presence of PWM generated higher numbers of RF-secreting cells; medians of 43 IgM-RF and 441 IgG-RF secreting cells/10⁶ were found. Thus, 8–9% of IgM- and IgG-secreting cells from synovial fluid were identified as RF-PFC. In co-cultures containing synovial fluid T cells, PWM-induced RF secretion was low. The data indicate that both blood and SF B cells have the potential for RF secretion.