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1.  Cytotoxic activity of rheumatoid and normal lymphocytes against allogeneic and autologous synovial cells in vitro. 
Journal of Clinical Investigation  1976;58(3):613-622.
The possibility that lymphocytes from patients with rheumatoid arthritis (RA) might be sensitized to RA synovial cell antigens was investigated with a 51Cr release cytotoxicity assay. Peripheral blood lymphocytes from rheumatoid and normal donors were tested for cytotoxic activity against their own synovial cells and against allogeneic rheumatoid and nonrhemuatoid synovial cells. In the allogeneic studies, the degree of cytotoxicity was significantly influenced by the age in culture (passage number) of the synovial target cells (P less than 0.001). When the passage number of the target cells was considered in the analysis, rheumatoid lymphocytes were found to have greater cytotoxic activity than normal lymphocytes against young cultures (low passage number) of both RA and non-RA synovial cells (P = 0.0042). Differences in susceptibility to lysis between RA and non-RA synovial cells were more susceptible to both RA and normal lymphocyte-induced lysis than were non-RA synovial cells (P = 0.0048). No evidence of cytotoxicity was detected when lymphocytes from nine RA patients and two osteoarthritis patients were reacted against their own synovial cells. Although the data demonstrated an increased cytotoxic activity of peripheral blood lymphocytes from some RA patients against allogeneic synovial cells, the fact that this reactivity was seen against both non-RA and RA synovial cells and was not demonstrated against autologous synovial cells argues against the presence of an immunospecific response of RA lymphocytes to RA synovial cell antigens.
PMCID: PMC333220  PMID: 956390
2.  The cytotoxicity of leukocytes and lymphocytes from patients with rheumatoid arthritis for synovial cells. 
Journal of Clinical Investigation  1976;58(3):690-698.
Unseparated peripheral blood leukocytes obtained from patients with rheumatoid arthritis (RA) were cytotoxic for synovial cells. The cytotoxic reactions produced by RA leukocytes were more frequent and of greater magnitude than cytotoxicity induced by leukocytes from normal persons and patients with other diseases, primarily connective tissue diseases. Furthermore, the cytotoxic activity of RA leukocytes was greater for RA synovial cells than for nonrheumatoid synovial cells, in contrast to the cytotoxicity of other leukocytes, which did not discriminate between synovial cells according to their origin. Tests with purified lymphocytes showed that the cytotoxicity of unseparated leukocytes directed against RA synovial cells was due to lymphocyte cytotoxicity. These data are consistent with the possibility that sensitized lymphocytes from patients with RA recognize a distinctive antigen present on rheumatoid synovial cells.
PMCID: PMC333227  PMID: 956395
3.  Autoantibody to an Immunoregulatory Inducer Population in Patients with Juvenile Rheumatoid Arthritis 
Journal of Clinical Investigation  1981;67(3):753-761.
The human inducer (T4+) and reciprocal cytotoxic/suppressor (T5+/T8+) subsets have been defined by monoclonal antibodies. In the present study, we examined the relationship of naturally occurring anti-T cell autoantibodies found in patients with active juvenile rheumatoid arthritis (JRA) to these subsets. In one approach, normal T cells were treated with anti-T4 or anti-T8 to eliminate the corresponding subset of cells and then analyzed for reactivity with JRA sera. It was found that JRA sera were reactive with only 15% of an enriched cytotoxic/suppressor population, whereas they reacted with 37% of an enriched inducer population. In reciprocal studies, JRA+ T cells were eliminated with JRA sera and complement and the residual T cells (JRA−) reacted with monoclonal antibodies and indirect immunofluorescence on a fluorescence-activated cell sorter. As expected, the JRA sera and complement treatment of unfractionated T cells markedly diminished the T4+ subset, whereas there was a concomitant increase in T cells reactive with anti-T5 and anti-T8. A similar diminution in T4+ T cells was found in the circulating peripheral T cell compartment of patients with active JRA who possessed the JRA antibody.
Functional studies demonstrated that removal of the JRA+ population of T cells diminished phytohemagglutinin and soluble antigen proliferative responses, both of which were previously shown to be functions of T4+ T cells. More importantly, in the absence of JRA+ T cells, pokeweed mitogen-stimulated immunoglobulin production was markedly enhanced, despite the concomitant increase in T5+/T8+ cytotoxic/suppressor cells. These results suggest that the JRA serum may define a Qal-like antigen found predominantly on the human inducer population which could activate suppressor and/or other feedback regulatory cells.
PMCID: PMC370626  PMID: 6451634
4.  Lymphocyte surface marker expression in rheumatic diseases: evidence for prior activation of lymphocytes in vivo. 
Expression of major histocompatibility complex (MHC) class II and other lymphocyte activation markers on peripheral blood and synovial fluid T lymphocytes from patients with rheumatoid arthritis (RA), psoriatic arthritis, and Reiter's syndrome were measured and the mean fluorescence intensities of these antigens were assessed. Increased expression of MHC class II antigens of synovial fluid T lymphocytes is not unique to RA, though it is quantitatively greater on RA synovial fluid T cells. There was less expression of other lymphocyte activation markers (4F2, transferin receptor) and a marked discordance between the expression of these markers and the interleukin 2 receptor (IL2r). Synovial fluid T lymphocytes contain a subpopulation of larger cells expressing MHC class II and other lymphocyte activation antigens with the exception of the IL2r. Mean fluorescence intensity of CD3 and CD4 antigens on synovial fluid T lymphocytes was decreased in all three patient groups, suggesting prior in vivo exposure of synovial fluid T lymphocytes to an unknown antigen.
PMCID: PMC1003983  PMID: 2138450
5.  Analysis of T cell subsets in the peripheral blood and synovial fluid of patients with rheumatoid arthritis by means of monoclonal antibodies. 
Annals of the Rheumatic Diseases  1983;42(4):357-361.
In an attempt to define the immunoregulatory mechanisms operating in rheumatoid arthritis we have enumerated T cell subsets in the peripheral blood and synovial fluid of patients with this disease. The peripheral blood analysis revealed an elevation of the ratio of inducer T cells (OKT4 positive) to suppressor/cytotoxic T cells (OKT5 positive) in patients with clinically active rheumatoid arthritis when compared with normal persons. This was due to a reduction in the percentage of suppressor/cytotoxic T lymphocytes in these patients. The synovial fluid in rheumatoid arthritis differed from the peripheral blood in 2 respects. Firstly, synovial fluid was characterised by a lower helper: suppressor ratio due to an increased number of suppressor/cytotoxic cells, and, secondly, it contained an increased number of activated T cells bearing HLA DR antigens. The majority of these activated T cells belonged to the helper/inducer T cell subset.
PMCID: PMC1001241  PMID: 6224467
6.  Very late activation antigens on rheumatoid synovial fluid T lymphocytes. Association with stages of T cell activation. 
Journal of Clinical Investigation  1986;78(3):696-702.
Lymphocytes from the synovial fluid of eight out of eight rheumatoid arthritis (RA) patients had elevated very late activation antigen-1 (VLA-1) expression (10-36% positive cells), whereas peripheral blood lymphocytes (PBL) from RA patients and healthy controls had low VLA-1 expression (0-6% positive cells). During 1-2 wk of in vitro culture, VLA-1 increased on synovial fluid cells but remained low on PBL. In comparison, the interleukin 2 receptor (IL-2 R) was less prominent than VLA-1 on fresh synovial fluid cells, did not increase on cultured synovial fluid T cells, but did increase greatly on cultured PBL. The mitogen PHA reversed or prevented the appearance of VLA-1+, IL-2 R- synovial fluid cells during in vitro culture, thus giving IL-2 R+, VLA-1- cells. These results emphasize that VLA-1+ SF cells are different from resting cells or IL-2 R+ activated PBL T cells, and VLA-1 on synovial fluid T cells may be incompatible with mitogen stimulation. In addition, the VLA-2 heterodimer (165,000/130,000 relative molecular mass [Mr]) was regulated opposite to the VLA-1 heterodimer (130,000/210,000 Mr) on synovial lymphocytes, and thus the VLA-1/VLA-2 ratio is another indicator of the stage of T cell activation.
PMCID: PMC423654  PMID: 3018043
7.  Expression of CD44 on rheumatoid synovial fluid lymphocytes. 
Annals of the Rheumatic Diseases  1995;54(7):566-570.
OBJECTIVES--To investigate the involvement of the adhesion molecule CD44 in the homing of lymphocytes to synovial tissue, by examining the density of expression and molecular mass of CD44 on rheumatoid synovial fluid lymphocytes. METHODS--Twenty patients with rheumatoid arthritis were studied. Peripheral blood and synovial fluid lymphocytes were isolated by Ficoll-Hypaque sedimentation. CD44 expression was analysed by two colour flow cytometry of CD3 positive T lymphocytes with calculation of mean fluorescence intensity. Expression of activation markers M21C5, M2B3, interleukin (IL)-2 receptor and transferrin receptor was quantitated. In addition, CD44 molecular mass was examined by Western blot in six patients. RESULTS--CD44 expression was markedly increased on synovial fluid T lymphocytes of rheumatoid patients relative to peripheral blood lymphocytes from the same individuals. CD44 molecular mass on peripheral blood mononuclear cells was 88 kDa, but that on synovial fluid lymphocytes was only 83 kDa. CD44 expression correlated significantly with expression of activation markers M21C5, M2B3, and the IL-2 receptor. CONCLUSIONS--Alterations in density of expression or of the molecular mass of CD44 could contribute to local tissue injury, either directly by facilitating adhesion, or indirectly through effects on other adhesion molecules.
PMCID: PMC1009936  PMID: 7545382
8.  Spontaneous production of fibroblast-activating factor(s) by synovial inflammatory cells. A potential mechanism for enhanced tissue destruction 
A characteristic feature of rheumatoid arthritis is hyperplasia of the synovial lining cells and fibroblasts, the source of tissue-degrading mediators, in association with the appearance and persistence of lymphocytes in affected joints. Diseased synovial tissue obtained at arthroscopy from 10 of 12 rheumatoid arthritis patients was found to release a factor(s) that could stimulate quiescent fibroblasts to proliferate in vitro. Mononuclear cells isolated from this synovial tissue and from the synovial fluid spontaneously produced fibroblast- activating factor(s) (FAF). In contrast, synovial tissue from patients with noninflammatory joint disease did not release FAF. By gel filtration, FAF was detected in two peaks (40,000 and 15,000 mol wt) that were consistent with the previously described peripheral blood T lymphocyte- and monocyte-derived factors with identical activity. The mononuclear cells were predominantly OKT3+/Leu-1+ T lymphocytes and OKM1+ cells of monocyte/macrophage lineage that expressed HLA-DR antigens, suggesting prior activation of these cells. Mononuclear cells isolated from the peripheral blood of these patients did not spontaneously secrete FAF. Lymphocytes and monocytes from the site of synovial inflammation appear to be activated in situ to produce factors that may contribute to the hyperplasia and overgrowth of the synovial membrane in rheumatoid arthritis.
PMCID: PMC2187543  PMID: 3968518
9.  Lymphocyte studies in rheumatoid arthritis. II. Antibody-mediated and mitogen-induced lymphocyte cytotoxicity in synovial fluid and peripheral blood. 
Annals of the Rheumatic Diseases  1978;37(5):410-415.
A comparison was made of the activity of synovial fluid (SF) lymphocytes with peripheral blood lymphocytes in antibody-mediated and mitogen-induced lymphocyte cytotoxicity in patients with a variety of inflammatory joint diseases. SF lymphocytes consistently showed little or no antibody-mediated cytotoxicity (AMC) although mitogen-induced cytotoxic activity was comparable with that of the peripheral blood lymphocytes. Blocking substances on the cell surface were not responsible for the lack of AMC by SF lymphocytes as preincubation at 37 degrees C and enzyme treatment (trypsin, neuraminidase) of the cells did not restore activity. The lack of AMC by SF cells from a variety of inflammatory joint fluids demonstrates that this may be a consequence of inflammation in the joint and excludes the possibility that this is a specific property of fluids from certain conditions such as rheumatoid arthritis. Lymphocytes thought to be involved in AMC have a characteristic surface morphology (Fc receptor positive, E rosette negative, surface immunoglobulin negative). Such lymphocytes are present in synovial fluid in comparable proportions to those in blood. Hence the absence of AMC indicates that functional assays must be used in determining the presence or absence of cells with special functions.
PMCID: PMC1000267  PMID: 718273
10.  Immunoblasts in synovial fluid and blood in the rheumatic diseases. 
Annals of the Rheumatic Diseases  1980;39(4):318-322.
Synovial fluid studies have been made on 43 patients with rheumatic disease. Lymphocytes separated by a 2-stage procedure were examined for the presence of activated large lymphoid cells or immunoblasts. Such immunoblasts were found in 19 of 21 patients with classical rheumatoid arthritis and 7 of 10 patients with seronegative polyarthritis, including patients with Still's disease, psoriatic arthritis, and ankylosing spondylitis. No immunoblasts were seen in synovial fluid from osteoarthrosis or in the inflammatory but nonimmune synovial fluid from crystal-induced arthritis. The presence of immunoblasts showed a correlation with the lymphocyte count in the synovial fluid but not with the total white cell count. Preliminary studies confirm the spontaneous metabolic activity of these cells by autoradiography and show them by scanning electron microscopy to have a villous surface membrane. Simultaneous peripheral blood studies showed a lower incidence of immunoblasts than in the synovial fluid. It is suggested that these cells originate in the synovial membrane. In view of the known migration characteristic of immunoblasts these cells may be important in the spread of immune arthritis as well as being markers of disease activity.
PMCID: PMC1000549  PMID: 6969065
11.  Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome 
Arthritis Research & Therapy  2004;7(1):R30-R37.
Macrophage activation syndrome (MAS) has been reported in association with many rheumatic diseases, most commonly in systemic juvenile rheumatoid arthritis (sJRA). Clinically, MAS is similar to hemophagocytic lymphohistiocytosis (HLH), a genetic disorder with absent or depressed natural killer (NK) function. We have previously reported that, as in HLH, patients with MAS have profoundly decreased NK activity, suggesting that this abnormality might be relevant to the pathogenesis of the syndrome. Here we examined the extent of NK dysfunction across the spectrum of diseases that comprise juvenile rheumatoid arthritis (JRA). Peripheral blood mononuclear cells (PBMC) were collected from patients with pauciarticular (n = 4), polyarticular (n = 16), and systemic (n = 20) forms of JRA. NK cytolytic activity was measured after co-incubation of PBMC with the NK-sensitive K562 cell line. NK cells (CD56+/T cell receptor [TCR]-αβ-), NK T cells (CD56+/TCR-αβ+), and CD8+ T cells were also assessed for perforin and granzyme B expression by flow cytometry. Overall, NK cytolytic activity was significantly lower in patients with sJRA than in other JRA patients and controls. In a subgroup of patients with predominantly sJRA, NK cell activity was profoundly decreased: in 10 of 20 patients with sJRA and in only 1 of 20 patients with other JRA, levels of NK activity were below two standard deviations of pediatric controls (P = 0.002). Some decrease in perforin expression in NK cells and cytotoxic T lymphocytes was seen in patients within each of the JRA groups with no statistically significant differences. There was a profound decrease in the proportion of circulating CD56bright NK cells in three sJRA patients, a pattern similar to that previously observed in MAS and HLH. In conclusion, a subgroup of patients with JRA who have not yet had an episode of MAS showed decreased NK function and an absence of circulating CD56bright population, similar to the abnormalities observed in patients with MAS and HLH. This phenomenon was particularly common in the systemic form of JRA, a clinical entity strongly associated with MAS.
PMCID: PMC1064882  PMID: 15642140
juvenile rheumatoid arthritis; macrophage activation syndrome; natural killer cells; perforin; reactive hemophagocytic lymphohistiocytosis
12.  Membrane and transformation characteristics of lymphocytes isolated from the synovial membrane and paired peripheral blood of patients with rheumatoid arthritis 
Membrane and transformation characteristics of lymphocytes isolated from the synovial membrane and from paired peripheral blood samples, obtained from patients with classical rheumatoid arthritis, were studied. Synovial tissue lymphocytes were isolated by a new technique. Two suspensions of peripheral blood lymphocytes were studied: one isolated by Ficoll-Isopaque density gradient centrifugation, the other enriched in T cells by an additional step of 1 hour nylon wool column filtration. All suspensions were characterised by the percentages of mononuclear phagocytic cells, and T and B lymphocytes. The spontaneous 3H-thymidine uptake of synovial tissue lymphocyte suspensions always exceeded that of the peripheral blood lymphocyte suspensions. The in-vitro responsiveness of synovial tissue lymphocytes to PHA, Con-A, and PWM, as measured by 3H-thymidine uptake, was always consistently lower than that of paired peripheral blood lymphocytes whether or not enriched in T cells. The responsiveness to antigens, including PPD, varidase, and an antigen cocktail consisting of varidase, trychophyton, and Staphylococcus aureus antigen, showed the same effect. No dissociation was found between the response to PPD and the other antigens studied. These results suggest that the relative unresponsiveness to mitogens and antigens of synovial tissue lymphocytes in comparison with blood lymphocytes is not caused by mononuclear phagocyte contamination, but either by different subsets of T lymphocytes or by different functional states of T lymphocytes present in the synovial membrane and peripheral blood of patients with rheumatoid arthritis.
PMCID: PMC1000474  PMID: 6445717
13.  Activation pathways of synovial T lymphocytes. Expression and function of the UM4D4/CDw60 antigen. 
Journal of Clinical Investigation  1990;86(4):1124-1136.
Accumulating evidence implicates a central role for synovial T cells in the pathogenesis of rheumatoid arthritis, but the activation pathways that drive proliferation and effector function of these cells are not known. We have recently generated a novel monoclonal antibody against a rheumatoid synovial T cell line that recognizes an antigen termed UM4D4 (CDw60). This antigen is expressed on a minority of peripheral blood T cells, and represents the surface component of a distinct pathway of human T cell activation. The current studies were performed to examine the expression and function of UM4D4 on T cells obtained from synovial fluid and synovial membranes of patients with rheumatoid arthritis and other forms of inflammatory joint disease. The UM4D4 antigen is expressed at high surface density on about three-fourths of synovial fluid T cells and on a small subset of synovial fluid natural killer cells; in synovial tissue it is present on more than 90% of T cells in lymphoid aggregates, and on approximately 50% of T cells in stromal infiltrates In addition, UM4D4 is expressed in synovial tissue on a previously undescribed population of HLA-DR/DP-negative non-T cells with a dendritic morphology. Anti-UM4D4 was co-mitogenic for both RA and non-RA synovial fluid mononuclear cells, and induced IL-2 receptor expression. The UM4D4/CDw60 antigen may represent a functional activation pathway for synovial compartment T cells, which could play an important role in the pathogenesis of inflammatory arthritis.
PMCID: PMC296841  PMID: 2212003
14.  Phagocytic Function of Polymorphonuclear Leukocytes in Rheumatic Diseases 
Journal of Clinical Investigation  1973;52(7):1632-1635.
Phagocytosis of yeast particles by peripheral blood and synovial fluid neutrophils was compared in the sera and synovial fluids from 16 osteoarthritis, 23 rheumatoid arthritis, and 12 miscellaneous arthritis patients. Phagocytosis by normal peripheral blood neutrophils was decreased equally and significantly in all synovial fluids. All synovial fluid neutrophils demonstrated decreased phagocytic capacity in all media. Rheumatoid arthritis synovial fluid neutrophils showed significantly less phagocytosis than miscellaneous arthritis synovial fluid neutrophils. Normal peripheral blood neutrophils which in vitro had previously ingested monosodium urate crystals or oil red O, subsequently exhibited a normal yeast phagocytic capacity. Normal peripheral blood neutrophils, which had ingested preformed immunoglobulin G-rheumatoid factor complexes exhibited significantly less yeast phagocytic capacity than control cells or cells preincubated with the individual complex components. There was a significant correlation between the log of the reciprocal of the rheumatoid factor titer in sera used to produce complexes and the phagocytic capacity exhibited by test neutrophils. Ingestion of immunoglobulin G-rheumatoid factor complexes may be important in the production of the cellular phagocytic defect which this study has demonstrated in rheumatoid arthritis synovial fluid neutrophils.
PMCID: PMC302436  PMID: 4578156
15.  Lymphocytes in rheumatoid and nonrheumatoid synovial fluids 
Annals of the Rheumatic Diseases  1976;35(5):451-455.
van de Putte, L. B. A., Meijer, C. J. L. M., Lafeber, G. J. M., Kleinjan, R., and Cats, A. (1976).Annals of the Rheumatic Diseases, 35, 451-455. Lymphocytes in rheumatoid and nonrheumatoid synovial fluids. Nonspecificity of high T-cell and low B-cell percentages. Lymphocytes were studied in paired peripheral blood and synovial fluid samples from patients with various forms of arthritis, including rheumatoid arthritis (group I) and other polyarthritides of unknown origin (group II), as well as arthritides generally considered not to be immunologically mediated, such as crystal synovitis, traumatic arthritis, osteoarthrosis, and pigmented villonodular synovitis (group III). In all 3 groups the percentages of T lymphocytes wee significantly higher in synovial fluids than in the peripheral blood, whereas those of the synovial fluid B lymphocytes were consistently very low and occasionally nil. Absolute numbers of synovial fluid lymphocytes were significantly higher in groups I and II as compared with group III, and in the peripheral blood absolute numbers of lymphocytes in groups I and II were significantly lower than in controls. No correlation was found between absolute numbers of lymphocytes and complement activity in the synovial fluid. The characteristic pattern of high T-cell and very low B-cell percentages in synovial fluids is a general feature of inflammatory exudates and cannot be considered an expression of cell-mediated immunity in itself.
PMCID: PMC1006579  PMID: 1086654
16.  Imbalance in distribution of functional autologous regulatory T cells in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2007;66(9):1151-1156.
Regulatory T cells (Tregs) exert their anti‐inflammatory activity predominantly by cell contact‐dependent mechanisms. A study was undertaken to investigate the regulatory capacity of autologous peripheral blood Tregs in contact with synovial tissue cell cultures, and to evaluate their presence in peripheral blood, synovial tissue and synovial fluid of patients with rheumatoid arthritis (RA).
44 patients with RA and 5 with osteoarthritis were included in the study. The frequency of interferon (IFN)γ‐secreting cells was quantified in synovial tissue cell cultures, CD3‐depleted synovial tissue cell cultures, synovial tissue cultures co‐cultured with autologous CD4+ and with CD4+CD25+ peripheral blood T cells by ELISPOT. Total CD3+, Th1 polarised and Tregs were quantified by real‐time PCR for CD3ε, T‐bet and FoxP3 mRNA, and by immunohistochemistry for FoxP3 protein.
RA synovial tissue cell cultures exhibited spontaneous expression of IFNγ which was abrogated by depletion of CD3+ T cells and specifically reduced by co‐culture with autologous peripheral blood Treg. The presence of Treg in RA synovitis was indicated by FoxP3 mRNA expression and confirmed by immunohistochemistry. The amount of FoxP3 transcripts, however, was lower in the synovial membrane than in peripheral blood or synovial fluid. The T‐bet/FoxP3 ratio correlated with both a higher grade of synovial tissue lymphocyte infiltration and higher disease activity.
This study has shown, for the first time in human RA, the efficacy of autologous Tregs in reducing the inflammatory activity of synovial tissue cell cultures ex vivo, while in the synovium FoxP3+ Tregs of patients with RA are reduced compared with peripheral blood and synovial fluid. This local imbalance of Th1 and Treg may be responsible for repeated rheumatic flares and thus will be of interest as a target for future treatments.
PMCID: PMC1955165  PMID: 17392348
17.  Lymphocyte subpopulations in rheumatoid synovial tissue. 
Annals of the Rheumatic Diseases  1977;36(2):176-180.
Synovial tissue obtained at synovectomy of the knee joint in 21 patients with rheumatoid arthritis contained a significantly lower proportion of T lymphocytes as measured by spontaneous rosette formation with nonsensitized sheep red blood cells than did synovial fluid or blood from the same patients. There was on concomitant increase in synovial tissue lymphocytes with B-cell markers such as surface immunoglobulin or Fc fragment receptors. Removal of lymphocyte receptors with trypsin followed by culture to allow new receptors to form, led to an increase in rosette forming cells, suggesting that part of the synovial cells without B- or T-cell markers may be T lymphocytes with blocked receptors.
PMCID: PMC1006655  PMID: 301013
18.  Markedly raised synovial fluid leucocyte counts not associated with infectious arthritis in children. 
Annals of the Rheumatic Diseases  1978;37(5):404-409.
Synovial fluid leucocyte counts greater than 50 000 cells/mm3 (50 X 10(9)/1) are usually associated with infectious arthritis. Six children, 3 of whom meet the criteria for juvenile rheumatoid arthritis (JRA), are described with synovial fluid white blood cell counts greater than 88 000 cells/mm3 (88 X 10(9)/1). Two had synovial fluid leucocyte counts greater than 100 000 cells/mm3 (100 X 10(9)/1). The diagnosis of infectious arthritis was unlikely in these 6 children since the synovial fluid smears and cultures for infectious agents were negative and their histories atypical for infection. While in most instances such markedly raised synovial fluid leucocyte counts indicate infection, this finding is not diagnostic of septic arthritis.
PMCID: PMC1000266  PMID: 718272
19.  Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis. 
Journal of Clinical Investigation  1994;94(3):1012-1018.
We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs in the milieu of the inflamed joint of RA patients.
PMCID: PMC295150  PMID: 8083342
20.  Expression of CD69 antigen on synovial fluid T cells in patients with rheumatoid arthritis and other chronic synovitis. 
Annals of the Rheumatic Diseases  1993;52(6):457-460.
OBJECTIVES--The expression of the CD69 antigen on synovial fluid and peripheral blood lymphocytes was studied in 12 patients with rheumatoid arthritis (RA), five subjects with other forms of chronic synovitis, and on the peripheral blood lymphocytes of 15 patients with systemic lupus erythematosus (SLE) and immune vasculitis. METHODS--The CD69 antigen and other activation markers (HLA-DR, interleukin 2 receptor (IL-2R), transferrin receptor) were measured by cytometric analysis. In patients with RA soluble IL-2R was determined by enzyme linked immunosorbent assay (ELISA). RESULTS--The percentage of T cells bearing CD69 was significantly increased in synovial fluid from patients with RA (30.3 (13)%) and other chronic synovitis (18 (9)%). The expression of CD69 on peripheral blood lymphocytes of patients with RA, other chronic synovitis, and SLE and immune vasculitis was within the normal range 2.1 (1.2)%. According to previously published work, a high proportion of synovial fluid T cells are HLA-DR positive (64.2 (12.4)% in synovial fluid from patients with RA and 61 (1.2)% in synovial fluid from patients with other chronic synovitis). Transferrin receptor expression on synovial fluid was up-regulated compared with that on peripheral blood. The increase of IL-2R expression on synovial fluid lymphocytes v peripheral blood was not significant; the quantitative determination of soluble IL-2R levels gave a mean value of 921 (351) U/ml in synovial fluid of patients with RA, 672 (229) U/ml in the serum of the same patients, and 273 (100) U/ml in serum from normal subjects. CONCLUSIONS--Synovial fluid lymphocytes are in a different functional state than peripheral blood lymphocytes. CD69 antigen is an interesting cellular marker which should be studied in patients with chronic synovitis. The unusual expression of the activation antigens and the sequence of their appearance require further study.
PMCID: PMC1005072  PMID: 8323399
21.  Behaviour of effector cells, synovial fluids, and sera from rheumatoid arthritis patients in antibody-dependent cell-mediated cytotoxicity. 
Annals of the Rheumatic Diseases  1979;38(3):252-256.
Antibody-dependent cell-mediated cytotoxicity (ADCC) was examined in patients with rheumatoid arthritis (RA). The cytotoxicity of peripheral blood leucocytes from patients with RA was similar to that found in normal persons, whereas ADCC was less effective in RA synovial fluid cells. It is possible that the activity in these cells is lower because of immune complexes and other factors being absorbed from the synovial fluid itself. Although patients' sera had little effect on normal peripheral blood leucocytes, synovial fluid from RA patients was markedly inhibitory in ADCC. The degree of inhibition correlated significantly with the clinical status of the patients.
PMCID: PMC1000447  PMID: 485583
22.  TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis 
TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.
TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.
TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts.
The expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.
PMCID: PMC3132040  PMID: 21435232
23.  Characterization of CCL19 and CCL21 in Rheumatoid Arthritis 
Arthritis and rheumatism  2011;63(4):914-922.
The aim was to characterize the expression of CCL19 and CCL21 in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages and RA synovial tissue fibroblasts.
Expression of CCL19 and CCL21 was demonstrated in RA and normal (NL) synovial tissues employing immunohistochemistry. CCL19 and CCL21 levels were quantified in fluids from osteoarthritis (OA), juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA) and RA using ELISA. Regulation of CCL19 and CCL21 expression was determined in RA peripheral blood in vitro differentiated macrophages as well as RA synovial tissue fibroblasts by real-time RT-PCR. CCL19 and CCL21 activated peripheral blood in vitro differentiated macrophages and RA synovial tissue fibroblasts were examined for proangiogenic factor production employing ELISA.
CCL19 and CCL21 were elevated in RA synovial tissue compared to NL controls. Levels of CCL19 and CCL21 were greatly increased in RA and PsA synovial fluid versus OA synovial fluid. In RA macrophages and fibroblasts, expression of CCL19 was increased by LPS, TNF-α and IL-1β stimulation. However, CCL21 expression was modulated by IL-1β in RA fibroblasts as well as TNF-α and RA synovial fluid in RA macrophages. CCL19 and CCL21 activation induced VEGF and Ang-1 production from RA synovial tissue fibroblasts and secretion of IL-8 and Ang-1 from macrophages.
We identify, for the first time, regulators of CCL19 and CCL21 in RA fibroblasts and RA peripheral blood in vitro differentiated macrophages and we document a novel role of CCL19/21 in RA angiogenesis.
PMCID: PMC3079365  PMID: 21225692
CCL19; CCL21; RA synovial tissue fibroblast; macrophages and proangiogenic factors
24.  Rheumatoid factor-producing cells detected by direct hemolytic plaque assay. 
Journal of Clinical Investigation  1976;58(4):933-941.
Lymphocytes secreting anti-IgC antibodies, rheumatoid factors (RF), can be detected in the peripheral bloods, synovial fluids, and bone marrows of patients with seropositive rheumatoid arthritis by using a direct plaque-forming cell (PFC) assay with sheep erythrocytes sensitized with reduced and alkylated rabbit IgG hemolysin. The autospecific nature of the RF produced by RF-PFC was indicated by inhibition studies in which the order of patency was human IgG greater than rabbit IgG greater than bovine IgG. In metabolic studies puromycin, cycloheximide, and venblastine suppressed RF-PFC. Cyclic AMP and cyclic GMP were without effect. A need was recognized for using full tissue culture media during the cell separation and plaquing procedures to optimize detection of the RF-PFC. RF-PFC may appear in the blood of patients intermittently despite their continuing presence in the bone marrow. They have been found in the peripheral blood, especially during acutely exacerbating polyarticular synovitis, generalized vasculities, or generally active, aggressive disease. RF-PFC were found in synovial effusions of new or recrduescent acute synovitis. RF-PFC were observed to disappear from the peripheral circulation and the bone marrow during therapy with cytotoxic drugs. The data are consistent with the hypothesis that the appearance of RF-PFC in the peripheral blood represents an anamnestic response to transiently appearing antigen. The nature of the antigen is not specified. The bone marrow may be a site of origin of RF-PFC.
PMCID: PMC333256  PMID: 787010
25.  Peripheral blood but not synovial fluid natural killer T cells are biased towards a Th1-like phenotype in rheumatoid arthritis 
Arthritis Research & Therapy  2005;7(3):R493-R502.
Natural killer T (NKT) cells have been implicated in the regulatory immune mechanisms that control autoimmunity. However, their precise role in the pathogenesis of rheumatoid arthritis (RA) remains unclear. The frequency, cytokine profile and heterogeneity of NKT cells were studied in peripheral blood mononuclear cells (PBMCs) from 23 RA patients and 22 healthy control individuals, including paired PBMC–synovial fluid samples from seven and paired PBMC–synovial tissue samples from four RA patients. Flow cytometry revealed a decreased frequency of NKT cells in PBMCs from RA patients. NKT cells were present in paired synovial fluid and synovial tissue samples. Based on the reactivity of PBMC-derived NKT cells toward α-galactosylceramide, RA patients could be divided into responders (53.8%) and nonresponders (46.2%). However, NKT cells isolated from synovial fluid from both responders and nonresponders expanded upon stimulation with α-galactosylceramide. Analysis of the cytokine profile of CD4+ and CD4- PBMC derived NKT cell lines from RA patients revealed a significantly reduced number of IL-4 producing cells. In contrast, synovial fluid derived NKT cell lines exhibited a Th0-like phenotype, which was comparable to that in healthy control individuals. This suggests that synovial fluid NKT cells are functional, even in patients with nonresponding NKT cells in their blood. We conclude that, because the number of Vα24+Vβ11+CD3+ NKT cells is decreased and the cytokine profile of blood-derived NKT cells is biased toward a Th1-like phenotype in RA patients, NKT cells might be functionally related to resistance or progression of RA. Providing a local boost to the regulatory potential of NKT cells might represent a useful candidate therapy for RA.
PMCID: PMC1174940  PMID: 15899036

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