Involvement of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis

OBJECTIVE: To clarify the mechanism by which osteoclasts are formed in culture of rheumatoid synoviocytes by exploring the involvement of receptor activator of nuclear factor kappaB ligand (RANKL)/osteoclast differentiation factor (ODF). METHODS: Osteoclast formation was evaluated in cocultures of rheumatoid synovial fibroblasts and peripheral blood mononuclear cells (PBMC) in the presence of macrophage colony stimulating factor and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) utilizing separating membrane filters. RANKL/ODF expression was examined by Northern blotting in synovial tissues from 5 rheumatoid arthritis (RA) patients and tissues from patients with giant cell tumor (GCT), osteosarcoma (OS), and osteoarthritis (OA). RANKL/ODF expression and the ability of synovial fibroblasts to support osteoclastogenesis were investigated in coculture with PBMC in the presence or absence of 1,25(OH)2D3, and soluble RANKL/ODF and osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) were measured by enzyme-linked immunosorbent assay. The effects of OPG/OCIF on the osteoclastogenesis in the primary culture of rheumatoid synoviocytes and the coculture system were determined. RESULTS: Synovial fibroblasts did not induce osteoclastogenesis when separately cocultured with PBMC. Northern blotting revealed that RANKL/ODF was highly expressed in all tissues from RA and GCT patients, but not from OA or OS patients. Cultured rheumatoid synovial fibroblasts efficiently induced osteoclastogenesis in the presence of 1,25(OH)2D3, which was accompanied by up-regulated expression of RANKL/ODF and decreased production of OPG/OCIF. Osteoclastogenesis from synoviocytes was dose-dependently inhibited by OPG/OCIF. CONCLUSION: RANKL/ODF expressed on synovial fibroblasts is involved in rheumatoid bone destruction by inducing osteoclastogenesis and would therefore be a good therapeutic target.     

Osteoprotegerin and receptor activator of nuclear factor-kappaB ligand mRNA expression in patients with rheumatoid arthritis and healthy controls

OBJECTIVE: To further understand the role of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANK-L) in rheumatoid arthritis (RA), we studied the levels of RANK-L and OPG mRNA in peripheral blood mononuclear cells (PBMC) and synovial tissue of patients with RA and controls. METHODS: RANK-L and OPG mRNA levels were measured in PBMC and CD4+/CD8+ T cell subsets of patients with chronic RA, osteoarthritis (OA), and healthy controls, using quantitative real-time polymerase chain reaction. OPG and RANK-L mRNA levels were measured in paired blood and synovial tissue samples of patients with early, untreated RA at 2 timepoints with an interval of 16 weeks. RESULTS: RANK-L mRNA levels were significantly higher in PBMC of patients with early and chronic RA compared to healthy controls. Contrary to healthy controls, RANK-L mRNA levels in patients with chronic RA were mainly of CD4+ T cell origin. OPG mRNA was observed in the blood of all (17/17) early RA patients, but could not be detected in chronic RA patients (0/14) or in patients with OA (0/8). Three out of 17 healthy controls showed measurable levels of OPG mRNA. The OPG/RANK-L ratio tended to be higher in the synovium than in the PBMC of early RA patients. RANK-L mRNA in synovial tissue was mainly of non-T cell origin. CONCLUSION: Since RANK-L and OPG mRNA levels are elevated in PBMC of RA patients, and CD4+ T cells are the major contributors to RANK-L mRNA expression, mononuclear cells in patients with RA may be involved in the pathways that regulate bone metabolism.     

Osteoprotegerin and the receptor activator of NF-kappa B ligand in the serum and synovial fluid. A comparison of patients with longstanding rheumatoid arthritis and osteoarthritis

We examined OPG and soluble RANKL in the serum (sOPG, sRANKL) and synovial fluid (synOPG, synRANKL) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). OPG and RANKL were measured in 85 patients (44 with RA, 41 patients with OA) in serum and synovial fluid as well. For measuring of OPG and RANKL ELISA tests were used. The results of OPG and RANKL were compared with clinical and radiological scores. We found a negative correlation for OPG and RANKL in synovial fluids: not only for the whole group of patients (P< 0.003, r=–0.32), but also for the subgroups (RA: P<0.04, r=–0.28, OA: P<0.002, r=–0.54). SRANKL and synRANKL were positively correlated in the whole group (P<0.01, r=0.25) and in the OA group (P<0.02, r=0.35); the RA group was showing a trend (P<0.063, r=0.24), however. Serum OPG was lower in RA, synOPG higher in OA. The difference between the two patient groups was only significant for synOPG (P<0.03, r=0.056), but not for sOPG (P<0.09, r=0.19), sRANKL (P<0.43, r=0.85) or synRANKL (P<0.11, r=0.22). The synOPG:synRANKL ratio was significantly correlated with the Larsen score (P<0.004, r=0.38). Synovial OPG is significantly decreased in rheumatoid joints, whereby synovial RANKL is increased. Lower synOPG could reflect a lower protective effect on bone, thus leading to an earlier and more pronounced bone destruction in RA. However, the effect of different mediators for joint destruction in RA and OA seems not to be important to the pathophysiological changes in the joints. The upregulation of serum OPG might be the result of the inflammation; in contrast, an upregulation of RANKL could not be found in the serum of patients with RA and OA.     

High levels of osteoprotegerin and soluble receptor activator of nuclear factor kappa B ligand in serum of rheumatoid arthritis patients and their normalization after anti-tumor necrosis factor alpha treatment

OBJECTIVE: To test the hypotheses that 1) proinflammatory cytokines affect osteoprotegerin (OPG) and soluble receptor activator of nuclear factor kappa B ligand (sRANKL) production and therefore the OPG and sRANKL levels differ in rheumatoid arthritis (RA) patients in comparison with healthy individuals; and 2) anti-tumor necrosis factor alpha (anti-TNF alpha) therapy influences OPG and sRANKL levels. METHODS: Sera were obtained from healthy individuals or RA patients receiving the combination of infliximab and methotrexate. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were isolated from RA patients. Fibroblast-like synoviocytes (FLS) were isolated from synovial tissue obtained at total knee replacement in RA patients. Supernatants from cells stimulated with cytokines were collected after culture in vitro. Concentrations of OPG and sRANKL were determined by enzyme-linked immunosorbent assays. RESULTS: A strong positive correlation between OPG concentration and age was observed in healthy individuals but not in RA patients. The OPG and sRANKL levels were higher in RA patients than in healthy controls. Cultured FLS spontaneously secreted much higher amounts of OPG than PBMCs or SFMCs. Proinflammatory cytokines enhanced OPG production. Anti-TNF alpha treatment resulted in the normalization of serum OPG and sRANKL levels in RA patients without influencing the OPG:sRANKL ratio. CONCLUSION: Although higher serum levels of OPG and sRANKL are present in RA patients than in healthy individuals, the ratio of OPG:sRANKL is similar. There is an age-dependent increase of OPG but not sRANKL levels in healthy subjects. Anti-TNF alpha treatment results in the normalization of elevated levels of OPG and sRANKL in RA patients.     

Interleukin (IL) 18 stimulates osteoclast formation through synovial T cells in rheumatoid arthritis: comparison with IL1 beta and tumour necrosis factor alpha

OBJECTIVE: To determine whether IL18 has any indirect effects on osteoclastogenesis mediated by T cells in RA synovium, and compare its effects with those of IL1 beta and TNF alpha. METHODS: Resting T cells were isolated from peripheral blood of healthy donors, and stimulated with 2 microg/ml phytohaemagglutinin (PHA) and 0.5 ng/ml IL2 for 24 hours. Synovial T cells were isolated from RA synovial tissue. The levels of soluble receptor activator of the NF-kappa B ligand (RANKL), osteoprotegerin (OPG), IFN gamma, M-CSF, and GM-CSF were determined by ELISA. Membrane bound RANKL expression was analysed by flow cytometry. Commercially available human osteoclast precursors were cocultured with T cells to induce osteoclast formation, which was determined with tartrate resistant acid phosphatase staining and pit formation assay. RESULTS: In PHA prestimulated T cells or RA synovial T cells, IL18, IL1 beta, or TNFalpha increased soluble RANKL production and membrane bound RANKL expression in a dose dependent manner. IL18, IL1 beta, and TNF alpha did not induce M-CSF, GM-CSF, IFN gamma, or OPG production in PHA prestimulated T cells or RA synovial T cells. IL18 increased the number of osteoclasts and bone resorption area on dentine slices in the coculture of human osteoclast precursors with PHA prestimulated T cells or RA synovial T cells; its ability was equivalent to that of IL1 beta, but less potent than that of TNF alpha. In the coculture system, OPG completely blocked osteoclast induction by IL18 or IL1 beta, and greatly inhibited induction by TNF alpha. CONCLUSION: IL18, IL1 beta, or TNF alpha can indirectly stimulate osteoclast formation through up regulation of RANKL production from T cells in RA synovitis; IL18 is as effective as IL1 beta, but less potent than TNF alpha.     

T cells support osteoclastogenesis in an in vitro model derived from human multiple myeloma bone disease: the role of the OPG/TRAIL interaction

The development of multiple myeloma (MM) bone disease is mediated by increased number and activity of osteoclasts (OCs). Using an in vitro osteoclastogenesis model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from patients with MM, we showed that T cells support the formation of OCs with longer survival. Different from T-cell-depleted MM PBMC cultures, exogenous macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL) were necessary for the formation of OCs; however, they did not exhibit longer survival. We found up-regulated production of RANKL, osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) by fresh MM T cells. Despite high OPG levels, the persistence of osteoclastogenesis can be related to the formation of the OPG/TRAIL complex demonstrated by immunoprecipitation experiments and the addition of anti-TRAIL antibody which decreases OC formation. OCs overexpressed TRAIL decoy receptor DcR2 in the presence of MM T cells and death receptor DR4 in T-cell-depleted cultures. In addition, increased Bcl-2/Bax (B-cell lymphoma-2/Bcl2-associated protein X) ratio, following Bcl-2 up-regulation, was detected in OCs generated in the presence of T cells. Our results highlight that MM T cells support OC formation and survival, possibly involving OPG/TRAIL interaction and unbalanced OC expression of TRAIL death and decoy receptors.     

A defect in immunoregulatory synovial fluid T cells in Behcet's syndrome

Summary Synovial fluid mononuclear cells from 5 patients with active Behcet's syndrome who were receiving no treatment were observed to have: decreased proportions of CD4⁺ and CD8⁺ cells, decreased proliferative responses of T cells in AMLR, poor capacity of non-T cells to stimulate in AMLR, decreased production of IL-2 upon PHA stimulation as compared both to their own peripheral blood mononuclear cells and to synovial fluid and peripheral blood mononuclear cells from 6 patients with RA and 6 patients with synovial effusions due to noninflammatory conditions. These findings provide further evidence of an immunoregulatory disturbance in Behcet's syndrome and indicate that synovial fluid, may be an important site where this derangement occurs in the active stage of the disease.     

Increased intraarticular interleukin-7 in rheumatoid arthritis patients stimulates cell contact-dependent activation of CD4(+) T cells and macrophages

OBJECTIVE: To determine the level of intraarticular expression of interleukin-7 (IL-7) in patients with rheumatoid arthritis (RA) and to investigate the mechanisms by which IL-7 facilitates activation of CD4(+) T cells and monocyte/macrophages in RA. METHODS: IL-7 levels were measured in synovial fluid obtained from patients with RA and patients with osteoarthritis (OA). Immunohistologic analysis was used to assess the expression of IL-7 in synovial tissue from patients with RA. Proliferation and activation markers were determined in order to measure the effect of IL-7 on mononuclear cells, isolated CD4(+) T cells, and monocyte/macrophages from the peripheral blood and synovial fluid. Cocultures of CD4(+) T cells and monocytic cells in the absence or presence of a semipermeable membrane were performed to assess the extent to which IL-7 induces its effects, either contact dependently or via soluble mediators. RESULTS: IL-7 levels were increased in synovial fluid from patients with RA compared with the levels in synovial fluid from patients with OA. Macrophages, fibroblasts, and endothelial cells in the joint lining tissue expressed abundant IL-7. In vitro, synovial fluid CD4(+) T cells and macrophages were hyperresponsive to IL-7 when compared with peripheral blood cells. Furthermore, IL-7 enhanced cell contact-dependent activation of CD4(+) T cells and monocyte/macrophages. CONCLUSION: The abundant intraarticular expression of IL-7 and the stimulation by IL-7 of contact-dependent activation of CD4(+) T cells and monocytic cells indicate that this cytokine plays an important proinflammatory role in RA synovitis. Further identification of IL-7-induced pathways may improve understanding of the important interactive role of CD4(+) T cells and monocytic cells in RA.     

Enhanced T cell proliferative response to type II collagen and synthetic peptide CII (255-274) in patients with rheumatoid arthritis.

OBJECTIVE: To determine the presence of specific immune recognition of type II collagen (CII) and its immunodominant epitope CII (255-274) in patients with rheumatoid arthritis (RA). METHODS: T cell proliferative responses to bovine CII and a synthetic peptide encompassing CII (255-274) in peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from RA patients, and in PBMC from osteoarthritis (OA) patients and healthy controls were assayed by mixed lymphocyte culture. RESULTS: The stimulation index (SI) and the number of positive (SI > or = 2) T cell responses to CII were higher in RA patients (n = 106) than in OA patients (n = 26) and healthy controls (n = 34). T cell responses to CII (255-274) were also enhanced in RA patients and correlated well with those to CII. In SFMC, positive responses to CII or CII (255-274) were detected in 61.9% of 42 RA patients. T cell responses to CII in SFMC were stronger and more prevalent than peripheral responses. The SI and positive responses to CII were higher in early RA than in late RA. Levels of IgG antibodies to CII in synovial fluid inversely correlated with T cell responses to CII. CONCLUSION: T cell responses to CII or CII (255-274) were enhanced in RA, especially in early disease. Synthetic peptide CII (255-274), as well as native CII, could be recognized as immunogenic antigens by T cells, particularly in the synovial fluid. These observations suggest that CII-reactive T cells play an important role in the pathogenesis of RA. Peripheral tolerance induction using CII (255-274) might be useful in the treatment of RA.     

High levels of osteoprotegerin and soluble receptor activator of nuclear factor κB ligand in serum of rheumatoid arthritis patients and their normalization after anti–tumor necrosis factor α treatment

Objective

To test the hypotheses that 1) proinflammatory cytokines affect osteoprotegerin (OPG) and soluble receptor activator of nuclear factor κB ligand (sRANKL) production and therefore the OPG and sRANKL levels differ in rheumatoid arthritis (RA) patients in comparison with healthy individuals; and 2) anti–tumor necrosis factor α (anti–TNFα) therapy influences OPG and sRANKL levels.

Methods

Sera were obtained from healthy individuals or RA patients receiving the combination of infliximab and methotrexate. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were isolated from RA patients. Fibroblast‐like synoviocytes (FLS) were isolated from synovial tissue obtained at total knee replacement in RA patients. Supernatants from cells stimulated with cytokines were collected after culture in vitro. Concentrations of OPG and sRANKL were determined by enzyme‐linked immunosorbent assays.

Results

A strong positive correlation between OPG concentration and age was observed in healthy individuals but not in RA patients. The OPG and sRANKL levels were higher in RA patients than in healthy controls. Cultured FLS spontaneously secreted much higher amounts of OPG than PBMCs or SFMCs. Proinflammatory cytokines enhanced OPG production. Anti‐TNFα treatment resulted in the normalization of serum OPG and sRANKL levels in RA patients without influencing the OPG:sRANKL ratio.

Conclusion

Although higher serum levels of OPG and sRANKL are present in RA patients than in healthy individuals, the ratio of OPG:sRANKL is similar. There is an age‐dependent increase of OPG but not sRANKL levels in healthy subjects. Anti‐TNFα treatment results in the normalization of elevated levels of OPG and sRANKL in RA patients.

     

Peripheral blood T lymphocytes from patients with early rheumatoid arthritis express RANKL and interleukin-15 on the cell surface and promote osteoclastogenesis in autologous monocytes

OBJECTIVE: To investigate the osteoclastogenic potential of T cells from the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) on autologous monocytes, and to study the cytokines implicated in this process. METHODS: T cells and monocytes were isolated from the PB of 20 healthy subjects and 20 patients with early RA, and from the SF of 20 patients with established RA. Autologous T cell/monocyte cocultures were established in the absence of exogenous cytokines or growth factors in order to examine spontaneous ex vivo osteoclast differentiation by tartrate-resistant acid phosphatase staining and calcified matrix resorption activity. RESULTS: Surface RANKL was expressed on freshly isolated T cells from the PB of patients with early RA and the SF of patients with established RA. In addition, surface interleukin-15 (IL-15) was detected on freshly isolated T cells and monocytes from the PB of patients with early RA and the SF of patients with established RA. Autologous T cell/monocyte cocultures derived from the SF of patients with established RA and from the PB of patients with early RA, but not from the PB of healthy controls, resulted in osteoclast differentiation that was significantly inhibited by osteoprotegerin (OPG) and by neutralizing monoclonal antibodies to IL-15, IL-17, tumor necrosis factor alpha (TNFalpha), and IL-1beta. OPG, anti-TNFalpha, and anti-IL-1beta demonstrated a cooperative inhibitory effect. At 1-year followup, surface RANKL and IL-15 and ex vivo osteoclastogenesis were no longer observed on PB T cells or monocytes from patients with early RA in whom clinical remission had been achieved with treatment. CONCLUSION: T cells are important contributors to the pathogenesis of bone erosions in RA through interaction with osteoclast precursors of the monocyte/macrophage lineage.     

Peripheral blood T lymphocytes from patients with early rheumatoid arthritis express RANKL and interleukin‐15 on the cell surface and promote osteoclastogenesis in autologous monocytes

Objective

To investigate the osteoclastogenic potential of T cells from the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) on autologous monocytes, and to study the cytokines implicated in this process.

Methods

T cells and monocytes were isolated from the PB of 20 healthy subjects and 20 patients with early RA, and from the SF of 20 patients with established RA. Autologous T cell/monocyte cocultures were established in the absence of exogenous cytokines or growth factors in order to examine spontaneous ex vivo osteoclast differentiation by tartrate‐resistant acid phosphatase staining and calcified matrix resorption activity.

Results

Surface RANKL was expressed on freshly isolated T cells from the PB of patients with early RA and the SF of patients with established RA. In addition, surface interleukin‐15 (IL‐15) was detected on freshly isolated T cells and monocytes from the PB of patients with early RA and the SF of patients with established RA. Autologous T cell/monocyte cocultures derived from the SF of patients with established RA and from the PB of patients with early RA, but not from the PB of healthy controls, resulted in osteoclast differentiation that was significantly inhibited by osteoprotegerin (OPG) and by neutralizing monoclonal antibodies to IL‐15, IL‐17, tumor necrosis factor α (TNFα), and IL‐1β. OPG, anti‐TNFα, and anti–IL‐1β demonstrated a cooperative inhibitory effect. At 1‐year followup, surface RANKL and IL‐15 and ex vivo osteoclastogenesis were no longer observed on PB T cells or monocytes from patients with early RA in whom clinical remission had been achieved with treatment.

Conclusion

T cells are important contributors to the pathogenesis of bone erosions in RA through interaction with osteoclast precursors of the monocyte/macrophage lineage.

     

Suppression of osteoclastogenesis in rheumatoid arthritis by induction of apoptosis in activated CD4+ T cells

OBJECTIVE: To examine the suppressive effect of anti-human Fas monoclonal antibody (mAb) on osteoclastogenesis in rheumatoid arthritis (RA) both in vitro and in vivo. METHODS: For in vitro analysis, activated CD4+ T cells derived from peripheral blood mononuclear cells were left untreated or were treated with humanized anti-human Fas mAb (R-125224) and cocultured with human monocytes. On day 12, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells was counted. For in vivo analysis, tissue derived from human RA pannus was implanted with a slice of dentin subcutaneously in the backs of SCID mice (SCID-HuRAg-pit model). R-125224 was administered intravenously once a week for 3 weeks. The implanted tissue and dentin slice were removed, and the pits formed on the dentin slice were analyzed. RESULTS: In vitro, coculture of activated CD4+ T cells and peripheral monocytes induced osteoclastogenesis. The number of TRAP-positive multinucleated cells was reduced when activated CD4+ T cells were treated with R-125224. We established a new animal model for monitoring osteoclastogenesis, SCID-HuRAg-pit. We found that with R-125224 treatment, the number of pits formed on the implanted dentin slices was significantly reduced and the number of lymphocytes in the implanted RA synovial tissue was dramatically reduced in this model. CONCLUSION: This is the first study to demonstrate the suppressive effect of anti-human Fas mAb on osteoclastogenesis in RA synovial tissues through the induction of T cell apoptosis. Induction of apoptosis of infiltrated lymphocytes could be a useful therapeutic strategy for RA, in terms of suppressing both inflammation and bone destruction.     

Increment of CD8S6F1 cells in synovial fluid from patients with rheumatoid arthritis.

OBJECTIVE--To investigate the role of CD8 cell subsets in the pathogenesis of rheumatoid arthritis (RA) and the phenotypes of T cells adherent or non-adherent to the target cells (endothelial cells and synovial cells) pre-treated with IL-1 beta. METHODS--The expression of S6F1 on CD8 cells and that of an activation marker on CD8 cells and CD8 cell subsets was evaluated in specimens of peripheral blood and synovial fluid obtained from 15 patients with RA and 10 with osteoarthritis (OA) using a two- or three-colour immunofluorescence method for analysis. RESULTS--The percentage of CD8S6F1 cells among CD8 cells in synovial fluid was significantly greater than that of peripheral blood. Synovial fluid from RA patients had a greater percentage of CD8S6F1 cells compared with either peripheral blood of matched patients or synovial fluid of OA patients. The percentage of CD8HLA-DR cells in synovial fluid was markedly greater than that in paired samples of peripheral blood in patients with RA. In the CD8S6F1 cells from both groups of patients, synovial fluid showed an increased percentage of HLA-DR cells compared with peripheral blood. Similar results were observed in CD8 cells lacking S6F1 expression (CD8S6F1-) from both groups of patients. There was no significant difference in the percentage of HLA-DR cells between CD8S6F1 and CD8S6F1- cell populations in peripheral blood. In contrast with peripheral blood, in synovial fluid of RA patients the percentage of HLA-DR cells in the CD8S6F1 cell population was markedly greater than that in the CD8S6F1- population. However, the percentage of HLA-DR cells in both cell populations was similar in synovial fluid of OA patients. In both the endothelial and the synovial cell adhesion assays, the percentage of CD8S6F1 among CD8 cells and the mean fluorescence intensity of S6F1 antigen on CD8S6F1 cells were significantly greater in the adherent T cell population than that in the non-adherent T cell population. CONCLUSION--These results suggest that increased expression of S6F1 antigen and the increased percentage of HLA-DR cells on CD8 cells in synovial fluid may be responsible for the migration of these cells into inflamed synovial tissues, and for cellular interactions between these cells and synovial cells or the extracellular matrix.     

The TNF superfamily member LIGHT contributes to survival and activation of synovial fibroblasts in rheumatoid arthritis

OBJECTIVES: The TNF superfamily member LIGHT has a T-cell co-stimulatory role and has previously been associated with inflammation and autoimmunity. To investigate its role in rheumatoid arthritis (RA), a disease where activated T cells contribute in a prominent way, we have analysed the expression of LIGHT and its receptors in RA and analysed its effects on synovial fibroblasts in vitro. METHODS: The expression of LIGHT was measured in synovial tissues and fluids and the receptors of LIGHT were detected on synovial fibroblasts derived from patients with RA and osteoarthritis (OA). The effects of recombinant LIGHT on the production of proinflammatory cytokines and proteases and on the apoptosis of synovial fibroblasts was assessed. RESULTS: LIGHT mRNA was present in synovial tissues of patients with RA but not with OA. Correspondingly, soluble LIGHT protein could be detected in RA synovial fluid samples at much higher levels than in synovial fluid from patients with OA. Immunohistochemical detection of LIGHT and analysis of synovial fluid cells by flow cytometry revealed CD4 T cells as the major source of LIGHT in the rheumatoid joint. Synovial fibroblasts from RA patients were found to express the LIGHT receptors HVEM and LTbetaR. Recombinant LIGHT induced RA synovial fibroblasts to upregulate MMP-9 mRNA, CD54 and IL-6 in an NF-kappaB-dependent fashion. In vitro, exposure of cultured synovial fibroblasts to LIGHT reduced FAS-mediated apoptosis significantly, without affecting the rate of spontaneous apoptosis. CONCLUSIONS: The results provide evidence for a novel T-cell-dependent activation of synovial fibroblasts by LIGHT in joints of patients with RA, contributing to an inflammatory and destructive phenotype.     

Cytokine production by synovial T cells in rheumatoid arthritis.

OBJECTIVE: To investigate the production of cytokines by T cells in patients with rheumatoid arthritis (RA), reactive arthritis (REA) and osteoarthritis (OA). METHODS: The lymphokines interleukin (IL)-2, IL-4, interferon gamma (IFN-gamma) and tumour necrosis factor beta (TNF-beta), as well as the monokines IL-1, IL-6 and TNF-alpha, were measured by immunoassays in sera and synovial fluid (SF) from patients with RA, REA and OA. In addition, cytokine expression was studied by immunohistochemistry in synovial membrane tissue sections from patients with RA and OA. RESULTS: Almost 60% of RA sera contained at least one of the cytokines investigated, though in low concentrations, whereas cytokines were generally not detectable in sera from REA and OA patients. In contrast, cytokines were found in virtually all SF; thus, the majority of SF from RA patients contained IFN-gamma (median level 17 pg/ml) in addition to the monokines IL-6 (4700 pg/ml) and TNF-alpha (157 pg/ml). IFN-gamma and IL-6 (but not TNF-alpha) were also frequently measured in SF from REA patients, whereas OA samples typically contained only IL-6. Immunohistochemical analysis of tissue sections from RA patients revealed lymphokine expression in 0.1-0.3% of T cells, particularly IL-2 and IFN-gamma, and to a lesser extent also IL-4. Interestingly, the expression of TNF-alpha and IL-6 by synovial T cells was also observed. The majority of cytokine-expressing T cells were CD4-positive T-helper cells typically found in perivascular areas, whereas cytokine-producing CD8-positive T cells were found distributed throughout the synovium. As expected, in specimens from OA patients, T cells were much less abundant and expression of cytokines could not be detected. CONCLUSION: These data clearly demonstrate production of cytokines by T cells in RA synovial tissue, indicating that activated T cells play a role in the pathophysiological events of RA.     

Comparison of blood and synovial fluid lymphocyte subsets in rheumatoid arthritis and osteoarthritis

Summary Immunoregulatory T-cell deficiency is thought to underlie pathogenesis of rheumatoid arthritis (RA) as a systemic autoimmunopathy. The aim of this study was a simultaneous analysis of peripheral blood and synovial lymphocyte subsets (Ly-SS) of RA patients as compared to patients with locally active osteoarthritis (OA). Peripheral blood Ly-SS and paired synovial fluid Ly-SS from 87 RA patients were analysed by two dimensional flow cytometry (Simulset Becton Dickinson) as compared to 15 OA patients. The control group consisted of 32 healthy subjects. The peripheral blood analysis from RA and OA patients revealed a significant decrease of CD8+T-cells and increase of CD4+:CD8+ ratio when compared to the control group. The blood of RA patients showed a significant increase of HLA DR+ and IL 2R+T cells as compared to OA group. The synovial fluid from RA and OA patients showed a significant increase of CD3+, CD8+, HLA DR+ T-cells and decrease of CD4+:CD8+ ratio and CD19+ cells in comparison to the peripheral blood. This study shows, that the OA T-cell system seems not to be activated in peripheral blood in opposition to RA patients. Synovial fluid Ly-SS in OA, however, showed only quantitative but not qualitative differences. OA seems to be mainly a local inflammatory response depending on T-cells, when lymphocyte T activity in blood is diminished.