Cells In Synovial Tissue Research Articles

Antigen-presenting cells containing bacterial peptidoglycan in synovial tissues of rheumatoid arthritis patients coexpress costimulatory molecules and cytokines

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by intimal lining hyperplasia and massive infiltration of the synovial sublining by antigen-presenting cells (APCs), lymphocytes, and plasma cells. Peptidoglycan (PG), a major cell wall component of gram-positive bacteria, which is abundantly expressed in all mucosa, is believed to be involved in the pathogenesis of RA because of its ability to induce the production of proinflammatory cytokines as well as to induce arthritis in rodents. While PG has been detected in APCs in RA joints, little is known about the role of these cells in RA. In this study, the presence and immune competence of PG-containing cells in synovial tissues from 14 RA and 14 osteoarthritis (OA) patients were analyzed in situ. Using immunohistochemistry, we examined the coexpression of phenotypic markers, costimulatory molecules, and cytokines by PG-containing cells. PG was present in higher numbers in RA than in OA synovial tissues, although the difference was not significant. PG-containing cells were mainly macrophages, but some mature dendritic cells also contained PG. A high percentage of PG-containing cells in both RA and OA synovial tissues coexpressed HLA-DR. CD40, CD80, and CD86 expression by PG-containing cells was higher in RA than in OA tissues. Furthermore, PG-containing cells coexpressed cytokines, which modulate inflammatory reactions, in particular, tumor necrosis factor alpha and interleukins 6 and 10. The results suggest that PG-containing cells may contribute to inflammation within the microenvironment of the joint in RA patients.

Contribution of OX40/OX40 ligand interaction to the pathogenesis of rheumatoid arthritis.

OX40 ligand (OX40L) and OX40 (CD134) are a pair of cell surface molecules belonging to the TNF/TNF receptor family. Interaction of OX40L with its receptor OX40 is thought to be important in T cell activation through T cell/antigen-presenting cell interaction. However, involvement of these molecules in the pathogenesis of rheumatoid arthritis (RA) remains unclear. To explore the contribution of OX40/OX40L interaction to the pathogenesis of RA in vivo, we evaluated the effect of a neutralizing anti-OX40L monoclonal antibody (mAb) on the development of collagen-induced arthritis (CIA) in DBA/1 mice as an animal model for RA. Administration of anti-OX40L mAb into type II collagen (CII) -immunized DBA/1 mice dramatically ameliorated the disease severity. In vivo treatment with anti-OX40L mAb did not inhibit the expansion of CII-reactive T cells, but suppressed IFN-gamma and anti-CII IgG2a production. Therefore, OX40/OX40L interaction appears to play a critical role in the development of CIA by enhancing Th1-type autoimmune response. In addition, T lymphocytes in synovial fluid and synovial tissue from RA patients expressed OX40, while OX40L was expressed on sublining cells in synovial tissue. These results indicate that OX40/OX40L interaction may play a critical role in the development of RA.

Contribution of OX40/OX40 ligand interaction to the pathogenesis of rheumatoid arthritis

OX40 ligand (OX40L) and OX40 (CD134) are a pair of cell surface molecules belonging to the TNF/TNF receptor family. Interaction of OX40L with its receptor OX40 is thought to be important in T cell activation through T cell/antigen-presenting cell interaction. However, involvement of these molecules in the pathogenesis of rheumatoid arthritis (RA) remains unclear. To explore the contribution of OX40/OX40L interaction to the pathogenesis of RA in vivo, we evaluated the effect of a neutralizing anti-OX40L monoclonal antibody (mAb) on the development of collagen-induced arthritis (CIA) in DBA/1 mice as an animal model for RA. Administration of anti-OX40L mAb into type II collagen (CII) -immunized DBA/1 mice dramatically ameliorated the disease severity. In vivo treatment with anti-OX40L mAb did not inhibit the expansion of CII-reactive T cells, but suppressed IFN-gamma and anti-CII IgG2a production. Therefore, OX40/OX40L interaction appears to play a critical role in the development of CIA by enhancing Th1-type autoimmune response. In addition, T lymphocytes in synovial fluid and synovial tissue from RA patients expressed OX40, while OX40L was expressed on sublining cells in synovial tissue. These results indicate that OX40/OX40L interaction may play a critical role in the development of RA.

Transcription factor decoy for NFkappaB inhibits cytokine and adhesion molecule expressions in synovial cells derived from rheumatoid arthritis.

Numerous cytokines are expressed in lesions of synovial hyperplasia of patients with rheumatoid arthritis (RA), and their pathophysiological contributions have been the subject of speculation. These genes are regulated by the transcription factor NFkappaB which in turn is activated by tumour necrosis factor-alpha (TNF-alpha) and cytokines. In this study we examined the inhibition of the production of pro-inflammatory cytokines, adhesion molecule and matrix metalloproteinase (MMP) from synovial tissue of patients with RA by the introduction of synthetic double-stranded DNA with high affinity for the NFkappaB binding site. NFkappaB decoy oligonucleotides (ODN) were introduced with the aid of the haemagglutinating virus of Japan (HVJ)-liposome method into synovial tissue or synovial cells derived from patients with RA. The levels of interleukin-1beta (IL-1beta), IL-6, TNF-alpha, intercellular adhesion molecule-1 (ICAM-1) and MMP-1 were determined by means of enzyme-linked immunosorbent assay (ELISA) and Northern blotting analysis. A cell counting kit was used to study the effect of NFkappaB decoy ODN on synovial cell proliferation. The production of these mediators was significantly inhibited by the introduction of NFkappaB decoy ODN compared with the effect of scrambled decoy ODN. Transfection of NFkappaB decoy ODN resulted in a significant inhibition of synovial cell proliferation as compared with that of scrambled decoy ODN. The results demonstrated in this study suggest the potential usefulness of NFkappaB decoy ODN for gene therapy of inflammatory synovitis of RA.

RANTES and Chemotactic Activity in Synovial Fluids From Patients With Rheumatoid Arthritis andOsteoarthritis

A massive accumulation of inflammatory cells in synovial tissues is a major pathological feature of rheumatoid arthritis (RA). Neutrophiles dominate synovial fluid while rheumatoid synovium is infiltrated with mononuclear cells. Mechanisms regulating influx of particular subpopulations of leukocytes into articular cavity and synovium compartment are not completely defined. An increasing amount of data supports a crucial role of a C-C chemokine RANTES in the RA pathogenesis. Our objective is to evaluate chemotactic activity for neutrophils (NCA), lymphocytes (LCA), and monocytes (MoCA) in SFs obtained from patients with RA and osteoarthritis (OA). We also aimed to characterise the relation between chemotactic activity, RANTES, and percentage distribution of leukocytes in SF. SFs from 11 patients with RA and 6 with OA were included in the study. Modified microchamber Boyden method was employed to assess chemotactic activity. Cytological and biochemical analysis of SF was performed. RANTES was measured with ELISA. Rheumatoid SFs were rich in cells with predominance of neutrophiles while osteoarthritic fluids were lymphocytic. RA SFs were also characterised by increased lactoferrin level. Both NCA and LCA were higher in SF from patients with RA (62 ± 12 and 24 ± 6 cells/HPF, resp) as compared to patients with OA (23 ± 6; P < .05 and 6 ± 2 cells/HPF; P < 0.05). The chemoattractive effect of RA SF was more pronounced on neutrophiles than on lymphocytes. RA SF expressed high RANTES levels (145 ± 36 pg/mL), while OA SF was characterised by only trace amount of this chemokine (2 ± 1 pg/mL). We found positive correlation of RANTES with chemotactic activity for mononuclear cells (LCA+MoCA; R = 0.61; P < .05). Surprisingly, RANTES correlated also positively with neutrophiles number (R = 0.77; P < 0.001). Rheumatoid SF possesses strong chemotactic potency for leukocytes. RANTES is overexpressed in RA SF and is a potential mediator influencing intensity and composition of cellular infiltration in joints affected with inflammatory arthritis.

Expression of peroxisome proliferator-activated receptorγ (PPARγ) in synovial tissues of inflammatory arthritis

Peroxisome proliferator-activated receptor γ (PPARγ) is transcription factors belong to the nuclear receptor gene family and binds to specific response elements termed PPRE, thus regulating the expression of target genes. PPARγ triggers adipocyte differentiation in adipose tissue and inhibits the cytokine production in macrophage. We analyzed the expression of PPARγ in human synovial tissues of patient with rheumatoid arthritis (RA) or osteoarthritis (OA) . PPARγ mRNA strongly expressed in synovial tissues with active RA synovitis more than that of OA. Immunohistological analysis demonstrated PPARγ localized in nuclear area of synovial lining macrophages and vascular endothelial cells in RA synovial tissues. PPARγ expression correlated with the rate of macrophage infiltration and hypervas-cularization in synovial tissues. We measured the expression of PPARγ mRNA in rat arthritic synovial tissues. PT-PCR analysis demonstrated the strong expression of PPARy in rat arthritic joints.PPARγ ligand, antidiabetic thiazolidinediones (troglitazone) inhibited the synovitis of adjuvant arthritis. These data suggest PPARγ ligands may have therapeutic application in inflammatory arthritis, such as RA.

Chondroprogenitor cells of synovial tissue.

To assess the chondrogenic potential of cells within the synovium. Explants of synovium taken from various sites in the joint were embedded in agarose and cultured with transforming growth factor beta1 (TGFbeta1) to assess their chondrogenic potential. Isolated synovial cells were also tested for their chondrogenic potential by culturing them as aggregates in a chemically defined medium with TGFbeta1. Cartilage formation was determined with histologic staining and immunohistochemistry. The osteochondral potential of the isolated cells was also assessed after subcutaneous implantation of the cells, loaded into porous calcium phosphate ceramic cubes, in athymic mice. A total of 48 synovial explants were cultured in agarose with TGFbeta1. The formation of cartilage was observed in the outer region of 21 explants, and type II collagen was localized in that region by immunohistochemistry. A larger percentage of TGFbeta1+ explants from the inner synovium sites formed cartilage compared with those from the outer synovium sites. Chondrogenesis occurred in aggregates incubated with TGFbeta1 as early as day 7, and by day 14, all TGFbeta1+ aggregates demonstrated chondrogenesis. In contrast with the results of the in vitro aggregate assay for chondrogenesis, no formation of cartilage or bone was evident in any section containing synovial cell-loaded ceramic cubes that were harvested at either 3 or 6 weeks after implantation subcutaneously in athymic mice. Synovial explants and isolated synovial cells will undergo chondrogenesis when cultured in the presence of TGFbeta1. The data indicate a possible synovial origin for the chondrocytic cells found in rheumatoid pannus. Furthermore, these data are consistent with the clinical findings of synovial chondrogenesis leading to synovial chondromatosis.

Production of interleukin-7 and interleukin-15 by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

To examine the ability of fibroblast-like synoviocytes in rheumatoid arthritis (RA) to produce interleukin-7 (IL-7) and IL-15, and the ability of these cytokines to induce the proliferation of synovium-infiltrating T cells. Messenger RNA (mRNA) and protein levels of IL-7 and IL-15 in synovial tissue cells and fibroblast cell lines were determined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. T cell-enriched populations from RA synovial tissues were isolated by deleting adherent cells after a 14-hour incubation in plastic dishes or by expanding T cells during a 14-day incubation of tissue cells with IL-2 alone, and their proliferative responses to IL-7, IL-15, and IL-2 were measured by 3H-thymidine incorporation. Freshly isolated cells from RA synovial tissues more strongly expressed mRNA for both IL-7 and IL-15 compared with the cells from osteoarthritis tissues, and could spontaneously release greater amounts of these cytokine proteins in culture. Fibroblast cell lines prepared from RA patients were able to produce large amounts of IL-15 and small amounts of IL-7 at both the transcriptional and protein levels, and their cytokine production was significantly elevated when stimulated with IL-1 and tumor necrosis factor alpha. Purified synovial tissue macrophages spontaneously released IL-15 but not IL-7, and synovial T cells did not produce either cytokine. IL-7 and IL-15, similar to IL-2, stimulated the proliferation of synovial tissue T cells from RA patients; IL-7 was less potent than IL-15 or IL-2. These results indicated that fibroblast-like synoviocytes are an important source of the cytokines with IL-2-like activity, IL-15 and IL-7, in RA joints, and that IL-15 may be mainly responsible for local T cell activation and expansion in the presence of deficient IL-2 production by T cells.

Apomodulation as a novel therapeutic concept for the regulation of apoptosis in rheumatoid synoviocytes.

Fas-mediated apoptosis is observed in synoviocytes of patients with rheumatoid arthritis (RA). This process may be involved in the pathophysiology of RA. We have recently found that Fas-mediated apoptosis of RA synoviocytes is associated with activation of two signaling pathways, the c-Jun amino-terminal kinase (JNK)/activator protein-1 (AP-1) pathway, and the FADD (Fas-associated death domain protein)/Caspase-8/Caspase-3/PARP (poly(ADP-ribose)polymerase) pathway. The latter appears to be one of the major signaling pathways required for Fas-mediated apoptosis in RA synoviocytes. Interestingly, Fas-mediated apoptosis in synoviocytes may be induced at least in part by tumor necrosis factor-alpha. Paradoxically, tumor necrosis factor-alpha also causes proliferation of synoviocytes. Employing these molecular processes in the treatment of RA, we have recently shown that ex vivo gene transfer of human Fas ligand (hFasL) induced apoptosis of synoviocytes and infiltrated mononuclear cells of RA synovial tissue through cell-to-cell interaction via the Fas/FasL system. We believe that further understanding of the complex regulatory mechanisms of apoptosis in RA synoviocytes would uncover further aspects of the pathophysiologic mechanisms of RA and contribute to the development of new and effective therapies for RA.

Expression of Receptor Tyrosine Kinase Axl and its Ligand Gas6 in Rheumatoid Arthritis: Evidence for a Novel Endothelial Cell Survival Pathway

Angiogenesis and synovial cell hyperplasia are characteristic features of rheumatoid arthritis (RA). Many growth and survival factors use receptors belonging to the tyrosine kinase family that share conserved motifs within the intracellular catalytic domains. To understand further the molecular basis of cellular hyperplasia in RA, we have used degenerate primers based on these motifs and RNA obtained from the synovium of a patient with RA to perform reverse transcriptase-polymerase chain reaction. We report detection of the receptor tyrosine kinase (RTK) Axl in RA synovium and we document the expression pattern of Axl in capillary endothelium, in vascular smooth muscle cells of arterioles and veins, and in a subset of synovial cells in RA synovial tissue. Gas6 (for growth arrest-specific gene 6), which is a ligand for Axl and is related to the coagulation factor protein S, was found in synovial fluid and tissue from patients with RA and osteoarthritis. Axl expression and function was studied in human umbilical vein endothelial cells (HUVECs). Gas6 bound to HUVECs; soluble Axl inhibited this binding. Exogenous Gas6 protected HUVECs from apoptosis in response to growth factor withdrawal and from TNFalpha-mediated cytotoxicity. These findings may reveal a new aspect of vascular physiology, which may also be relevant to formation and maintenance of the abnormal vasculature in the rheumatoid synovium.

Cytokine production by synovial T cells in rheumatoid arthritis

To investigate the production of cytokines by T cells in patients with rheumatoid arthritis (RA), reactive arthritis (REA) and osteoarthritis (OA). 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. 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. 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.

Flow cytometric single-cell analysis of cytokine production by CD4+ T cells in synovial tissue and peripheral blood from patients with rheumatoid arthritis.

To determine the cytokine profile of CD4+ T cells in the synovial tissue (ST) of rheumatoid arthritis (RA) patients at the single-cell level. Unseparated ST cells and paired CD4+ T cells separated from the peripheral blood (PB) and ST of RA patients were stimulated for 4 hours with phorbol myristate acetate (PMA) plus calcium ionophore A23187, or for 6 hours with immobilized anti-CD3 plus anti-CD28, in the presence of brefeldin A. Cells were stained for intracellular cytokines such as interferon-gamma (IFNgamma), interleukin-2 (IL-2), IL-4, IL-10, and IL-13, in combination with cell surface markers. The percentages of cytokine-producing T cells were analyzed by flow cytometry. When ST cells were stimulated with PMA plus A23187 in bulk culture, IFNgamma-producing T cells were more frequently detected in the CD8+ subset, but cells producing other cytokines were found in the CD4+ subset. Purified ST CD4+ T cells, after stimulation with PMA plus A23187, were able to produce higher levels of IFNgamma but lower levels of IL-4 and IL-13, by analysis at the single-cell level, as compared with the PB CD4+, CD45RO+ T cells. The majority of IL-4- or IL-13-producing ST CD4+ cells produced IFNgamma, although PB CD4+ T cells rarely showed this cytokine pattern. IL-10-producing CD4+ T cells were more frequently found in the ST than in the PB. Of interest, most of the IL-10-producing ST CD4+ T cells were able to produce IFNgamma. IL-2-producing CD4+ T cells were similarly present in both compartments. Similar intracellular cytokine patterns were observed with anti-CD3 plus anti-CD28 stimulation, although the number of detected cells was lower. These data indicate that CD4+ T cells present within the inflamed synovium have apparently distinct cytokine profiles from those of memory CD4+ T cells in the PB, as typified by their ability to secrete both IFNgamma and IL-10.

Analysis of the cellular infiltrates and expression of cytokines in synovial tissue from patients with rheumatoid arthritis and reactive arthritis.

The cellular infiltrates and cytokine patterns in synovial tissue (ST) from patients with rheumatoid arthritis (RA) and reactive arthritis (ReA) were compared in order to determine the mechanisms responsible for the chronic and destructive course of RA. Since the results could be influenced by differences in disease duration, ST was studied from patients in both early and late stages of the disease. Ten patients had early RA (< 1 year), ten long-standing RA (> 1 year), six early ReA (< 1 year), and five long-standing ReA (> 1 year). Histological analysis demonstrated that the scores for infiltration by lymphocytes and plasma cells, and the scores for inflammation, were significantly higher in RA than in ReA. Immunolabelling studies showed that in particular, the scores for infiltration by CD38+ plasma cells, granzyme B+ cells, and interferon-gamma (IFN gamma)+ cells were significantly higher in RA than in ReA. The results were independent of the disease duration. The increased number of lymphocytes, plasma cells, and granzyme B+ cells in rheumatoid synovial tissue supports the paradigm that RA is the result of specific immune recognition in the joint and that granzyme B+ cells play an important role in joint destruction.

Poor Expression of T Cell-Derived Cytokines and Activation and Proliferation Markers in Early Rheumatoid Synovial Tissue

We compared the state of activation and proliferation of T cells in synovial tissue (ST) from rheumatoid arthritis (RA) patients in early and late stages of the disease to find out whether T-cell-driven immune responses vary during the course of the disease. ST was obtained from 12 patients with early RA (< 1 year) and 12 patients with longstanding RA (> 5 years). T cells and interferon-γ (IFN-γ)-positive cells were detected in ST using immunohistologic methods. To determine the percentage of T cells expressing the interleukin-2 receptor, IFN-γ, or the proliferation associated antigen Ki-67, immunofluorescence double-staining techniques were used. The scores for the number of T cells and for the expression of IFN-γ as well as the percentages of T cells expressing CD25, IFN-γ, or Ki-67 in rheumatoid synovium were not dependent on disease duration. These results do not support the assumption that the responsiveness of T cells in ST of RA patients differs between early and late stages of the disease. The data indicate that at present no arguments exist that the effect of T-cell-directed interventions on synovial inflammation might vary in different stages of the disease.

Interleukin 15 is produced by endothelial cells and increases the transendothelial migration of T cells In vitro and in the SCID mouse-human rheumatoid arthritis model In vivo.

The capacity of endothelial cells (EC) to produce IL-15 and the capacity of IL-15 to influence transendothelial migration of T cells was examined. Human umbilical vein endothelial cells expressed both IL-15 mRNA and protein. Moreover, endothelial-derived IL-15 enhanced transendothelial migration of T cells as evidenced by the inhibition of this process by blocking monoclonal antibodies to IL-15. IL-15 enhanced transendothelial migration of T cells by activating the binding capacity of the integrin adhesion molecule LFA-1 (CD11a/CD18) and also increased T cell motility. In addition, IL-15 induced expression of the early activation molecule CD69. The importance of IL-15 in regulating migration of T cells in vivo was documented by its capacity to enhance accumulation of adoptively transferred human T cells in rheumatoid arthritis synovial tissue engrafted into immune deficient SCID mice. These results demonstrate that EC produce IL-15 and imply that endothelial IL-15 plays a critical role in stimulation of T cells to extravasate into inflammatory tissue.

Expression of interleukin-12 in synovial tissue from patients with rheumatoid arthritis.

To examine the importance of interleukin-12 (IL-12) as a factor in the interferon-gamma (IFNgamma)-dominant T cell cytokine response in the synovial tissue of patients with rheumatoid arthritis (RA). The expression of IL-12 in synovial tissue samples from patients with chronic RA (> or = 2 years) was compared with that in samples from osteoarthritis (OA) patients by detection of IL-12 p40 messenger RNA (mRNA) using reverse transcriptase-polymerase chain reaction, measurement of IL-12 p70 protein in culture supernatants of tissue cells by immunoassay, and immunostaining of tissue sections with anti-IL-12 p70. The production of IFNgamma by RA synovial tissue cells cultured with or without IL-12 was determined. In addition, T cells were obtained 14 days after culturing RA synovial tissue cells with IL-2 alone or with IL-2 plus IL-12, neutralizing anti-IL-12, or IL-4, and cytokine patterns (i.e., IFNgamma and IL-4 levels) were determined by stimulating cells for 24 hours with anti-CD3 plus phorbol myristate acetate. Synovial tissues from RA patients more strongly expressed IL-12 p40 mRNA than did OA tissues. Dissociated tissue cells from 21 of 37 RA patients spontaneously released detectable amounts of IL-12 p70 (> or = 12.5 pg/ml) in culture, whereas production of IL-12 by OA tissues was limited. By immunohistochemical analysis, IL-12-producing cells were localized mainly in the sublining layer of RA synovium, and mostly expressed the CD68 antigen. Levels of IFNgamma production by RA synovial tissue cells were potently and selectively enhanced by IL-12. The ability of IL-2-expanding synovial T cells to produce IFNgamma was augmented by costimulation with IL-12 and diminished by anti-IL-12, while it was not affected by IL-4. These data suggest that IL-12, produced mainly by macrophage-lineage cells, may be involved in IFNgamma-dominant cytokine production by infiltrating T cells in joints with chronic RA.

T Cell Receptor Gene in Synovial Tissues of Rheumatoid Arthritis

Rheumatoid arthritis is a chronic and destructive autoimmune joint disease characterized by inflammation of synovial tissue of unknown aetiology. Studies on TCR genes expressed by infiltrating T cells in synovial tissues have attempted to identify mechanism and specificity of the recruitment. T cell infiltrate in rheumatoid arthritis appears to be an association of a polyclonal non specific infiltrate with dominant clones or clonotypes. T cell repertoire in synovial tissue is biased compared to peripheral blood but no TCR V gene can be identified as commonly over-used. Comparison of motifs found in the CDR3 region of dominant clones from different studies has currently failed to identified a commonly motif. The fact that a number of dominant clones or clonotypes is present in different joints and at different times of the disease suggests a selective expansion of T lymphocytes in rheumatoid arthritis synovial membrane. Further investigations are needed to characterize the specificity of these dominant clonotypes.Synovial T cell infiltrate in rheumatoid arthritis appears to be polyclonal and the majority of T cells are non-specifically recruited into sites of inflammation following tissue injury. But almost all the studies show the presence of a large number of dominant clones in the synovial tissue, using different TCR V genes, which certainly play an important role in the perpetuation of the disease. This, and the fact that a number of dominant clones or clonotypes is present in different joints and at different times of the disease, strongly suggest a selective expansion of T lymphocytes in synovial membrane. Comparison of motifs found in the CDR3 region of dominant clones from different studies is difficult as of the number of TCRs analysed is generally low and the HLA-DR differs from patient to other. To further investigate TCR in RA, identification of the pathogenic clonotypes would be the first step before characterization of CDR3 regions of dominant clonotypes. Although the target or the auto-antigens involved in the disease have not yet been identified, sequence analyses of the CDR3 regions of dominant clonotypes could, in the future, give important informations about peptides that are the targets of auto-reactive T cells.

Low-Level Production of Interleukin-13 in Synovial Fluid and Tissue from Patients with Arthritis

Rheumatoid arthritis (RA) is a chronic, aggressive disease characterized by inflammatory cells in the synovial tissue (ST) and synovial fluid (SF). Interleukin (IL)-13 inhibits the production of proinflammatory cytokines, chemokines, and hematopoietic growth factors by activated human monocytes. The aim of this study was to determine the production of IL-13 in various forms of arthritis. The presence of IL-13 in RA was found to be low, in that 18 of 26 RA SF samples and 10 of 14 RA peripheral blood (PB) samples had nondetectable levels (≤12 pg/ml). Similar low levels were found in SF and PB from patients with osteoarthritis (OA) and other arthritides. In contrast, RANTES, IL-8, monocyte chemotactic protein-1, and soluble P-selectin were found at levels of 13-, 120-, 1200-, and 2000-fold excess of IL-13, respectively. Mononuclear cells isolated from RA SFs did not produce significant levels of IL-13 in culture (≤12 pg/ml) but were able to do so when stimulated with phytohemagglutinin. Likewise, tissue explants from RA synovium cultured for 24 or 48 hr with or without serum did not produce appreciable quantities of IL-13 (≤12 pg/ml). Immunohistochemical data were in accordance with this result in that antigenic IL-13 was not detected on the majority of RA, OA, and normal (NL) ST cells. These results demonstrate a paucity of IL-13 within the joints of RA, OA, NL, and other arthritic patients by comparison with levels of other cytokines.

Detection of p53 in inflammatory tissue and lymphocytes using immunohistology and flow cytometry: a critical comment.

AIMS: To analyse the expression of p53 in lymphatic cells found in inflammatory tissues and the peripheral blood by immunological methods. METHODS: Immunohistological analysis of synovial tissues from patients with...

Comparative analysis of CD45RA- and CD45RO-positive CD4+T cells in peripheral blood, synovial fluid, and synovial tissue in patients with rheumatoid arthritis and osteoarthritis.

To determine whether the predominant infiltration with memory CD4+T cells in joints is specific to the local immune and inflammatory response in rheumatoid arthritis (RA), the proportions of CD45RA+ or CD45RO+ cells in the CD4+T cell populations in three different compartments (i.e., peripheral blood, synovial fluid, and synovial tissue) from patients with RA and osteoarthritis (OA) were compared by two-color flow-cytometric analysis. In the CD4+T cell population of peripheral blood, the number of CD45RO+ cells was relatively higher than CD45RA+ cells in both RA and OA patients, but their percentages did not differ from those found in healthy individuals. However, the great majority of CD4+T cells present in synovial fluid and synovial tissue were CD45RO-positive and CD45RA-negative in both patient groups; although CD4+T cells infiltrating both the disease compartments were markedly greater in RA joints, their mean percentages of CD45RO+ cells were not significantly different from those in OA joints. These data indicate that an accumulation of CD45RO+ memory CD4+T cells is a generalized phenomenon during local inflammatory responses in both RA and OA joints, and may be due mainly to the propensity of these cells to preferentially transmigrate into the inflamed joint via adhesion molecules as compared with CD45RA+ naive CD4+T cells.