Enrichment of differentiated CD45RBdim,CD27 – memory T cells in the peripheral blood,synovial fluid,and synovial tissue of patients with rheumatoid arthritis

Objective. To delineate in greater detail the phenotype of T cells that reside in the synovial tissue (ST) and synovial fluid (SF) of patients with rheumatoid arthritis (RA), in order to determine their precise differentiation status, and to determine whether the accumulation of these specific T cell subsets in these synovial compartments could be related to their capacity for transendothelial migration. Methods. Lymphocytes from normal subjects or from the peripheral blood (PB), ST, and/or SF of RA patients were phenotypically analyzed by flow cytometry. Normal PB CD4+ T cells were also characterized using an in vitro assay of transendothelial migration. Results. ST and SF were found to be enriched with memory (CD45RA-, CD45RO+, CD11a^bright, CD44^bright) and activated (CD69+) T cells. Moreover, ST and SF cells from RA patients were enriched in differentiated CD4+, CD45RB^dim, CD27- T cells, a subset of mature memory T cells that develops after prolonged antigenic stimulation. In addition, PB of some RA patients contained an increased number of CD4+, CD45RB^dim, CD27- T cells. The CD4+, CD11a^bright, CD44^bright memory T cells, which included the CD45RB^dim, CD27- more mature memory cells, exhibited an enhanced capacity for transendothelial migration that is likely to contribute to their enrichment in the rheumatoid synovium. Conclusion. RA patients manifest an increased number of mature memory T cells in the SF and ST, and some also have an increased number of these cells in PB that is likely to reflect chronic antigenic stimulation. The enrichment of these cells in the SF and ST reflects, in part, an enhanced capacity to migrate from the vascular space into inflamed tissue.     

CD4+ PD-1+ T cells accumulate as unique anergic cells in rheumatoid arthritis synovial fluid

OBJECTIVE: The PD-1 receptor, whose deficiency in mice causes autoimmune diseases such as arthritis, is considered to be a negative regulator of activated T cells and to play a crucial role in peripheral tolerance. To clarify the involvement of the PD-1 system in rheumatoid arthritis (RA), we investigated PD-1 expression on synovial fluid (SF) T cells from patients with RA. METHODS: FACS analysis for PD-1 was performed on SF T cells from 44 patients with RA and 6 with osteoarthritis (OA), and also on peripheral blood (PB) T cells from 12 RA patients and 7 healthy controls. Two-color analysis of cell surface PD-1 expression and the intracellular concentration of cytokine production was used to investigate CD4+ T cells from SF of patients with RA and PB from controls. RESULTS: Scarcely any PD-1 expression was detected on control PB T or OA SF T cells. In contrast, PD-1+ cells made up 20.9 +/- 8.6% (mean +/- SD) of RA SF T cells. In RA SF, PD-1 was expressed more predominantly on CD4+ T cells than on CD8+ T cells. As well as expressing CD45RO and CXCR3, CD4+ PD-1+ T cells were mostly CTLA-4 positive and CD26 negative, and were enriched in CD45RB(low) cells. Intracellular cytokine staining revealed that CD4+ PD-1+, but not CD4+ PD-1-, T cells produced interleukin 10 (IL-10), and that CD4+ PD-1+ T cells produced less IL-2 than CD4+ PD-1- T cells. CONCLUSION: PD-1+ T cells in RA SF are enriched, and phenotypic analysis suggests that these cells constitute a unique anergic T cell subset in RA SF.     

The transsynovial lymphocytic ratio. Characterization of blood and synovial fluid lymphocytes from patients with arthritic diseases

Monoclonal antibodies and flow cytometry techniques were used to analyze and compare the distribution of lymphocyte subpopulations of peripheral blood and synovial fluid (SF) from 70 patients, 43 with rheumatoid arthritis (RA), 10 with ankylosing spondylitis (AS) or reactive synovitis, 10 with psoriatic arthritis and 7 with other inflammatory arthritic diseases. Patients with RA had significantly reduced number of CD8+ T cells and greater CD4/CD8 ratios in peripheral blood, a greater number of CD4+ T cells and lower CD4/CD8 ratio in SF. No significant difference was found between the groups with AS, reactive synovitis and psoriatic arthritis. The simultaneous analysis of peripheral blood and SF lymphocyte subpopulations allowed us to establish a transsynovial lymphocytic ratio which reflects CD4/CD8 variations on both peripheral blood and SF, 2 easily accessible compartments for physicians. This new ratio may distinguish RA from other inflammatory arthritic diseases.     

Increased frequency of vβ17-positive t cells in patients with rheumatoid arthritis

Objective. To identify the T lymphocytes that mediate disease in rheumatoid arthritis (RA). Methods. A panel of monoclonal antibodies reactive with T cell receptor (TCR) Vβ gene products was used to analyze the RA T cell repertoire. Results. Of 5 TCR Vβ gene products studied, only Vβ17-positive T cells were increased in peripheral blood and synovial fluid (SF) from RA patients, compared with controls (P < 0.01 and P = 0.0006, respectively). Thirty-one percent of the 49 RA SF samples and none of the 19 non-RA SF samples contained >10% Vβ17-positive T cells. Activated (Tac-positive) T cells were enriched among Vβ17-positive synovial T cells. Conclusion. The selective increase of Vβ17-positive T cells suggests a role for those T cells in the pathogenesis of RA.     

Rheumatoid synovium is enriched in CD45RBdim mature memory T cells that are potent helpers for B cell differentiation

Objective. To delineate the phenotype and function of synovial T cells in rheumatoid arthritis (RA). Methods. T cells from normal subjects or from RA peripheral blood (PB), synovial fluid (SF), or synovial tissue (ST) were analyzed phenotypically and functionally. Results. RA SF and ST T cells were found to be markedly enriched in CD45RA^dim, CD45RO+, CD45RB^dim mature memory cells, whereas in the PB, CD45RA^bright naive T cells were more frequent than CD45RO+ memory T cells, and only a minority were CD45RB^dim. SF and ST T cells proliferated less well and produced less interleukin-2 in response to mitogenic stimuli than did PB T cells. However, synovial T cells effectively promoted the production of Ig from normal B cells. Moreover, PB and synovial T cells differed in their capacity to down-regulate immunoglobulin production. Anti-CD3—stimulated PB T cells suppressed Ig production unless their proliferation was prevented with mitomycin C. In contrast, synovial T cells were potent helpers of B cell Ig production regardless of antecedent treatment with mitomycin C. To examine the relationship between the CD45RB^dim phenotype and B cell help, CD45RB^dim T cells were sorted from PB. As opposed to the findings with synovial T cells, suppression by control PB CD45RB^dim T cells was observed, but only when large numbers were employed. B cell Ig production was enhanced after treatment of PB CD45RB^dim T cells with mitomycin C. In contrast, healthy control sorted CD45RB^bright or sorted CD4+, CD45RO+, CD45RB^bright T cells did not support Ig secretion. After treatment with mitomycin C, both of these populations were more effective helpers of Ig production. Conclusion. RA synovium is enriched in differentiated CD45RB^dim memory T cells with potent helper activity and diminished capacity to down-regulate B cells, strongly implying an active role for these cells in the production of Ig in the synovium, and thus in the propagation of disease.     

Inhibition of synovial fluid T cell proliferation by anti-CD5 monoclonal antibodies. A potential mechanism for their immunotherapeutic action in vivo

Objective. Monoclonal antibodies (MAb) directed against the T cell surface molecule CD5 are able to provide accessory stimulatory signals to resting T cells. The potential role of CD5 as an immunoregulatory molecule in inflammatory synovitis was examined. Methods. Synovial fluid and peripheral blood T cells of patients with active rheumatoid arthritis (RA) were purified and stimulated with interleukin-2 (IL-2), and the effect of MAb directed against CD5 on IL-2 responsiveness was examined. Results. IL-2—induced proliferation of synovial fluid T cells was strongly inhibited by anti-CD5 MAb, but not by anti-CD28 or anti-CD3 MAb. In RA peripheral blood T cells, MAb directed against CD5, CD3, and CD28 induced IL-2—dependent T cell growth, similar to findings in healthy controls. The difference in activity of anti-CD5 MAb on synovial fluid T cells compared with peripheral blood T cells was not due to different surface expression of CD5. Conclusion. Anti-CD5 has an inhibitory effect on in vivo—activated synovial fluid T cells. The disease-ameliorative effects of anti-CD5 immunotoxin treatment of RA may be partly due to “switching-off” of T cell activation in the joints.     

Outcome of intensive immunosuppression and autologous stem cell transplantation in patients with severe rheumatoid arthritis is associated with the composition of synovial T cell infiltration

Objective: To determine clinical and immunological correlates of high dose chemotherapy (HDC) + autologous stem cell transplantation (ASCT) in patients with severe rheumatoid arthritis (RA), refractory to conventional treatment. Methods: Serial samples of peripheral blood and synovial tissue were obtained from seven patients with RA treated with HDC and autologous peripheral blood grafts enriched for CD34+ cells. Disease activity was assessed with the Disease Activity Score (DAS), serum concentrations of C reactive protein (CRP), and human immunoglobulin (HIg) scans, and the extent of immunoablation was determined by immunophenotyping of peripheral blood mononuclear cells, and immunohistochemistry and double immunofluorescence of synovium. Results: Clinical responders (n = 5) had a larger number of cells at baseline expressing CD3, CD4, CD27, CD45RA, CD45RB, and CD45RO in synovium (p<0.05), higher activity on HIg scans (p = 0.08), and a trend towards higher concentrations of CRP in serum than non-responders (n = 2). Subsequent remissions and relapses in responders paralleled reduction and re-expression, respectively, of T cell markers. A relatively increased expression of CD45RB and CD45RO on synovial CD3+ T cells was seen after HDC + ASCT. No correlations were found between DAS and changes in B cells or macrophage infiltration or synoviocytes. Conclusions: HDC + ASCT results in profound but incomplete immunoablation of both the memory and naïve T cell compartment, which is associated with longlasting clinical responses in most patients. The findings provide strong circumstantial evidence for a role of T cells in established RA, and demonstrate a role for the synovium in post-transplantation T cell reconstitution.     

Increased usage of vβ2 and vβ6 in rheumatoid synovial fluid t cells

Objective. To determine if the T cell antigen receptor V_β usage of unstimulated rheumatoid arthritis (RA) synovial fluid (SF) T cells is biased compared with those in peripheral blood (PB). Methods. Freshly isolated, matched synovial fluid and peripheral blood T cells were analyzed for V_β gene expression using quantitative polymerase chain reaction (PCR) methods. Ten synovial fluid samples from the knees of 7 patients with RA were studied. The PCR assay used 26 V_β primers with a constant region C_β primer, and 2 C_α primers that co-amplified a product that served as an internal standard. Cycle number and complementary DNA content were controlled to ensure the linear accumulation of PCR products. Labeled products were separated on 10% polyacrylamide gels and counted with a Betascope blot analyzer. Results. There were consistent differences between the V_β gene usage of SF and PB T cells directly isolated from patients with RA, regardless of HLA–DR haplotype. In all synovial specimens, V_β2 was increased relative to the peripheral blood, while V_β13.1 and V_β13.2 were decreased. V_β6 and V_β21 were increased in 9 of the 10 synovial samples. Analyses of bilateral SF specimens from 2 subjects and serial specimens from the same knee of 1 subject revealed virtually identical patterns in each patient. The SF V_β bias was not solely due to differences in the proportion of CD4+ and CD8+ cells, because the CD4:CD8 ratios in SF and PB were similar. However, V_β gene usage of separated CD4+ and CD8+ synovial T cells showed that V_β2 and V_β6 were more highly expressed on CD4 cells. Conclusion. Freshly isolated synovial T cells from inflamed (not end-stage) knees of patients with RA have a remarkably consistent biased V_β gene usage compared with PB T cells. V_β2 and V_β6 are uniformly increased, and this increase is primarily in CD4+ T cells. The same V_β bias in the SF T cells of several RA patients suggests that shared antigens may be stimulating the T cell response.     

Cd28 expression on t cell subsets in vivo and cd28-mediated t cell response in vitro in patients with rheumatoid arthritis

Objective. In view of the critical importance of the CD28–CD80 (B7/BB1) costimulatory pathway in antigen-specific T cell activation and clonal expansion, we examined CD28 surface molecule expression in vivo, and T cell receptor/CD3-mediated and B7/BB1-costimulated T cell proliferation in vitro, in rheumatoid arthritis (RA). Methods. Two-color immunofluorescence analyses of peripheral blood and synovial fluid–derived T cells, as well as ³H-thymidine incorporation assays, were performed. Results. In the peripheral blood of 31 patients with active, untreated RA, a mean of 91% (range 48–100%) of CD4+ and 46% (range 13–82%) of CD8+ T cell subsets were CD28+, which was not significantly lower than normal. Although an overall decrease in the number of T cells was not observed, the numbers of CD28+CD8+ T cells were significantly lower in RA patients (mean 233/μl, versus 292/μl in controls), and this decrease was more pronounced in patients with severe disease (mean 172/μl). CD28 expression on peripheral CD8+ T cells in RA patients, but not in healthy individuals, correlated inversely with T cell activation as assessed by HLA–DR antigen expression. In contrast to the peripheral blood, RA synovial fluid T cells were almost exclusively CD28+, suggesting that migration of CD28+CD8+ T cells to active sites of inflammation may occur. In vitro proliferative responses of peripheral blood T cells to B7/BB1 costimulation in the presence of mitogenic doses of anti-CD3 monoclonal antibody were identical in patients with RA and healthy individuals. Conclusion. Functionally intact CD28+ T cells may contribute to the abnormal immunoregulation and joint inflammation in RA.     

The response of T cells to interleukin‐6 is differentially regulated by the microenvironment of the rheumatoid synovial fluid and tissue

Objective

Interleukin‐6 (IL‐6) is a proinflammatory cytokine with regulatory effects on the survival and differentiation of T cells. It exerts its biologic function in 2 ways: by directly binding to the IL‐6 receptor (IL‐6R; CD126) or via trans‐signaling, in which soluble IL‐6R/IL‐6 complexes bind to the signaling component CD130. This study was undertaken to assess the expression and regulation of CD126 and CD130 and determine how these affect the response of CD4+ T cells to IL‐6 in the joints of patients with rheumatoid arthritis (RA).

Methods

Flow cytometry and immunofluorescence microscopy were used to determine the expression, function, and regulation of CD126 and CD130 in CD4+ T cells from the peripheral blood (PB), synovial fluid (SF), and synovial tissue of RA patients.

Results

Compared to the findings in RA PB, CD4+ T cells in the SF and synovial tissue expressed low levels of CD126. In contrast, whereas CD4+ T cell expression of CD130 was minimal in the SF, its level in the synovial tissue was high. Consistent with this phenotype, synovial tissue T cells responded to trans‐signaling by soluble IL‐6R/IL‐6 complexes, whereas no response was evident in CD4+ T cells from the SF. Down‐regulation of both receptor components in SF T cells could be explained by exposure to high levels of IL‐6. Increased levels of CD130 messenger RNA and protein in synovial tissue CD4+ T cells suggested that CD130 is up‐regulated locally. Among a range of cytokines tested, only IL‐10 induced CD130 expression in T cells.

Conclusion

The inflamed microenvironment in the synovial tissue maintains responsiveness to IL‐6 trans‐signaling through the up‐regulation of CD130 expression in CD4+ T cells, and this process may be driven by IL‐10.

     

Expression of L-Selectin,CD43, and CD44 in Synovial Fluid Neutrophils from Patients with Inflammatory Joint Diseases

Objective. To study the expression of L-selectin, CD43, and CD44 on peripheral blood (PB) and synovial fluid (SF) neutrophils from patients with inflammatory joint diseases, and to investigate the presence of soluble L-selectin in both SF and plasma from patients with acute and chronic arthritis. Methods. PB and SF neutrophils were isolated from 13 patients with rheumatoid arthritis (RA) and 17 patients with various inflammatory joint diseases other than RA. Expression of L-selectin, CD43, CD44, CD11a, and CD11b was determined in both unstimulated and in vitro—activated cells by immunofluorescence flow cytometry. Soluble L-selectin levels were estimated in SF and plasma by a semiquantitative radioimmunoassay. Results. Neutrophils from SF showed diminished expression of L-selectin compared with PB neutrophils; CD43 expression and CD44 expression were decreased in SF neutrophils from most patients. In contrast, SF neutrophils exhibited significantly increased expression of CD11b, to an extent similar to that seen with in vitro—activated PB neutrophils. Soluble L-selectin was detected at similar levels in SF and PB. Conclusion. The phenotypic profile of SF neutrophils (low levels of L-selectin, CD43, and CD44, and high levels of CD11b) from most patients with RA or other inflammatory joint conditions resembles that observed in in vitro—activated neutrophils. Our results suggest that SF neutrophils are activated to a similar degree in inflammatory joint diseases with different pathogenic mechanisms.     

Increased percentage of cd3+, cd57+ lymphocytes in patients with rheumatoid arthritis. correlation with duration of disease

Objective. To determine whether a small CD3+ lymphocyte population expressing 110-kd CD57 antigens (HNK1) is expanded in patients with rheumatoid arthritis (RA), as it is in patients who have undergone bone marrow transplantation and patients with the acquired immunodeficiency syndrome, and to investigate whether it is involved in the pathogenesis of RA. Methods. The phenotype of CD3+, CD57+ lymphocytes was analyzed by flow cytometry, and correlations between the percentage of these cells in the blood and various clinical and biologic parameters were investigated. Results. The percentage of CD3+, CD57+ lymphocytes was increased in RA patients compared with controls. These lymphocytes expressed T cell receptor α/β. Eighty percent expressed the CD8 accessory molecule, and 20% expressed the CD4 accessory molecule. The leukocyte common antigen CD45RA isoform was expressed by these CD3+, CD57+ lymphocytes in blood. The HLA–DR antigen was expressed in synovial fluid but not in blood. Finally, the percentage of these lymphocytes in the blood correlated with the duration of RA. Conclusion. The expansion of the CD3+, CD57+ lymphocyte population and their activation in the synovial fluid of RA patients suggest that these cells are involved in the inflammatory process.     

Predominance of CD8+ T lymphocytes in psoriatic arthritis.

OBJECTIVE: To characterize the synovial fluid (SF) derived T cell populations in psoriatic arthritis (PsA) and compare with similar populations from rheumatoid arthritis (RA). METHODS: Paired peripheral blood (PB) and SF samples were analyzed by 3 color flow cytometry using monoclonal antibodies to CD3, CD4, CD8, HLA-DR, CD25, CD45RA, and CD45RO. RESULTS: There was a significantly increased CD8+ T cell population in PsA SF compared to RA: PsA mean 61% (range 35-93), RA mean 46% (range 6-72) (p < 0.005). This resulted in a reversal of the CD4:CD8 ratio in PsA SF compared to RA SF (p < 0.001). Patients with oligoarticular PsA had the most pronounced differences in SF derived T cell populations compared to RA (p < 0.0005) but these results were not significantly different from PsA patients with a polyarticular disease pattern. PB PsA T cell populations were not different from controls, in contrast to RA, where the CD4+ T cell population was increased (p < 0.0026), giving an exaggerated PB CD4:CD8 ratio. The majority of PsA SF CD8+ T cells expressed CD45RO, mean 73% (range 58-95), and HLA-DR antigen: mean 72% (range 38-94). Low levels of CD25 were detectable in this population, indicating a nonclassical activation pattern: mean 2% (range 0.3-4.4). CONCLUSION: In PsA, activated (HLA-DR+) and mature (CD45RO+) CD8+ T cells predominate in SE Analysis of this population may uncover clues to pathogenesis in this HLA class I mediated disease.     

T lymphocytes in the synovial fluid of patients with active rheumatoid arthritis display CD134-OX40 surface antigen.

OBJECTIVE: To assess the percentage of T lymphocytes, bearing CD134, a member of the TNF receptor superfamily, primarily found on autoreactive CD4+ T cells in the peripheral blood (PB) and synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS: The surface expression of CD134 on SF and PB mononuclear cells was performed by flow cytometry in 25 RA patients and correlated to the disease activity. RESULTS: CD134 expression on CD3+, CD4+, CD8+ and CD25+ cells was higher in SF than in PB of RA patients (P < 0.001). No differences were observed in the percentage of CD134+/CD4+ T lymphocytes in the PB of RA patients and controls. Patients with active RA had significantly higher percentage of CD3+/CD134+, CD4+/CD 134+, CD8+/CD134+ and CD25+/CD 134+ than those with inactive disease. CONCLUSION: These findings suggest that CD134+ T cells are involved in the immunopathological process of RA synovitis, maybe mirroring some other autoimmune disease in which autoreactive T cell infiltrating the target tissues largely coexpress CD134.     

Analysis of T cell subsets present in the peripheral blood and synovial fluid of reactive arthritis patients

OBJECTIVE—Reactive arthritis (ReA), a HLA-B27 associated arthropathy, develops in susceptible people after infection with certain bacteria. T cells have been implicated in the pathogenesis of the arthritis but which of the different subsets is involved is still debated. This study has further elucidated the role of the CD4⁺ and CD8⁺ T cells by examining the expression of various surface markers associated with activation.
METHODS—Three colour flow cytometry was used to examine the phenotype of the T cells within the synovial fluid (SF) and peripheral blood (PB) of ReA patients.
RESULTS—ReA SF, compared with paired PB, contained a higher percentage of CD69⁺, CD25⁺, and HLA-DR⁺ CD3⁺ T cells. The majority of SF T cells also expressed the putative memory marker CD45RO. Within the T cell subsets, CD25 was expressed primarily on the CD4⁺ T cells; however more CD8⁺ T cells were HLA-DR⁺.
CONCLUSION—The results show that both CD4⁺ and CD8⁺ T cell populations demonstrate evidence of recent activation. Whether these cells are involved in inducing inflammation, regulating the inflammation, or have become active as a result of migration through the endothelium, remains to be determined by functional studies.

Keywords: reactive arthritis; T cell; peripheral blood; synovial fluid     

Local anti—type ii collagen antibody production in rheumatoid arthritis synovial fluid

Objective. To investigate the production of type II collagen (CII) antibodies in the synovial fluid (SF) of rheumatoid arthritis (RA) patients, and to examine the HLA dependence of this local production. Methods. The ELISPOT method was used for enumerating anti-CII—reactive cells. Serologic tissue typing was performed. Results. Anti-CII—reactive cells were found in the SF of 16 of 31 patients, but not in any of the peripheral blood samples obtained in parallel. SF anti-CII antibody production showed no correlation with clinical parameters, but its frequency increased significantly with age. The IgG anti-CII response occurred exclusively in patients who were positive for HLA—DR4 and was significantly associated with DR4. Conclusion. Anti-CII production may be important in local immune complex formation. The indirect demonstration of a DR4-restricted T cell response to CII is an indication of a pathogenetic role of collagen autoimmunity in RA.     

Characterization of plasmacytoid dendritic cells in inflammatory arthritis synovial fluid

OBJECTIVE: To examine the phenotype of dendritic cell subsets in synovial fluid and peripheral blood from patients with rheumatoid arthritis (RA) or spondyloarthropathy (SpA). METHODS: Multiparameter flow cytometry was used to identify and characterize dendritic cells in mononuclear cell populations isolated from synovial fluid and peripheral blood. RESULTS: Synovial fluid contained two subsets of dendritic cells (DC), myeloid and plasmacytoid. These subsets could also be identified in peripheral blood, but there were lower numbers of DC in peripheral blood compared with synovial fluid. Plasmacytoid DC were distinguished from the myeloid subset by high expression of CD123 and lack of expression of CD11c. In comparison with myeloid dendritic cells, the plasmacytoid subset were less mature, similar to those in peripheral blood. They failed to express CD83 and DC-LAMP, and had relatively low levels of CD40 and CD86. Comparison of dendritic cells in synovial fluid from RA and SpA patients showed increased numbers of the plasmacytoid subset in SpA. CONCLUSIONS: This is the first demonstration of the plasmacytoid subset of dendritic cells in synovial fluid. Since these cells are major producers of type I interferons, their increased numbers in SpA might be relevant to pathogenesis, but the immature phenotype in SpA synovial fluid may also indicate that conditions for maturation of this subset do not pertain in SpA synovium.     

Selective recruitment of polarized T cells expressing CCR5 and CXCR3 to the inflamed joints of children with juvenile idiopathic arthritis

OBJECTIVE: To study the expression of chemokine receptors CCR5 and CXCR3 and the Th1/Th2 cytokine balance in children with oligoarticular or polyarticular juvenile idiopathic arthritis (JIA). METHODS: Using 3-color immunofluorescence, we studied the expression of CCR5 and CXCR3 on, and T cell cytokine production by, paired samples of synovial fluid (SF) and peripheral blood (PB) T cells from 20 patients with oligoarticular- or polyarticular-onset JIA. Chemokine and cytokine phenotypes were also compared within the CD45RO+,CD3+ subsets. CCR5 genotypes were confirmed by polymerase chain reaction typing and sequencing. RESULTS: In the majority of samples, the number of T cells that were CCR5+ and CXCR3+ was higher in SF than in PB, and this difference was significant. One child was homozygous for the null A32 CCR5 allele; 4 others had lower expression of CXCR3 in SF than in blood. All samples showed strongly Th1-type cytokine production by synovial T cells compared with that by PB T cells. Both features were also markedly polarized within the synovial CD45RO+ subset compared with PB CD45RO+ T cells. CONCLUSION: The high expression of CCR5 and CXCR3 and high interferon-gamma:interleukin-4 ratios suggest a type 1 phenotype of SF T cells in JIA. The difference between CD45RO+ T cells from SF and from PB suggests that specific activation events have occurred in synovial T cells. We suggest that the highly activated, Th1-type phenotype of T cells within the chronically inflamed joints of children with JIA may reflect specific recruitment events that contribute to the polarization of these cells.     

The CD69 activation pathway in rheumatoid arthritis synovial fluid t cells

Objective. To study the CD69 activation pathway in synovial fluid (SF) T lymphocytes from patients with rheumatoid arthritis (RA). Methods. Peripheral blood mononuclear cells (PBMC) or SF mononuclear cells (SFMC) were used in proliferation assays with anti-CD69, anti-CD28, anti-CD3, phorbol myristate acetate (PMA), and/or recombinant interleukin-2 (IL-2). CD69+, CD69—, and resting SF T cells were also proliferated. CD25 expression and production of IL-2 after CD69 activation were assessed by flow cytometry and in a bioassay with the IL-2-dependent cell line CTLL-2. Results. RA SFMC did not proliferate either in the presence of anti-CD69 monoclonal antibodies alone or with concomitant PMA activation, when compared with paired or control PBMC. Similar low proliferative responses via the CD3 or CD28 pathway with PMA were observed. This defective proliferation of RA SFMC after stimulation through the CD69 molecule was explained in part by a failure to express CD25 and to produce IL-2. SF CD69- T cells and resting SF T cells had higher rates of proliferation through the alternative costimulatory pathway CD28 than did SF CD69+ T cells or freshly isolated SF T cells. Conclusion. Freshly isolated SF T cells present a profound state of hyporesponsiveness through the CD69 and CD28 costimulatory pathways. This state appears to be dependent on the activation status of SF T cells, since CD69— and resting SF T cells showed recovery of the ability to proliferate through the CD28 activation pathway.