CD69 expression on neutrophils from patients with rheumatoid arthritis

OBJECTIVE: Since the early activation antigen CD69 has been implicated in the pathogenesis of some inflammatory diseases, we evaluated the expression of the molecule on peripheral blood (PB) and synovial fluid (SF) neutrophils obtained from RA patients and its possible correlation with PB and SF cytokine concentration. METHODS: CD69 membrane expression (and CD11b as control marker) was assessed by indirect immunofluorescence and flow cytometry analysis on purified PB and SF neutrophils. Cytokine levels (GM-CSF, IFN-gamma, TNF-alpha) in plasma and SF supernatants were measured by ELISA. RESULTS: CD69 was absent on control neutrophils, while it was expressed on PB neutrophils from RA patients although no detectable GM-CSF, IFN-gamma or TNF-alpha was observed in their plasma. CD69 expression was still more evident on SF neutrophils from RA patients; 59% had detectable levels of INF-gamma in their SF while GM-CSF and TNF-alpha were detectable in SF from 95% and 33% of RA patients, respectively. However, no correlation was observed between cytokine concentrations and CD69 expression on SF neutrophils. SF but not PB neutrophils from RA patients expressed increased amounts of CD11b when compared to control PB neutrophils without any correlation with CD69 membrane expression. CONCLUSION: The activation antigen CD69 is significantly expressed on PB and SF neutrophils from RA patients. However, the mechanism(s) of induction and its possible role in the pathogenesis of RA remain to be defined.     

Expression of adhesion molecules on synovial fluid and peripheral blood monocytes in patients with inflammatory joint disease and osteoarthritis

OBJECTIVE: To determine the presence of adhesion molecules on monocytes/macrophages (Mphi) from peripheral blood (PB) and synovial fluid (SF) in patients with osteoarthritis (OA) and inflammatory joint diseases (rheumatoid (RA) and reactive arthritis (ReA)) in order to improve our understanding of the possible mechanisms underlying the inflammatory process. METHODS: Whole blood and SF cells were stained with monoclonal antibodies against CD11a (LFA-1), CD15 s (sialyl-Lewis X), CD44, CD54, VLA-4, and HLA-DR counterstained with anti-CD14 antibodies as a Mphi marker for dual fluorescence analysis by flowcytometry. RESULTS: On PB-Mphi, CD15s was markedly increased in both RA as well as ReA compared with OA. Furthermore, in the PB LFA-1, CD44, and HLA-DR showed a higher surface density on Mphi in ReA than in OA. Comparison between SF and PB showed significantly higher CD44 and CD54 expression on SF-Mphi. These molecules play an important part in lymphocyte-Mphi interaction. CONCLUSION: In PB from patients with inflammatory joint diseases, Mphi are activated, allowing recruitment into the synovial compartment. These disorders, in contrast with OA seem to be "systemic" in nature. Within the SF, different adhesion molecules are expressed on CD14(+) Mphi as compared with PB.     

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.     

Increased basal phosphorylation of mitogen-activated protein kinases and reduced responsiveness to inflammatory cytokines in neutrophils from patients with rheumatoid arthritis

OBJECTIVE: We studied the functions of peripheral blood (PB) and synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA), focusing the molecular basis for the activated state and the functional responsiveness of RA neutrophils to inflammatory cytokines. METHODS: Paired samples of PB neutrophils and SF neutrophils from the inflamed knee joint were obtained from 18 RA patients (5 males and 13 females). RESULTS: RA neutrophils exhibited increased spontaneous superoxide (O2-) release and adherence, increased basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase, accelerated spontaneous apoptosis, and enhanced O2- release in response to N-formyl-methionyl-leucyl-phenylalanine as compared with healthy normal PB neutrophils. When challenged with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) or tumor necrosis factor alpha (TNF-alpha), RA neutrophils exhibited reduced responses to these cytokines, which included O2- release, adherence, priming for enhanced O2- release, and phosphorylation of ERK and p38. The functional alterations were greater in SF neutrophils than in PB neutrophils from RA. Reduced responsiveness to cytokines in RA neutrophils was closely associated with increased serum and SF levels of GM-CSF and TNF-alpha. RF and RAHA titers were closely correlated with increased TNF-alpha level in SF. CONCLUSION: These findings indicate that RA neutrophils are in the activated state with increased basal phosphorylation of ERK and p38, and exhibit reduced responsiveness to inflammatory cytokines (G-CSF, GM-CSF and TNF-alpha) and accelerated spontaneous apoptosis.     

Proinflammatory mediator-induced reversal of CD4+,CD25+ regulatory T cell-mediated suppression in rheumatoid arthritis

OBJECTIVE: We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg-mediated suppression. METHODS: Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme-linked immunosorbent assay. Magnetically sorted CD4+,CD25- and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti-CD3 monoclonal antibody (mAb) and autologous antigen-presenting cells, in the absence or presence of anti-CD28 mAb or the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), or IL-7. RESULTS: Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB-derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti-CD28 mAb to cocultures of CD4+,CD25- and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg-mediated suppression in both PB and SF. Furthermore, IL-7 and, to a limited extent, TNFalpha, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg-mediated suppression. In contrast, IL-6 did not influence Treg-mediated suppression. CONCLUSION: Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.     

Increased expression of CD23 in rheumatoid synovitis

OBJECTIVE: Soluble (s)CD23 is a potent macrophage stimulator. High levels of this molecule have been reported in rheumatoid arthritis (RA) serum. We investigated the expression of CD23 and its ligands in rheumatoid synovial fluid and cells. METHODS: Levels of sCD23, and cellular expression of CD23 and its ligands CD21, CD11b, and CD11c were measured in synovial fluid (SF) of RA patients and in blood of RA patients and controls. RESULTS: SF contained higher levels of sCD23 than either rheumatoid or normal sera (median 4.8, 3.16,and 1.13 ng/ml respectively, p <0.01). Synovial CD23 was found to be expressed principally on macrophages. While little CD21 expression was detected, CD11b and CD11c were both expressed at high levels, particularly on macrophages. CONCLUSIONS: Soluble CD23 is present in high levels in RA synovial fluid. Macrophages appear to be the principal source. Macrophages also express ligands for sCD23, and may therefore also be the targets of this potent pro-inflammatory molecule.     

CD14+,CD16+ blood monocytes and joint inflammation in rheumatoid arthritis

OBJECTIVE: CD14+,CD16+ monocytes, identified as a minor population of monocytes in human peripheral blood (PB), have been implicated in several inflammatory diseases. We undertook this study to investigate the relevance of this phenotype to joint inflammation in rheumatoid arthritis (RA). METHODS: The expression of CD14, CD16, CC chemokine receptor 1 (CCR1), CCR5, and intercellular adhesion molecule 1 (ICAM-1) on monocytes was measured by flow cytometric analysis. Concentrations of the cytokines known to induce CD16 (including transforming growth factor beta1 [TGFbeta1], macrophage colony-stimulating factor [M-CSF], and interleukin-10 [IL-10]) and concentrations of the soluble form of CD14 (sCD14) in plasma and synovial fluid (SF) samples were measured by enzyme-linked immunosorbent assay. The induction of CD16 on RA blood monocytes cultured for 18 hours with 1 or with all 3 cytokines was determined. RESULTS: The mean +/- SD frequency of CD14+,CD16+ blood monocytes was significantly increased in RA patients (11.7 +/- 5.6%; n = 105) compared with healthy controls (9.5 +/- 2.2%; n = 15) (P < 0.01), and the patient group with an increased frequency of CD16+ monocytes (> or =13.9%) had active disease, as defined by increased counts of tender and swollen joints, levels of acute-phase reactants, and titers of rheumatoid factor. The response to drug therapy correlated with changes in the frequency of this phenotype. The expression of CD16 on SF monocytes from RA patients was markedly elevated compared with the expression on PB monocytes. CD16 expression on RA blood monocytes was augmented in vitro by IL-10, M-CSF, and TGFbeta1. Plasma concentrations of these cytokines and of sCD14 were significantly higher in RA patients with high CD16+ monocyte frequencies than in those with low CD16+ monocyte frequencies or in healthy controls. CD14+,CD16+ monocytes expressed higher levels of CCR1, CCR5, and ICAM-1 than did regular CD14++,CD16- monocytes, particularly in active RA. CONCLUSION: These results indicate that the maturation of blood monocytes into tissue-infiltrative CD16+ cells before entry into the joint, induced by cytokine spillover from the inflamed joint, may contribute to the persistent joint inflammation of RA.     

CD4(+)CD25(+) regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid

OBJECTIVE: In mice, CD4(+)CD25(+) regulatory T cells play a pivotal role in preventing autoimmunity. Regulatory T cells are also present and functional in healthy humans. We investigated the presence, phenotype, and function of CD4(+)CD25(+) regulatory T cells in peripheral blood (PB) and synovial fluid (SF) from patients with rheumatoid arthritis (RA). METHODS: The presence and phenotype of CD4(+)CD25(+) regulatory T cells were determined by flow cytometry. Anergy and suppressive activity were assessed by culturing CD4(+)CD25(-) and CD4(+)CD25(+) T cells with anti-CD3 monoclonal antibodies and antigen-presenting cells, followed by proliferation and cytokine detection. RESULTS: The percentage of CD4(+)CD25(+) T cells in RA SF was significantly increased compared with that in RA PB, and both of these percentages were higher than that in PB from controls. The cells in RA PB were similar in phenotype and function to CD4(+)CD25(+) regulatory T cells from controls. In SF, however, approximately 40-50% of CD4(+)CD25(+) T cells expressed an activated phenotype, i.e., CD69+, class II MHC(+), OX-40(+), with high levels of CTLA-4 and glucocorticoid-induced tumor necrosis factor receptor. These synovial CD4(+)CD25(+) T cells displayed an increased suppressive capacity compared with blood CD4(+)CD25(+) T cells. However, this enhanced suppressive activity was counterbalanced, because activated responder T cells from SF were less susceptible to CD4(+)CD25(+) T cell-mediated suppression than were responder cells from PB. CONCLUSION: We demonstrate that CD4(+)CD25(+) regulatory T cells are present and functional in patients with RA, with higher numbers of regulatory T cells with increased suppressive activity found in SF compared with PB. These findings suggest a negative feedback system that is active at the site of inflammation. The balance between activated responder and regulatory T cells appears to influence the extent of immunoregulation in RA.     

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.     

Proinflammatory mediator–induced reversal of CD4+,CD25+ regulatory T cell–mediated suppression in rheumatoid arthritis

Objective

We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg–mediated suppression.

Methods

Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme‐linked immunosorbent assay. Magnetically sorted CD4+,CD25– and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti‐CD3 monoclonal antibody (mAb) and autologous antigen‐presenting cells, in the absence or presence of anti‐CD28 mAb or the proinflammatory cytokines interleukin‐6 (IL‐6), tumor necrosis factor α (TNFα), or IL‐7.

Results

Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB‐derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti‐CD28 mAb to cocultures of CD4+,CD25– and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg–mediated suppression in both PB and SF. Furthermore, IL‐7 and, to a limited extent, TNFα, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg–mediated suppression. In contrast, IL‐6 did not influence Treg‐mediated suppression.

Conclusion

Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.

     

Cr1, CD35 IN synovial fluid from patients with inflammatory joint diseases

Objective. To investigate synovial fluid (SF) for the presence of CR1 and to study its relationship to SF leukocytes and to serum levels of soluble CR1 (sCR1) in patients with rheumatic diseases. Methods. Synovial fluids were collected from 35 patients with rheumatoid arthritis (RA) and 26 patients with other inflammatory joint diseases. Total CR1 in the SF and serum were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) that recognized both soluble and transmembrane forms of CR1. The characteristics of CR1 in SF were analyzed by ultracen-trifugation and by a second ELISA specific for trans-membrane CR1. Results. CR1 was found in all SF samples tested (range 5-281 ng/ml). SF CR1 was higher in patients with RA (mean ± SD 81 ± 66 ng/ml) than in those with other inflammatory joint diseases (31.8 ± 23.8 ng/ml) (P < 0.001). Serum sCR1 was not significantly increased in the patients compared with the normal subjects. There was no correlation between serum sCR1 and SF CR1. In 44% of the patients, the SF CR1 level was higher than the serum sCR1 level. A fraction (30–80%) of SF CR1 was pelleted by ultracentrifugation and, unlike serum sCR1, it reacted in an ELISA specific for transmembrane CR1. Thus, SF contained 2 forms of CR1: a membrane-associated and a soluble form, which was confirmed by sucrose density-gradient ultracentrifugation. SF CR1 levels correlated directly with the number of SF total leukocytes and polymorphonuclear leukocytes (PMN). These 2 forms of CR1 were also found in the supernatant of in vitro—activated PMN from normal subjects. SF CR1 exhibited the capacity to act as a cofactor for the factor I degradation of C3b. Conclusion. CR1 is found in the SF of patients with joint inflammation. The data suggest that SF CR1 originates from the infiltrating leukocytes, which shed both a soluble and a membrane-associated form. Whether SF CR1 participates in the local regulation of complement activation remains to be examined.     

Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45ro+CD25+CD127low regulatory T cells

Objective

Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127^low FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin‐17 (IL‐17)–producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function.

Methods

The presence and phenotype of CD4+CD45RO+CD25+CD127^low T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence‐activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti‐CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme‐linked immunosorbent assay, and proliferation assays.

Results

In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127^low Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro–activated monocytes induced an increase in the percentage of IL‐17–positive, interferon‐γ (IFNγ)–positive, and tumor necrosis factor α (TNFα)–positive Treg cells as well as IL‐10–positive Treg cells. The observed increase in IL‐17–positive and IFNγ‐positive Treg cells was driven by monocyte‐derived IL‐1β, IL‐6, and TNFα and was mediated by both CD14+CD16− and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL‐17 production.

Conclusion

Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.

     

Differential expression of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages in rheumatoid arthritis

OBJECTIVE: Since it is likely that monocytes utilize chemokines to migrate to the rheumatoid arthritis (RA) joint, we investigated the expression of C-C chemokine receptors (CCR) 1-6 and C-X-C receptor 3 (CXCR3) in the peripheral blood (PB), synovial fluid (SF), and synovial tissue of patients with RA as well as in the PB of normal subjects. METHODS: We compared chemokine receptor expression on CD14+ monocytes from normal PB, RA PB, and RA SF using 2-color flow cytometry. Correlations with patient clinical data were determined. Chemokine and receptor expression were investigated in RA synovial tissue by immunohistochemistry and 2-color immunofluorescence to identify CD68+ macrophages. RESULTS: Most normal PB monocytes expressed CCR1 (87%) and CCR2 (84%), but not CCRs 3, 4, 5, or 6 or CXCR3. RA PB monocytes expressed CCR1 (56%) and CCR2 (76%), with significantly more expressing CCR3 (18%), CCR4 (38%), and CCR5 (17%) compared with normal PB monocytes. Significantly fewer SF monocytes from RA patients expressed CCR1 (17%), CCR2 (24%), and CCR4 (6%) while significantly more expressed CCR3 (35%) and CCR5 (47%) compared with RA and normal PB monocytes; CCR6 and CXCR3 were rarely detected. Clinically, the erythrocyte sedimentation rate was inversely correlated with the expression of CCR1 and CCR4 by RA PB, and CCR5 expression by RA SF was correlated with the SF white blood cell count. CCR1-, CCR2-, and CCR5-immunoreactive cells were found in RA synovial tissue and colocalized with CD68+ macrophages. RA synovial tissue RANTES (regulated upon activation, normally T cell expressed and secreted chemokine)- and monocyte chemoattractant protein 1-immunoreactive cells colocalized with CCR1 and CCR2, respectively, on serial sections. Macrophage inflammatory protein 1alpha (MIP-1alpha) was principally restricted to vascular endothelium, and MIP-1beta+ macrophages were found throughout the sections. CONCLUSION: Monocytes mainly express CCR1 and CCR2 in normal and RA PB, CCR3 and CCR5 in RA PB and RA SF, and CCR4 in RA PB. The differential expression of chemokine receptors suggests that certain receptors aid in monocyte recruitment from the circulation while others are important in monocyte retention in the joint.     

Mechanisms of CD23 hyperexpression on B cells from patients with rheumatoid arthritis.

OBJECTIVE: To analyze the mechanisms involved in the characteristic hyperexpression of CD23 on peripheral blood B cells from patients with rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from patients with active disease and activated during 18 h with an anti-CD3 monoclonal antibody in the presence or absence of blocking antibodies to CD154 or CD40. PBMC were further purified by rosetting and CD23 expression was assessed on B cells by flow cytometry after double staining (CD19/CD23). Lymphocytes were also isolated from synovial fluid (SF). CD154 expression was analyzed on PB or SF CD4+ T cells after double staining (CD4/CD154) by flow cytometry at basal conditions and after different stimuli [anti-CD3 or phorbol myristic acetate (PMA) plus ionomycin]. Co-culture experiments between SF and PB cells were performed to analyze the involvement of the CD40-CD154 interaction on CD23 expression. CD154 and CD23 expression was also analyzed on synovial membrane by immunohistochemical techniques. RESULTS: A high proportion of activated CD23 B cells was detected in patients with RA. Blocking experiments with both anti-CD40 and anti-CD154 Mab showed a significant reduction in the proportion of PB B cells expressing CD23. Following activation with anti-CD3 Mab or PMA plus ionomycin, CD154 expression was mainly induced on PB CD4+ T cells. In co-culture experiments, SF T cells were more efficient than PB T cells in inducing CD40 dependent CD23 expression on PB B cells. In addition, CD4+ T cells from synovial membrane clearly expressed CD154. CONCLUSION: Our results establish a link between CD154-CD40 pathway and CD23 expression on PB B cells from patients with RA. T cells from the synovial microenvironment were active participants in this CD23 expression, presumably in the context of cell recirculation.     

CD95-Mediated control of anti-citrullinated protein/peptides antibodies (ACPA)-producing plasma cells occurring in rheumatoid arthritis inflamed joints

OBJECTIVE: Serum anti-citrullinated protein/peptides antibodies (ACPA) are a valuable diagnostic parameter that might be involved in rheumatoid arthritis (RA) pathogenesis. CD95-dependent apoptosis is defective in RA synovium. The present study explores the occurrence of ACPA IgG, and the CD95-mediated control of ACPA IgG-secreting plasma cells (PC) in RA patients. METHODS: Mononuclear cells (MC) were purified from synovial fluid (SF) and peripheral blood (PB) of 15 RA patients. PC capable of secreting ACPA IgG were detected in MC cultures. ACPA IgG present in serum and SF, and PB and SF MC culture supernatant was measured by ELISA. CD95, CD27 and CD138 expression was examined on RA PC identified as CD19(low) CD38(high) cells by flow cytometry. CD95-ligation was obtained by treatment of cultured MC with the anti-CD95 Ab CH11. Apoptotic PC were identified as Annexin-V+. RESULTS: ACPA IgG level was found higher in patients' SF than in their serum. PC were detectable in SF and PB, and exhibited high CD95 and CD27 expression. In contrast, SF, but not PB, PC expressed elevated levels of CD138. SF, but not PB, PC actively secreted ACPA IgG in cultures, in a linear fashion for at least 14 days, and CD95-ligation markedly reduced this activity and provoked PC apoptosis. CONCLUSIONS: The results suggest that RA synovium is a prominent site for ACPA IgG formation and for the accumulation of ACPA IgG-secreting PC exhibiting prolonged survival, probably due to RA defective CD95-mediated control.     

CD4+CD25+ regulatory T cells in rheumatoid arthritis: Differences in the presence,phenotype, and function between peripheral blood and synovial fluid

Objective

In mice, CD4+CD25+ regulatory T cells play a pivotal role in preventing autoimmunity. Regulatory T cells are also present and functional in healthy humans. We investigated the presence, phenotype, and function of CD4+CD25+ regulatory T cells in peripheral blood (PB) and synovial fluid (SF) from patients with rheumatoid arthritis (RA).

Methods

The presence and phenotype of CD4+CD25+ regulatory T cells were determined by flow cytometry. Anergy and suppressive activity were assessed by culturing CD4+CD25− and CD4+CD25+ T cells with anti‐CD3 monoclonal antibodies and antigen‐presenting cells, followed by proliferation and cytokine detection.

Results

The percentage of CD4+CD25+ T cells in RA SF was significantly increased compared with that in RA PB, and both of these percentages were higher than that in PB from controls. The cells in RA PB were similar in phenotype and function to CD4+CD25+ regulatory T cells from controls. In SF, however, ∼40–50% of CD4+CD25+ T cells expressed an activated phenotype, i.e., CD69+, class II MHC+, OX‐40+, with high levels of CTLA‐4 and glucocorticoid‐induced tumor necrosis factor receptor. These synovial CD4+CD25+ T cells displayed an increased suppressive capacity compared with blood CD4+CD25+ T cells. However, this enhanced suppressive activity was counterbalanced, because activated responder T cells from SF were less susceptible to CD4+CD25+ T cell–mediated suppression than were responder cells from PB.

Conclusion

We demonstrate that CD4+CD25+ regulatory T cells are present and functional in patients with RA, with higher numbers of regulatory T cells with increased suppressive activity found in SF compared with PB. These findings suggest a negative feedback system that is active at the site of inflammation. The balance between activated responder and regulatory T cells appears to influence the extent of immunoregulation in RA.

     

Natural killer cells in the synovial fluid of rheumatoid arthritis patients exhibit a CD56bright,CD94bright,CD158negative phenotype

BACKGROUND: Natural killer (NK) cells play an important role in several animal models of autoimmunity by modulating T-cell responses, but it is unclear whether human NK cells have similar functions. METHODS: We characterized the phenotype of NK cells in synovial fluid (SF) and peripheral blood (PB) of patients with rheumatoid arthritis (RA) and in healthy control subjects using flow cytometry and quantitative PCR. RESULTS: The proportions of NK cells in PB and SF of RA patients were not significantly different from those in healthy PB. However, the SF NK cell phenotype was strikingly different, with increased CD94 and CD56 densities and greatly reduced proportions of cells expressing CD158a/b. These cells also had reduced mRNAs coding for CD158a/b and low perforin levels compared with RA PB and healthy PB NK cells. CONCLUSIONS: We identified a novel phenotype of SF NK cells that is of potential significance in RA. Experiments are now under way to determine the function of these SF NK cells and their potential role in RA.     

Relationship between interleukin-8 and neutrophil adhesion molecules in rheumatoid arthritis

To evaluate the role of interleukin-8 (IL-8) on the activation of neutrophils in rheumatoid arthritis (RA), we measured IL-8 and the adhesion molecules, L-selectin (CD62L), CD11b and CD18, on neutrophils in paired peripheral blood and synovial fluid of RA patients. Synovial fluid IL-8 levels were significantly increased compared to peripheral blood. L-selectin was split off and CD 11b and CD18 were upregulated on neutrophils in the synovial fluid. A positive correlation occurred between the IL-8 concentration and CD18 or CD11b densities on neutrophils in the synovial fluid (r=0.75,P <0.005 andr=0.60,P < 0.05, respectively). Peripheral blood neutrophils of the patients were desensitised with IL-8 in vivo, as shown by the significantly lower L-selectin shedding after in vitro IL-8 stimulation: 1.6 times decrease for patients vs 3.2 for controls (P < 0.05). In conclusion, these results add further evidence for the role of IL-8 in the activation of neutrophils in RA.