High percentage of CD8+, Leu-7+ cells in rheumatoid arthritis synovial fluid.

OBJECTIVE. While analyzing the phenotype of the synovial fluid mononuclear cells (SFMC) clustered about dendritic cells in rheumatoid arthritis (RA) joint effusions, it was noted that most of the clustering cells were CD8+ and coexpressed Leu-7. Therefore, the present study was conducted to investigate the frequency of CD8+, Leu-7+ cells in RA SF. METHODS. SFMC from 13 patients with RA and from 12 patients with non-RA inflammatory arthritides were examined for CD8 and Leu-7 expression using 2-color immunofluorescence flow cytometry. RESULTS. RA SFMC had statistically significantly greater percentages of total CD8+ cells, total Leu-7+ cells, and CD8+, Leu-7+ cells, compared with SFMC from the non-RA patients. These RA CD8+, Leu-7+ SFMC had a distinctive electron microscopic appearance compared with CD8+, Leu-7- SFMC. When peripheral blood mononuclear cells (PBMC) from 31 RA patients (including 7 from the SFMC group) were compared with PBMC from 15 normal controls, the percentage of CD8+, Leu-7+ cells was not significantly greater in the RA patients. However, the combination of a modest increase in CD8+, Leu-7+ cells and a decrease in total CD8 cells in RA PBMC altered the composition of the RA CD8 population compared with normal PBMC, such that over 40% of RA peripheral blood CD8 cells coexpressed Leu-7. CONCLUSION. The increased frequency of CD8+, Leu-7+ cells in RA SFMC may arise from the fact that a high percentage of the CD8+ PBMC in RA patients are also Leu-7+. This altered composition of CD8 cells in RA SF may have a role in the pathogenesis of the disease.     

Shift toward T helper 1 cytokines by type II collagen-reactive T cells in patients with rheumatoid arthritis

OBJECTIVE: To investigate the impact of type II collagen (CII)-reactive T cells on the Th1/Th2 cytokine balance in patients with rheumatoid arthritis (RA). METHODS: T cell proliferative responses to bovine CII were examined in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) by mixed lymphocyte culture. CII-reactive T cell lines were generated from the SFMC and PBMC. Interferon-gamma (IFNgamma), interleukin-12 (IL-12), and IL-4 were measured by enzyme-linked immunosorbent assay in the SF, sera, and culture supernatants of PBMC and SFMC that had been stimulated with CII. RESULTS: The frequency of CII-reactive T cells was higher in the PBMC from RA patients than in that from osteoarthritis patients and healthy control subjects. In RA patients, CII-reactive T cells were more prevalent in SFMC than in PBMC. The mean level of IFNgamma and the ratio of IFNgamma to IL-4 were significantly higher in the culture supernatants of T cells stimulated with CII; these differences were more prominent in SFMC. Levels of IL-12 in the culture supernatants of SFMC and PBMC stimulated with CII were significantly higher than those in unstimulated supernatants. T cell responsiveness correlated well with the level of type 1 cytokines in culture supernatants from RA T cells stimulated with CII. In the CII-reactive cell lines, the increased production of IFNgamma was consistent with clonal expansion. CONCLUSION: CII-reactive T cells are more abundant in SFMC than in PBMC and are strongly associated with a shift toward Thl cytokine in the inflamed joints of RA patients. Our results suggest that a skewing toward type 1 cytokines by CII-reactive T cells may play an important role in the chronic inflammatory process of RA.     

High percentage of CD8+, Leu-7+ cells in rheumatoid arthritis synovial fluid

Objective. While analyzing the phenotype of the synovial fluid mononuclear cells (SFMC) clustered about dendritic cells in rheumatoid arthritis (RA) joint effusions, it was noted that most of the clustering cells were CD8+ and coexpressed Leu-7. Therefore, the present study was conducted to investigate the frequency of CD8+, Leu-7+ cells in RA SF. Methods. SFMC from 13 patients with RA and from 12 patients with non-RA inflammatory arthritides were examined for CD8 and Leu-7 expression using 2-color immunofluorescence flow cytometry. Results. RA SFMC had statistically significantly greater percentages of total CD8+ cells, total Leu-7+ cells, and CD8+, Leu-7+ cells, compared with SFMC from the non-RA patients. These RA CD8+, Leu-7+ SFMC had a distinctive electron microscopic appearance compared with CD8+, Leu-7– SFMC. When peripheral blood mononuclear cells (PBMC) from 31 RA patients (including 7 from the SFMC group) were compared with PBMC from 15 normal controls, the percentage of CD8+, Leu-7+ cells was not significantly greater in the RA patients. However, the combination of a modest increase in CD8+, Leu-7+ cells and a decrease in total CD8 cells in RA PBMC altered the composition of the RA CD8 population compared with normal PBMC, such that over 40% of RA peripheral blood CD8 cells coexpressed Leu-7. Conclusion. The increased frequency of CD8+, Leu-7+ cells in RA SFMC may arise from the fact that a high percentage of the CD8+ PBMC in RA patients are also Leu-7+. This altered composition of CD8 cells in RA SF may have a role in the pathogenesis of the disease.     

血管内皮生长因子在脊柱关节病外周关节病变发病机制中的作用研究

目的 探讨血管内皮生长因子(VEGF)在脊柱关节病(SpA)发病机制中的作用以及与疾病活动性的相关性。方法 收集外周血单个核细胞(PBMC)和关节液中的单个核细胞(SFMC)，经脂多糖(LPS)刺激后，采用反转录聚合酶链反应(RT-PCR)检测VEGF mRNA的表达，并与疾病活动性进行相关性比较，同时采用酶联免疫吸附试验(ELISA)方法测定关节液和血清中的VEGF(SF-VEGF／s-VEGF)，并与VEGF mRNA以及疾病活动性进行相关性比较。结果 未经任何刺激的PBMC无VEGF mRNA表达，经LPS刺激的SFMC其VEGF mRNA的表达比未经刺激的SFMC显著增高，未经刺激的SFMC VEGF mRNA表达与临床常用的疾病活动性指标C反应蛋白(CRP)显著相关，经LPS刺激后的SFMC VEGF mRNA表达还与Bath强直性脊柱炎(AS)疾病活动性指数(BASDAI)、外周关节肿胀数．SF-VEGF、s-VEGF显著相关。SF-VEGF水平较s-VEGF明显增高，SF-VEGF水平与BASDAI、外周关节肿胀数以及外周血白细胞计数的相关有显著性；s-VEGF水平显示出与BASDAI、Bath强直性脊柱炎功能指数(BASFI)、外周关节肿胀数、疲倦程度以及外周血小板计数有相关性。结论 SpA患者SFMC表达VEGF mRNA，LPS可上调其表达；SF-VEGF水平高于S-VEGF，与血沉(ESR)、CRP相比s-VEGF更能准确反应机体疾病活动性。     

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.     

Shift toward T helper 1 cytokines by type II collagen–reactive T cells in patients with rheumatoid arthritis

Objective

To investigate the impact of type II collagen (CII)–reactive T cells on the Th1/Th2 cytokine balance in patients with rheumatoid arthritis (RA).

Methods

T cell proliferative responses to bovine CII were examined in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) by mixed lymphocyte culture. CII‐reactive T cell lines were generated from the SFMC and PBMC. Interferon‐γ (IFNγ), interleukin‐12 (IL‐12), and IL‐4 were measured by enzyme‐linked immunosorbent assay in the SF, sera, and culture supernatants of PBMC and SFMC that had been stimulated with CII.

Results

The frequency of CII‐reactive T cells was higher in the PBMC from RA patients than in that from osteoarthritis patients and healthy control subjects. In RA patients, CII‐reactive T cells were more prevalent in SFMC than in PBMC. The mean level of IFNγ and the ratio of IFNγ to IL‐4 were significantly higher in the culture supernatants of T cells stimulated with CII; these differences were more prominent in SFMC. Levels of IL‐12 in the culture supernatants of SFMC and PBMC stimulated with CII were significantly higher than those in unstimulated supernatants. T cell responsiveness correlated well with the level of type 1 cytokines in culture supernatants from RA T cells stimulated with CII. In the CII‐reactive cell lines, the increased production of IFNγ was consistent with clonal expansion.

Conclusion

CII‐reactive T cells are more abundant in SFMC than in PBMC and are strongly associated with a shift toward Th1 cytokine in the inflamed joints of RA patients. Our results suggest that a skewing toward type 1 cytokines by CII‐reactive T cells may play an important role in the chronic inflammatory process of RA.

     

Interleukin 7 stimulates tumour necrosis factor alpha and Th1 cytokine production in joints of patients with rheumatoid arthritis

BACKGROUND: A large number of activated T cells are found in the joints of patients with rheumatoid arthritis (RA). Interleukin 7 (IL7), a T cell growth factor and a regulator of Th1 and Th2 cytokine production, is produced by synoviocytes from patients with RA. OBJECTIVE: To investigate the effect on proinflammatory cytokine production of synovial fluid mononuclear cells (SFMC) and the mechanism by which IL7 influences CD4+ T cell activity in patients with RA. METHODS: In a cross sectional group of patients with RA, IL7 levels were compared with those of healthy controls and related to disease activity. The effect of IL7 on cytokine production was tested by RA SFMC and on SF CD4+ T cells in the presence of mononuclear cells (MC). Production of tumour necrosis factor alpha (TNF alpha), IL1 beta, interferon gamma (IFN gamma), and IL4 was measured by enzyme linked immunosorbent assay (ELISA) and by single cell FACS analysis. Expression of the IL7 receptor alpha chain on CD4+ T cells (essential for IL7 signalling) was assessed. Direct effects of IL7 on isolated synovial fluid (SF) CD4+ T cells were studied by cytokine analysis. By neutralisation of IL12 in MC cultures, indirect effects of IL7 on T cells through accessory cells were studied. RESULTS: IL7 serum levels were higher in patients with RA than in healthy controls and correlated positively with C reactive protein levels. IL7 stimulated TNFalpha production by SFMC and very potently stimulated IFN gamma and TNF alpha production by SF CD4+ T cells. These effects were probably mediated through the IL7 receptor alpha chain, which was abundantly expressed on SF CD4+ T cells. Besides the direct stimulation of T cell cytokine production by IL7, its action was partly dependent on IL12, indicating that IL7 also stimulates accessory cell function, leading to T cell activation. CONCLUSION: IL7 stimulates proinflammatory cytokine production of intra-articular CD4+ T cells and accessory cells from patients with RA. The correlation with measures of disease activity indicates that IL7 might substantially contribute to the perpetuation of Th1 and TNF alpha mediated proinflammatory responses in patients with RA.     

Mononuclear cell response to enterobacteria and Gram-positive cell walls of normal intestinal microbiota in early rheumatoid arthritis and other inflammatory arthritides

OBJECTIVE: To study whether enterobacteria and Gram-positive bacterial cell walls (BCW) derivedfrom normal intestinal microbiota are involved in the etiopathogenesis of early rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) were isolatedfrom patients with early RA (the average duration of 5 months) and the controls (other types of inflammatory arthritis). The mononuclear cell proliferation and tumor necrosis factor-alpha (TNF-alpha) responses to heat-killed Salmonella enteritidis (SE). Yersinia enterocolitica (YE), and Escherichia coli (EC), and to Gram-positive BCW derived from four common intestinal indigenous bacteria, Eubacterium aerofaciens (EA), Eubacterium limosum (EL), Lactobacillus casei (LC), and Lactobacillus fermentum (LF), and a BCW derived from a pathogen, Streptococcus pyogenes (SP) were investigated. RESULTS: 39% or 56% of patients with early RA showed significant proliferation responses by PBMC or SFMC against enterobacteria, respectively. In other types of arthritis, corresponding figures were 59% or 66%. When BCW were used as antigens, 8.1% or 23% of patients with early RA showed proliferation responses by PBMC or SFMC, respectively. In other types of arthritis the corresponding figures were 7.5% or 35%, respectively. However, TNF-alpha production by SFMC stimulated by EA BCW, SE, YE or EC, was significantly higher in early RA than in other types of arthritis. CONCLUSION: These results suggest that SFMC reacting with enterobacteria or BCW exist in some patients with early RA, but also in other types of inflammatory arthritis. Intestinal bacterial agents may play a role in the etiopathogenesis of RA, but the effect appears to be non-specific.     

Quantitation of IL-2Rp75 (CD122) expression on mononuclear cells in rheumatic disease.

OBJECTIVE: To compare expression of the p75 chain of the interleukin-2 receptor (IL-2Rp75, CD122) on peripheral and synovial mononuclear cells in rheumatoid and non-rheumatoid inflammatory arthritis. METHODS: Peripheral blood (PBMC) and synovial (SFMC) mononuclear cells were isolated from subjects with rheumatoid arthritis (n = 16) and non-rheumatoid inflammatory arthritis (n = 12). PBMC were isolated from six healthy controls. Expression of CD122 was examined using indirect immunofluorescence and quantitative flow cytometry. RESULTS: There was no difference in IL-2Rp75 expression on PBMC from rheumatoid arthritis patients, non-rheumatoid arthritis patients, and controls. In subjects with rheumatoid arthritis there was no difference in IL-2Rp75 expression on PBMC and SFMC. However, in the non-rheumatoid arthritis group there was an increase in IL-2Rp75 expression on SFMC compared with PBMC (P = 0.0032). On SFMC there was a greater expression of IL-2Rp75 in non-rheumatoid arthritis than in rheumatoid arthritis (P = 0.0007). Expression was greater on CD8 positive cells and in subjects with shorter duration of disease. CONCLUSIONS: The p75 chain of the IL-2 receptor, an important T cell activation antigen, is not upregulated in synovial fluid. This appears to be a disease specific defect and provides further support for the concept of "frustrated" or incomplete T cell activation in this disease.     

Gene expression in juvenile arthritis and spondyloarthropathy: pro-angiogenic ELR+ chemokine genes relate to course of arthritis

OBJECTIVE: To evaluate the ability of microarray-based methods to identify genes with disease-specific expression patterns in peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) of juvenile arthritis patients and healthy controls. METHODS: Microarray data (Affymetrix U95Av2) from 26 PBMC and 20 SFMC samples collected from patients with active disease (classified by course according to ACR criteria) were analysed for expression patterns that correlated with disease characteristics. For comparison, PBMC gene expression profiles were obtained from 15 healthy controls. Real-time PCR was used for confirmation of gene expression differences. RESULTS: Statistical analysis of gene expression patterns in PBMC identified 378 probe sets corresponding to 342 unique genes with differing expression levels between polyarticular course patients and controls (t test, P<0.0001). The genes represented by these probe sets were enriched for functions related to regulation of immune cell functions, receptor signalling as well as protein metabolism and degradation. Included in these probe sets were a group of CXCL chemokines with functions related to angiogenesis. Further analysis showed that, whereas angiogenic CXCL (ELR+) gene expression was elevated in polyarticular PBMC, expression of angiostatic CXCL (ELR-) chemokines was lower in polyarticular SFMC compared with corresponding pauciarticular samples (t test, P<0.05). CONCLUSIONS: This pilot study demonstrates that juvenile arthritis patients exhibit complex patterns of gene expression in PBMC and SFMC. The presence of disease-correlated biologically relevant gene expression patterns suggests that the power of this approach will allow better understanding of disease mechanisms, identify distinct clinical phenotypes in disease subtypes, and suggest new therapeutic approaches.     

Sporadic enteric reactive arthritis and undifferentiated spondyloarthropathy: evidence for involvement of Salmonella typhimurium

OBJECTIVE: To define the candidate bacterial trigger and cytokine profile of synovial fluid mononuclear cells (SFMC) in patients with sporadic enteric reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA). METHODS: The study group comprised 10 patients with ReA and 23 with uSpA who fulfilled European Spondylarthropathy Study Group criteria. Ten patients with rheumatoid arthritis (RA) served as disease controls. IgG, IgA, and IgM antibodies to Shigella flexneri, Salmonella typhimurium, and Yersinia enterocolitica were measured in sera and SF by ELISA. Peripheral blood mononuclear cell (PBMC) and SFMC proliferation assays were done in the presence or absence of crude bacterial lysates. Bacterial antigens and DNA in synovial cells were detected by indirect immunofluorescence and polymerase chain reaction, respectively. Interferon-g (IFN-g), interleukin 10 (IL-10), and IL-4 were measured in 18 h SFMC culture supernatants in presence of bacterial lysate. RESULTS: Antibodies to S. typhimurium were significantly elevated in the sera of 8 of 25 patients compared to controls (0/22; p < 0.05). The ratio of SF:serum anti-salmonella IgA was significantly higher in patients compared to controls (p < 0.0002). The ratio of SF:serum IgA antibodies to S. typhimurium was higher than that for S. flexneri (p < 0.007) and Y. enterocolitica (p < 0.05). Out of 25 patients, 8, 2, and none had elevated antigen-specific SFMC proliferation response to S. typhimurium, S. flexneri, and Y. enterocolitica, respectively, whereas no control had elevated response. Salmonella antigens were detected in the synovial cells of 4 out of 14 patients. There was significantly higher IFN-g production from SFMC of patients who had increased proliferative response to Salmonella (LTT+) in the presence of Salmonella antigens compared to antigen control. The mean +/- SD of the ratio of IFN-g:IL-10 in the LTT+ patients was significantly lower compared to controls. Conclusion. S. typhimurium is probably one of the triggers for enteric ReA and uSpA in our cohort of patients, and the immune response is characterized by increased production of both IL-10 and IFN-g.     

Production of T-cell cytokines at the single-cell level in patients with inflammatory arthritides: enhanced activity in synovial fluid compared to blood

We used a newly developed, sensitive ELISPOT technique in order to estimate the number of cells producing interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) in patients with rheumatoid arthritis (RA) and other inflammatory arthritides, and to correlate the results with clinical and laboratory parameters of disease activity. SFMC and PBMC were cultured either without stimuli or with a standardized dose of phytohaemagglutinin (PHA) for 6 h. Twenty-nine patients, 16 with RA and 13 with other inflammatory joint diseases, were investigated and compared to PBMC from 25 healthy controls. The mean number of IFN-gamma-producing cells was 37.1/10(5) plated SFMC (range 0-121.5). The corresponding value for PBMC was 5.1 (0-39). The difference was highly significant (P = 0.0033 for RA patients, P = 0.0050 for non-RA patients and P < 0.0001 for all patients). Forty-five per cent of SFMC samples (range for all samples 0-38.5 SFC/10(5) MNC) and 25% of PBMC samples (0-20.5) exhibited spontaneous IL-4 production, yielding a significant difference for all patients treated collectively (P = 0.021). Although the cells that spontaneously secrete these cytokines are relatively few, quantification of these cells thus shows increased functional T-cell activation and decreased ratio of cells spontaneously producing IL-4 vs IFN-gamma in the joint fluid as compared to blood of arthritis patients.      

Shift toward T lymphocytes with a T helper 1 cytokine-secretion profile in the joints of patients with rheumatoid arthritis

Objective. To investigate whether T cells in the inflamed joints of patients with rheumatoid arthritis (RA) preferentially produce the T helper 1 (Th1) cytokines, interferon-γ (IFNγ) and interleukin-2 (IL-2), or the Th2 cytokine, IL-4, when compared with corresponding peripheral blood—derived T cells. Methods. Synovial fluid mononuclear cells (SFMC) and corresponding peripheral blood mononuclear cells (PBMC) from 10 patients with RA were analyzed, either directly or after in vitro stimulation, for the intracellular presence of Th1 and Th2 cytokines. The amount of secreted cytokine in the cell culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Results. IFNγ-containing cells were detected in the unstimulated SFMC, but not in the PBMC, of 3 patients with RA. Cells positive for IL-2 or IL-4 were not detected in the unstimulated samples. Following stimulation, the mean percentage of cells containing Th1 cytokines was significantly increased in the SFMC compared with the PBMC; no differences were found in the mean percentage of IL-4—containing cells. A comparable shift toward Th1 cytokines was observed when the amount of secreted cytokine was determined by ELISA. Conclusion. A shift toward T cells with a Th1 cytokine profile was observed in the joints of patients with RA. Since an imbalance between Th1 and Th2 cells is thought to be of pathogenic significance, this finding might have implications for the development of new therapies for RA.