T lymphocytes in synovia of patients with rheumatoid arthritis

Conclusions The observation that rheumatoid synovial-derived lymphocytes display clonal dominance may have important implications in the understanding of the underlying pathogenic processes in RA. Isolation of the relevant T cell clones will allow further characterization including the development of anti-clonotypic mAb which could be used for diagnostic and possibly even for therapeutic purposes. Functional analysis of the clones could be used as an approach to identify the antigen(s) that trigger the disease.Our observations, however, raise a number of points. First, the detection of clonal dominance in a T cell population within a tissue or in blood has been considered a marker of malignancy [9, 13]. Recent studies of several skin diseases suggest that T cell oligoclonality observed in some of these lesions represents a selected repertoire of responding T cells; our study shows that T cell clonality is not restricted to lymphoproliferative diseases but may indeed be a feature of certain inflammatory processes as well.     

改善病情抗风湿药对类风湿关节炎患者血脂谱的影响

类风湿关节炎(RA)患者心血管风险明显高于正常人群,血脂代谢异常与之密切相关。作为RA 治疗的基石,改变病情抗风湿药(DMARDs)对血脂水平的影响仍存在很大争议,部分药物可能促进动脉粥样硬化。本文总结分析了DMARDs 对RA 患者血脂谱的影响,提示抗风湿药的心血管安全性应引起重视。     

The B lymphocyte in rheumatoid arthritis: recirculation of B lymphocytes between different joints and blood

In order to search for further evidence for a pathogenetic role of recirculating, antigen-driven B cell clones in rheumatoid arthritis (RA) rearranged VH genes were analysed for clonal relationship and somatic mutations from synovial tissue and peripheral blood of a patient with RA undergoing synovectomy of several finger joints. DNA was prepared from the synovial tissue of two finger joints and blood. PCR for the different VH families was performed with one specific oligonucleotide for each VH family and a mixture of JH-specific oligonucleotides. The PCR products were separated on a high resolution acrylamide gel differentiating one base pair difference of length. Transfer of the products onto a nylon membrane and hybridization with an oligonucleotide specific for the FR3 region revealed a polyclonal representation of rearranged VH1, VH3, VH4 and VH5 genes. The VH6 family, which is encoded by a single germline gene, was represented by few distinct bands, with some bands of identical height for both joints and blood. DNA from these bands of interest was eluted, reamplified by PCR, cloned and sequenced. Sequence analysis of 27 independent bacterial colonies allowed distribution of the different VH genes to seven B cell clones (A-G). Members of clone A were found in both joints and blood, clones B and C in one joint and blood, clone D in both joints, and clones E, F and G only in one joint. The VH regions were somatically mutated with characteristic patterns for the different clones. In conclusion, our findings confirm the systemic character of RA, because they show that not only expansion and affinity maturation of B cells occur in synovial membranes but antigen-specific B cells recirculate between different joints and blood.     

共刺激分子与类风湿性关节炎患者T细胞的平衡失控

共刺激分子和凋亡受体CD95(Fas)分子的异常表达与T细胞的异常活化及其亚群平衡偏移密切相关，本研究拟通过对类风湿性关节炎(RA)患者T细胞亚群上表面CD30和CD95分子表达的检测，探讨细胞信号分子在参与RA免疫紊乱中的作用。     

Autoantibodies against Tmu and B lymphocytes in patients with rheumatoid arthritis.

Patients with rheumatoid arthritis have decreased numbers of T mu lymphocytes in their peripheral blood. To find out whether these low number of T mu lymphocytes were associated with the presence of anti-lymphocyte antibodies, the sera of 27 patients with definite or classical rheumatoid arthritis (RA) were investigated for the presence of autoantibodies against subsets of lymphocytes. In addition the numbers of T, T mu, T gamma and B lymphocytes in the peripheral blood of these patients were investigated. Patients with active RA showed lower numbers of T mu lymphocytes in their peripheral blood than patients with inactive RA. However, both groups of RA patients had significantly decreased numbers of T mu lymphocytes in their peripheral blood as compared with 22 age matched healthy donors. Moreover, mainly in patients with active RA cold reactive antibodies were found directed against T mu and B lymphocytes, but never against T gamma lymphocytes of healthy donors. Similar results were found in the indirect immunofluorescence procedure when tested for reactivity against T-cell subsets. This serum reactivity was not caused by rheumatoid factors or antinuclear antibodies. Since RA sera after precipitation with 2.5% polyethyleneglycol, still showed cytotoxicity against T and B lymphocytes, it is suggested that this serum reactivity is not caused by immune complexes but by antibodies.     

Altered T lymphocyte signaling in rheumatoid arthritis

Synovial and peripheral blood T cells from patients with rheumatoid arthritis are functionally deficient. This may be secondary to their reduced cytokine (e.g. interleukin-2) synthesis. We have investigated the possibility of an alteration in pathways common to interleukin-2 production and proliferation in peripheral blood T cells from patients with active rheumatoid arthritis. Intracellular calcium levels ([Ca²⁺]_i) were analyzed by flow cytometric methods in Indo 1-loaded T cells. These were purified by negative selection from patients or age/sex-matched controls, and stimulated with phytohemagglutinin-P or anti-CD3. Rheumatoid [Ca²⁺]_i responses to both stimuli were reduced (p < 0.005). Patient cell samples included a larger proportion of non-responding cells, but even in the responsive population the magnitude of the response in rheumatoid cells was impaired compared with those in normal cell samples (p < 0.0001) for both stimuli. Proliferation responses were also impaired (p < 0.005), and there was a positive correlation between the paired [Ca²⁺]_i elevation and proliferative responses for both stimuli. CD2 and CD3 expression were normal, and the proportions of CD4, CD8 and CD45RO and CD45RA subsets were also unaffected by disease. Thus a signaling defect downstream of CD2 or CD3 surface molecules may contribute to functional deficiencies in rheumatoid T lymphocytes. This effect is not due to non-steroidal anti-inflammatory drugs which some patients were taking. We have demonstrated similar alterations in [Ca²⁺]_i responses and proliferation in a smaller study of patients with inflammatory bowel disease, indicating that such changes might be present in other chronic inflammatory states.     

Antigen presentation by B lymphocytes to CD4+ T lymphocytes in vivo: importance for B lymphocyte and T lymphocyte activation.

B lymphocytes, like macrophages and dendritic cells, can present antigen to CD4+ T cells. Antigen presentation by B cells is essential for the generation of an in vivo T cell dependent antibody response, and repeated antigen presentation by B cells to T cells is necessary to induce B cell clonal expansion. Presentation of antigen by resting B cells to unprimed T cells tolerizes T cells, while anti-IgD antibody activates B cells and allows B cell antigen presentation that productively activates T cells. However, activation is not all that is required for B cells to productively present antigen to T cells.     

B lymphocyte activation by contact-mediated interactions with T lymphocytes.

T cell dependent B lymphocyte activation requires interactions between numerous receptor-ligand pairs on the two cell types. Recently, advances have been made both in understanding how these various signals regulate B cell effector functions and in identifying many new receptor-ligand pairs that contribute to the regulation of B cell function by T lymphocytes.     

Influence of Epstein-Barr virus infection on B lymphocyte responses in patients with rheumatoid arthritis

The influence of Epstein-Barr virus (EBV) infection on clinical and serological features of rheumatoid arthritis (RA) were studied. Patients with in-vivo activated, in-vitro spontaneously proliferating EBV-infected B lymphocytes had higher levels of serum IgG and IgA, and tended to have more extensive disease. The finding of in-vivo activated EBV-transformed B cells was not specific for RA but was also seen in patients with ankylosing spondylitis. When supernatants of spontaneously proliferating B-cell lines from patients with RA were studied, autoantibody reactivities comparable with those from patients with infectious mononucleosis were detected. These observations suggest that EBV infection might have a profound influence on B-lymphocyte responses and clinical course in patients with RA.     

类风湿性关节炎患者T细胞亚群失衡与炎性粘附分子的关系

目的：探讨类风湿性关节炎（RA）患者细胞免疫调节功能异常与参与介导免疫炎性损伤的细胞粘附分子之间的关系。方法：以ELISA方法检测40例RA患者外周血中IL－2、IL－10、 sICAM-1、sVCAM-1水平。结果：RA患者TH1，细胞因子IL－2水平明显低于健康对照组；TH2细胞因子IL－10及细胞粘附分子sICAM-1/sVCAM-1水平明显高于健康对照组，各组间比较均具有显著性差异（P＜0．01）。结论：RA患者高水平的 IL－10与低水平的IL－2提示TH2细胞功能亢进，进而TH1细胞被抑制，并导致细胞因子谱的偏移；高水平的sICAM-1、sVCAM-1是RA患者慢性炎症损伤的重要介质；异常升高的IL－10与细胞粘附分子的共同作用是导致RA患者病理损伤的重要原因。     

Increased EA-rosette formation by lymphocytes from patients with rheumatoid arthritis.

A modified method for estimating erythrocyte-antibody (EA) rosette formation of human peripheral blood lymphocytes (PBL) reveals consistent differences between rheumatoid arthritis (RA) patients tested and healthy control subjects. Using this method we find an average of 27 +/- 0-8% (standard error of mean) of PBL from 120 RA patients forming EA rosettes in contrast to only 6 +/- 0-6% of PBL from ninety-five healthy controls, and 7 +/- 0-9% from eighteen patients with systemic lupus erythematosus (SLE). This difference is not due to monocytes forming EA rosettes or to T-cell sheep red blood cell (SRBC) binding. The concentration of antibody used in our assay appears to highlight the RA-control differences--suggesting a possible qualitative difference in EA-binding capacity. We find no correlation between EA binding and disease duration or rheumatoid factor titre. The assay is susceptible to technical variation, and the effects of antibody concentration, lymphocyte to SRBC ratio, method of blood collection and lymphocyte-separation procedure have all been evaluated.     

Tracking antigen-specific human T lymphocytes in rheumatoid arthritis by T cell receptor analysis.

The aim of this study was to use TCR sequencing as a tool to address the frequency of antigen specific T cells in different T cell compartments from a rheumatoid arthritis patient. We have previously established a clear link between T cell recognition of a specific Mhsp60 epitope and the amino acid sequence in the CDR3 region of the TCRB chain. This information was used to determine the frequency of these characteristic sequences in unmanipulated synovial fluid (SF), peripheral blood (PB) and hyperplastic lymph node of the same patient by amplification and sequencing. TCRBV sequences identical to those seen in antigen-specific clones, and closely related sequences, were readily identified in SF, where they represented approximately 1% of all T cells, but were absent from PB or lymph node. The prevalence of putative Mhsp60 specific T cells within the SFMC is much greater than previously suggested by limiting dilution assays. Thus, amplification and sequencing may prove a superior technique for tracking the frequency of antigen-specific T cells in different tissues and in a longitudinal fashion.     

Human blood L lymphocytes in patients with active systemic lupus erythematosus, rheumatoid arthritis and scleroderma: a comparison with T and B cells.

Human blood lymphocytes with high affinity Fc receptors for IgG will bind small aggregates of this immunoglobulin at 4 degrees C. These cells have been named L lymphocytes because of membrane-labile IgG determinants. L cells possess a profile of surface markers and functional characteristics which differ from T and B cells. Immunofluorescence methods have been employed to quantify L lymphocytes in subjects with connective tissue diseases and certain infections, and these values have been compared with those for T and B cells. The mean values of L lymphocytes in groups of patients with systemic lupus erythematosus, rheumatoid arthritis and scleroderma ranged between 14% and 18%; values similar to normals. Groups with acute pneumonia and tuberculosis, however, had significantly increased percentages of L lymphocytes. The absolute number of L cells was decreased in subjects with connective tissue diseases, as was the number of T and B cells. L lymphocytes in those with infections were not significantly decreased. Only L lymphocytes were depleted by immobilized antigen--antibody complexes, another characteristic which distinguishes them from T and B cells.     

表达Foxp3的调节性T细胞在类风湿关节炎发病中的意义

目的：研究表达Foxp3的调节性T细胞在类风湿关节炎（RA）发病中的意义及其与类风湿关节炎临床特征的相关性。方法：提取外周血总RNA，并逆转录为cDNA。应用实时荧光定量PCR法对RA治疗组（n=25）、RA未治疗组（n=25）及健康对照组（n=30）外周血Foxp3 mRNA含量进行检测，并研究其与RA患者病情活动程度、抗环瓜氨酸（CCP）抗体、C反应蛋白（CRP）、血沉（ESR）及类风湿因子（RF）的关系。结果：RA患者的Foxp3 mRNA含量明显低于正常对照组（P〈0．01）；RA未治疗组Foxp3表达水平明显低于RA治疗组（P〈0．01）。RA患者Foxp3表达水平与DAS28评分、抗CCP抗体及ESR水平呈明显负相关（P〈0．05），而与CRP及RF无明显相关性。结论：RA患者存在表达Foxp3的调节性T细胞数量减少和／或功能降低，这种调节性T细胞亚群的异常可能参与了RA的发病和病变进展。     

Characterization of altered calcium signalling in T lymphocytes from patients with rheumatoid arthritis (RA)

Abnormal function of peripheral blood T lymphocytes is characteristic of RA; diminished proliferation and secretion of cytokines following in vitro mitogen stimulation are observed. We have investigated the calcium flux initiating T cell activation in rheumatoid peripheral blood mononuclear cells (PBMC) to determine whether abnormalities in signalling are also present. We have found that both phytohaemagglutinin (PHA-P)- and anti-CD3-stimulated calcium fluxes were much reduced in the patients’ PBMC compared with controls, with a mean six-fold difference (P < 0·01) in rate of Ca²⁺ flux with PHA-P stimulation. When purified T cells were examined with PHA and CD3 stimulation, a reduction in the peak and plateau [Ca²⁺]_i was observed in RA T cells, but the rate of rise of [Ca²⁺]_i was only reduced in those cells stimulated with PHA. These results suggest that alterations in the initiating signal may underlie the functional T cell abnormalities associated with RA, and that there may be an additional extrinsic influence from non-T cells in the PBMC population.     

类风湿关节炎前后肠道T淋巴细胞的分布变化

目的探讨在类风湿关节炎条件下大鼠小肠(十二指肠、空肠、回肠)上皮内T淋巴细胞(intraepithelial lymphocyte,IEL)、固有层T淋巴细胞(lamina propria lymphocyte,LPL)的变化规律。方法通过免疫组织化学染色方法,观察正常组和致病组肠管黏膜的T淋巴细胞的变化规律,然后分别对IEL和LPL计数,并进行统计学分析。结果大鼠致病组肠道各部分(十二指肠、空肠、回肠)上皮层和固有层可见T淋巴细胞数量增多。结论通过免疫组织化学方法可见在类风湿关节炎条件下,肠道黏膜的上皮层和固有层均可见T淋巴细胞的分布增多,肠道各段的IEL、LPL可能在黏膜免疫应答中起着关键的生物学作用。     

Induction of autologous lymphocyte transformation by synovial fluids from patients with rheumatoid arthritis

Appropriately diluted synovial fluids from thirteen of eighteen patients with rheumatoid arthritis induced in vitro transformation of autologous peripheral blood lymphocytes. By contrast, no significant transformation of autologous lymphocytes was induced by ten of eleven synovial fluids from patients without rheumatoid arthritis. These studies suggest that a similar blastogenic response in vivo may perpetuate subsynovial lymphoid hyperplasia and chronic synovitis in patients with rheumatoid arthritis.     

Immunopathologic role of B lymphocytes in rheumatoid arthritis: rationale of B cell-directed therapy

Although the immunopathogenesis of rheumatoid arthritis (RA) remains unclear, recent advances have paved the way for new therapies, such as anti-cytokine and cell-directed therapies. Here, B cells have re-gained interest concerning the pathogenesis of a number of autoimmune diseases after observing that patients with RA and non-Hodgkin lymphoma, who received anti-CD20 therapy leading to B cell depletion, demonstrated remarkable improvements. The underlying modes of action appear to be related to B cell functions, such as deletion of memory B cells, interruption of immune activation, antigen-presentation and production of inflammatory cytokines. In many RA patients, synovial extrafollicular germinal centers develop, where B cells play an intimate role in local inflammation and the generation of memory B cells and plasma cells. These local processes lead to activation of the immune system and ultimately to joint destruction in RA. Recent data demonstrating the clinical value of B cell depletion in refractory RA patients substantiate the notion that B cells are important players in the pathogenesis of the disease. Future studies should clarify which functions are affected by B cell depletion, providing the promise of new avenues to patient-tailored therapies.