The CD45 tyrosine phosphatase regulates Campath-1H (CD52)-induced TCR-dependent signal transduction in human T cells

Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo.The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.     

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.     

In vitro stimulation of lymphocytes from patients with rheumatoid arthritis

Peripheral blood lymphocytes from patients with rheumatoid arthritis (RA) and from normal controls were compared in 20 microliters droplet cultures following stimulation with phytohemagglutinin or concanavalin A. The dynamics of proliferation were significantly changed in RA. Higher numbers of cells in culture were needed to achieve the same response. This may explain the low proliferative responses of lymphocytes from some patients with RA, and apparent changes of in vitro suppressor effects, reported by other authors. Diurnal variations of lymphocytes in RA patients were also studied. No differences in the response to mitogen of lymphocytes taken at 7 AM and 7 PM were found.     

Reduced catecholamine response of lymphocytes from patients with rheumatoid arthritis

Catecholamines modulate lymphocyte function via stimulation of beta2-adrenergic receptors (beta2R). Previous investigations revealed a decreased density of beta2R on peripheral blood mononuclear cells (PBMC) in patients with chronic rheumatic diseases. Aim of the present study was to determine the impact of this decrease on catecholamine response of PBMC from patients with rheumatoid arthritis (RA) in vitro. PBMC from 17 patients with RA and 12 healthy blood donors (HD) were investigated. Beta2R were determined by a radioligand binding assay. The effects of epinephrine (E) and norepinephrine (NE) on PBMC proliferation were studied using cells activated with pokeweed mitogen (PWM) and monoclonal anti-CD3-antibodies (OKT3), respectively. In parallel, alpha1- or beta-receptor antagonist were added to the culture to determine the specificity of the catecholaminergic effects. The results showed that depending on the stimulus and the catecholamine concentration employed E and NE exert inhibitory (OKT3) or stimulatory signals (PWM) on lymphocyte proliferation. Inhibitory effects could be abolished by adding beta-antagonist, while stimulatory signals were diminished after addition of alpha1- of beta-antagonist. Patients with RA showed a significantly reduced density of beta2R compared to HD paralleled by a significantly reduced influence of catecholamines on lymphocyte function. The study demonstrates the intricate relationship between PBMC reactivity and catecholamine effects that are mediated via alpha1- and beta-adrenergic receptors. In this respect the reduced catecholamine response of PBMC from RA patients may contribute to the pathogenic process of RA.     

Soluble CD4 in patients with rheumatoid arthritis and osteoarthritis

An ELISA was used to measure the soluble form of the leukocyte surface antigen CD4 (sCD4) in the sera and synovial fluids (SF) of patients with rheumatic diseases. Patients with rheumatoid arthritis (RA) had raised levels of sCD4 in both their sera and synovial fluid compared to age-matched healthy controls. In patients with osteoarthritis levels of sCD4 in SF and sera were lower than in RA but higher than in sera of healthy individuals. Mononuclear cells from the synovial fluid of RA patients were found to produce spontaneously high levels of sCD4, but autologous blood cells only produced comparable levels after in vitro stimulation with mitogenic lectin. In individual RA patients with active disease, serial sCD4 levels fell preceding clinical improvement. In three patients where serum sCD4 levels fell and clinical improvement occurred, subsequent small increases in serum sCD4 preceded increased clinical disease activity by up to 5 days. Synovial fluid levels of sCD4 correlated positively with soluble interleukin 2 receptor levels but no correlation was found with sCD8 levels. We conclude that the release of sCD4 reflects the involvement of T helper cells and macrophages in the pathogenesis of joint inflammation, especially in RA.     

Regulation of p38 MAP kinase in CD4+ lymphocytes by infliximab therapy in patients with rheumatoid arthritis

Tumor necrosis factor alpha (TNFalpha) plays a key role in the pathogenesis of rheumatoid arthritis (RA) and most patients treated with anti-TNFalpha agents show significant improvement in both signs and symptoms. While TNFalpha inhibitors rapidly reduce joint inflammation, the mechanisms by which these agents exert their long-term effects remain unclear. The p38 MAP kinase pathway is one of the signaling pathways triggered by TNFalpha and pharmacological inhibitors of this kinase are being developed for use in RA. Since p38 MAP kinase is involved in interferon gamma (IFNgamma) production by CD4+ T helper 1 (Th1) cells and a Th1 immune response has been associated with RA, we investigated whether anti-TNFalpha therapy could affect the activation of this signaling pathway in the CD4+ T cells of RA patients. We show that in five patients, treatment with infliximab caused a marked reduction of activated p38 MAP kinase levels in CD4+ T cells, without affecting the total levels of p38 MAPK. In contrast to T cells, infliximab therapy did not affect the levels of active p38 MAP kinase in macrophages from the same patients. The selective effect of anti-TNFalpha therapy on the p38 MAP kinase signaling pathway of CD4+ T cells in patients with RA suggests that prolonged benefit with these agents may be mediated by their effect on CD4+ T cells.     

Tg cells in peripheral blood lymphocytes from patients with rheumatoid arthritis

Using combinations of methods for detecting Fc receptors and B lymphocytes, non-B, non-T lymphocytes and T lymphocytes we have shown T_G cells to be significantly increased in rheumatoid arthritis (RA) patients' peripheral blood lymphocytes (PBL). The possible significance of this finding to the disease process is discussed.     

Blood levels of CD11b+ memory T lymphocytes are selectively upregulated in patients with active rheumatoid arthritis

The adhesion molecules CD11b (a beta2-integrin component) and CD54 (ICAM-1) on blood leukocytes were studied by flow cytometry in patients with rheumatoid arthritis (RA). The fractions of CD4+ cells co-expressing CD11b were elevated in 16 patients with active RA compared with those in 16 RA patients who improved during therapy and 8 healthy controls: 0.8+/-0.12% (mean+/-SEM) versus 0.3+/-0.06% (p<0.002) and 0.3+/-0.06% (p<0.005), respectively. Increased levels of CD11b+CD45R0+ cells were observed in patients with active RA compared to those with improved RA and controls: 12.6+/-3.9% versus 4.8+/-2.7% (p<0.002) and 6.1+/-1.2% (p<0.003), respectively. Disease activity, determined by C-reactive protein, correlated with the numbers of CD11b+CD45R0+ cells: r=0.62 (p<0.001). Seven patients were followed during induction of remission with methotrexate and glucocorticoids. The numbers of CD11b+CD4+ and CD11b+CD45R0+ cells fell significantly after clinical improvement. The levels of CD11b+CD14+ cells (monocytes) did not differ between the groups. The number of CD11b+CD15+ cells (neutrophils) was elevated in patients with RA irrespective of disease activity. The levels of CD54+ cells were not different between the RA and control groups. We conclude that the increased numbers of CD11b+ memory T cells may arise from exposure to stimuli outside the synovial compartment.     

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.     

Expression and function of CD5 and CD28 in patients with rheumatoid arthritis.

To assess the role of CD5 and CD28 in the pathogenesis of the decreased cellular immune function in patients with rheumatoid arthritis (RA) we analysed the expression and function of these T-cell surface molecules. The expression of CD5 as well as of CD28 in synovial and peripheral blood T cells was similar to that of control T cells. Monoclonal antibodies (mAb) directed at CD28 and CD5 were able to provide an accessory signal to anti-CD3 activated T cells both from the synovial fluid and from the peripheral blood. However, the proliferation induced by anti-CD3 mAb in conjunction with anti-CD5 or anti-CD28 mAb was always higher in peripheral blood (PB) T cells compared to the paired synovial fluid T cells. After simultaneous ligation of CD5 and CD28, proliferation was induced in the PB T cells. However, when compared to control PB T cells, this proliferation was significantly lower in the RA patients. Purified normal memory (CD45RO+) T cells proliferated less strongly than naive (CD45RA+) T cells, but no difference was observed between rheumatoid and normal memory T-cell proliferative responses. However, enriched PB CD45RA+ T cells from rheumatoid patients proliferated less vigorously to CD5 and CD28 ligation when compared to normal enriched CD45RA+ T cells. Synovial fluid (SF) T cells, which are mainly of the memory cell type, did not proliferate after simultaneous ligation of CD5 and CD28. This refractory state of synovial T cells could not be explained by a difference in the surface expression of CD5 or CD28. Our data suggest that the cellular immune dysfunction in the PB from rheumatoid patients may be due to a decreased responsiveness of the naive T-cell subset to accessory signals provided by CD5 and CD28. In addition, SF T cells appear hyporesponsive to stimulating signals provided through CD5 and CD28.     

类风湿关节炎患者外周血淋巴细胞的凋亡及其意义

目的 研究类风湿关节炎(RA)患者外周血淋巴细胞的凋亡及其意义.方法 采用流式细胞术和激光共聚焦显微镜,检测35例RA患者和30例健康志愿者的外周血淋巴细胞抵抗凋亡的能力,包括分析新鲜分离和培养的淋巴细胞凋亡率及其与临床病情严重度的相关性.结果 两组间新鲜分离的淋巴细胞凋亡率差异无统计学意义[(2.63±1.47)%比(3.53±2.04)%,P＞0.05].活化淋巴细胞检测的结果显示,RA组的凋亡率明显低于对照组[(12.08±6.06)%比(18.66±7.27)%,P＜0.05],并与DAS28评分呈负相关(r=-0.617,P=0.008).结论 RA患者活化的外周血淋巴细胞出现凋亡缺陷,并与RA病情活动度相关.提示这些细胞募集到组织损害和自身免疫炎性反应进程异常的部位并对炎性反应进程的发展和维持起重要作用.     

类风湿关节炎患者外周血淋巴细胞的凋亡及其意义

目的研究类风湿关节炎(RA)患者外周血淋巴细胞的凋亡及其意义。方法采用流式细胞术和激光共聚焦显微镜,检测35例RA患者和30例健康志愿者的外周血淋巴细胞抵抗凋亡的能力,包括分析新鲜分离和培养的淋巴细胞凋亡率及其与临床病情严重度的相关性。结果两组间新鲜分离的淋巴细胞凋亡率差异无统计学意义[(2.63±1.47)%比(3.53±2.04)%,P>0.05]。活化淋巴细胞检测的结果显示,RA组的凋亡率明显低于对照组[(12.08±6.06)%比(18.66±7.27)%,P<0.05],并与DAS28评分呈负相关(r=-0.617,P=0.008)。结论RA患者活化的外周血淋巴细胞出现凋亡缺陷,并与RA病情活动度相关。提示这些细胞募集到组织损害和自身免疫炎性反应进程异常的部位并对炎性反应进程的发展和维持起重要作用。     

Effect of bioresonance therapy on antioxidant system in lymphocytes in patients with rheumatoid arthritis

We measured activities of superoxide dismutase, catalase, and glutathione peroxidase and content of nonprotein thiol groups (reduced glutathione) in blood lymphocytes from patients with rheumatoid arthritis before and during bioresonance therapy. The state of the antioxidant system in lymphocyte from patients receiving standard pharmacotherapy was characterized by activation of the key antioxidant enzymes and decreased content of thiol groups. Bioresonance therapy increased the content of thiol groups and normalized activities of superoxide dismutase and glutathione peroxidase. However, catalase activity remained above the control. Changes in the lymphocyte antioxidant system indicate that bioresonance therapy activates nonspecific protective mechanisms in patients with rheumatoid arthritis.     

NRAMP1 gene polymorphisms in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease associated with HLA-DR genes that share amino acid sequence motif QKRAA/QRRAA from position 70 to 74 in the third hypervariable region of DR1 molecule. The contribution of HLA in RA is however about 37%, suggesting a role for other genes. One such candidate is the gene that encodes natural resistance-associated macrophage protein (NRAMP1), which plays a crucial role in inflammation and tissue destruction. In the present study, we examined the role of NRAMP1 gene polymorphisms in susceptibility to RA. The results show that variation at position 543 in exon 15, which involves substitution of negatively charged aspartic acid (D) by uncharged asparagine (N), and the deletion of TGTG in the 3' UTR may confer protection from development of RA.     

淋巴细胞共刺激信号和凋亡信号异常与RA免疫效应亢进的研究

目的：探究T淋巴细胞表面多种细胞信号分子所介导的细胞活化或凋亡信号在RA患者免疫功能紊乱中的作用。方法：采用流式细胞术检测RA患者外周血T细胞亚群及其表面共刺激分子cD154（cD40L）、CD30和凋亡受体CD95（Fas）的表达。结果：RA患者外周血T细胞亚群偏移，CD4^＋T细胞增加，CD8^＋T细胞减少；共刺激分子CD154在CD4^＋和CD8^＋T细胞上的表达均上调，但CD30分子的表达均降低，并以CD4^＋T细胞降低更为明显。同时，凋亡受体CD95分子在T细胞亚群上的表达均明显增加。结论：RA患者T淋巴细胞表面多种信号分子表达异常，共同导致了RA患者免疫功能紊乱。     

Pre-B lymphocytes with intracytoplasmic mu chains in the peripheral blood of rheumatoid arthritis patients

A dysregulation of B-cell differentiation and activation has long been evidenced in rheumatoid arthritis (RA). Such analyses have, however, usually focused on the latest stages of B-cell development. Using a classical technique of immunofluorescence labeling on cytospins, we investigated the presence of peripheral pre-B lymphocytes in 92 RA patients and 23 controls. Cells with intracytoplasmic mu chains were evidenced in 58.7% of the RA patients studied, ranging between 0 and 30%, while small numbers of c-mu cells, never exceeding 6% of peripheral blood lymphocytes, were observed in 9 controls. Relationships between this feature and clinical or laboratory data were investigated, showing a negative correlation between the percentage of c-mu + lymphocytes and Ritchie's index (P = 0.05), the number of tender or swollen joints (P = 0.05), erythrocyte sedimentation rate (P = 0.005), seropositivity (P = 0.05), and disease duration (P = 0.01).     

Studies on the nature of EA binding by lymphocytes from rheumatoid arthritis patients

Investigation of the nature of the increased erythrocyte-antibody (EA) binding activity of peripheral blood lymphocytes (PBL) from rheumatoid arthritis (RA) patients reported in the preceding paper has revealed that IgG is the active class of antibody in this rosette formation. Some IgM binding also occurs. SRBC sensitized with F(ab)₂ preparations of IgG do not give rosette formation even at high concentrations. EA binding is inhibited by prior incubation of lymphocytes with heat-aggregated human IgG but antigen-antibody complexes did not give significant inhibition.The majority of these rosettes were found to be stable at 4°C and room temperature but labile at 37°C.Enzyme studies with pronase, trypsin, neuraminidase and treatment with sodium azide gave results strongly supporting the conclusion that the increased binding observed is increased Fc-receptor activity. This activity appears not to be a result of Fc binding by cell-bound rheumatoid factor.A range of titres of antibody and of IgG was used to sensitize erythrocytes to form EA and the enhanced EA-rosette formation by PBL from RA patients occurred throughout the range of concentrations of sensitizing antibody. Significantly more EA were bound by individual lymphocytes from RA patients than control subjects. This data suggest that the Fc receptors on RA lymphocytes are more avid for EA than receptors on lymphocytes from healthy people.