| BackgroundSystemic lupus erythematosus (SLE) is a chronic autoimmune disease which characterized by the formation of antibodies and the involvement with all organs in body. With complicated clinical manifestations and protraction course, it has a harmful effect on human health. However, the exactly etiology and etiopathogenesis still have not been fully elucidated until now. Dysregulation of cellular immunity is an important part in the pathogenesis, especially activation of lymphocytes and production of inflammatory cytokine, which may lead to activation of complement system, deposition of Immune complex and invasion of target organs, play a critical role in the pathogenesis of SLE. Therefore, it is necessary to research on the phenotype and function of immune regulatory T cells.Regulatory T cells (Tregs) which have been shown to have an effect on the maintenance of peripheral immunological tolerance and regulation of various kinds of immune response, play an important role in autoimmune disease, such as SLE and rheumatoid arthritis (RA). It is well known that Tregs include many kinds of T cell subsets. TCRαβ+CD4-CD8-Double-negative Treg (DNT) cells that were defined as a novel subset of Tregs. Normally, DNT cells compose less than1%of peripheral total lymphocytes. Interestingly, DNT cells as have been shown to expand in the peripheral blood of patients with SLE; moreover, it display an inflammatory effector phenotype and play an important in pathogenesis of disease. A researcher from Taiwan reported that Chinese patients with SLE had slightly increased levels of DNT cells, however, did not reach statistical significance. In our initial research, we found that DNT cells increased and related to the development of disease in patients with SLE from small sample. Therefore, in the present study, we expand the number of samples and try to find the relationdship between the percentages of DNT cells and disase activity index(SLEDAI).However, the precise mechanism whereby DNT cells increased in SLE patients is unknown. The Fas pathway, as a major regulator of T cell homeostasis, plays a critical role in regulating the tissue distribution of DNT cells. Decreased expression of Fas receptors result in reduced sensitivity of Fas-mediated apoptosis, which can make it escape from apoptosis mediated by Fas-FasLpathway. Up to now, the relationship that between DNT cells and Fas receptor expression and that between Fas receptor expression on DNT cells and disease activity have not been reported.It has been convinced that DNT cells show ability to inhibit immune responses in both mice and humans. Recently, DNT cells have been proven to contribute to the production of autoantibodies by the interaction with B cells. It plays a critical role in lupus nephiritis. In addition, DNT cells from MRL/lpr mice have been shown to expand and express high amounts of IL-17and that as disease progressively worsens. IL-17(Interleukine-17), as an inflammatory cytokine, mainly produced by activated T lymphocytes. It may lead to tissue damage in the condition of chronic inflammation. It is thus clear that IL-17plays an important role in inflammatory reaction of autoimmune diseases such as SLE. So far, to our knowledge, a study on foreign SLE patients suggests that expanded DNT cells produce IL-17and infiltrate the kidneys. However, the precise role and mechanism of DNT cells is unclear in Chinese SLE patients.ObjectiveAlthough many studies have been reported, the pathogenesis of SLE remains unclear. The reason of DNT increased is still not clear. In view of the important role of DNT cells, it is important to understand the related role and mechanism of DNT cells in SLE. SLE patients, RA patients and normal controls were included in our study. In order to find the reason why DNT cells increased in SLE patients, we are going to explore the sensitivity of Fas-mediated apoptosis of DNT cells by detecting the expression of Fas receptor on DNT cells. In order to explore the evidences that DNT cells involved in the pathogenesis of SLE, we are going to investigate the expression level of IL-17produced by different T cell subsets, especially by DNT cells. Our works will contribute to the pathogenesis of SLE; it can shed light on exploring new treatments of SLE.Methods1. Subjects1.1Patients:According to the American College of Rheumatology (ACR) revised criteria for the classification of SLE in1997, Fifty-five Patients (3male and53female) with SLE from Nanfang hospital were included. Mean age was28.07±10.84years. Evaluation of disease activity in patients with SLE was performed by systemic lupus erythematosus disease activity index (SLEDAI). On the basis of0～4scores means no activity;5～9scores means mild activity;10～14scores means moderate activity;(?)15scores means severe activity. Acoording to our objectives, patients were divieded into inactive group (<10scores) and active group ((?)10scores).1.2Controls:According to the American College of Rheumatology (ACR) revised criteria for the classification of RA in1987. Twenty-five Patients (2male and23female) with RA from Nanfang hospital were studied. Mean age was32.56±4.22years.Twenty-seven healthy volunteers (2male and25female) served as controls. Mean age was32.37±10.02years. There is no significant difference between these three groups in age and sex.2. Research methods2.1Flow Cytometry (FCM) was used to detect the percentage of DNT cells and its Fas receptor expression in56SLE patients,25RA patients and27normal controls.Removed fluorescence antibodies into the bottle of the tube, added some whole blood. Mixed well and incubated in the dark. After fixed and washed, cells were resuspended with PBS. At least0.1million cells were acquired for analysis using FACS Cell Quest software. Lymphocytes were gated according to FSC and SSC.2.2Flow Cytometry (FCM) was used to detect the percentage of subsets of T cells and the IL-17producing in13SLE patients and7normal controls.Sodium heparin peripheral bloods in RPMI1640were incubated with PMA, Ionomycin and BFA at37℃. Then cells were harvested for surface and intracellular staining with fluorescence antibodies. IL-17-prodution was detected in the subsets of T cells. At least0.1million cells were acquired for analysis using FACS Cell Quest software.2.3Flow Cytometry (FCM) was used to detect the percentage of subsets of T cells and the IL-17-prodution with or without5days anti-CD3-stimulated in13SLE patients and7normal controls.PBMCs were isolated by density gradient centrifugation. Cells were cultured for5days in12-well plates that have been precoated with anti-CD3or not.5hours before collection, PMA, Ionomycin and BFA were added. Then cells were harvested for surface and intracellular staining with fluorescence antibodies. IL-17-prodution was detected in the subsets of T cells. At least0.1million cells were acquired for analysis using FACS Cell Quest software.3. Statistical analysisResults are expressed as the mean±SD and analyzed with SPSS13.0. Mann-Whitney U test was used to detect in two independent samples tests, Wilcoxon test was used for two related samples test. Kruskal-Wallis H test was used for K independent samples test, and Bonferroni test was used for multiple comparisons. P value<0.05was considered significant. P value<0.017was considered significant for Bonferroni test. Results1. The percentages of DNT cells and the corelation with disease activity in SLE patients.1.1The percentages of DNT cells were significantly different in SLE patients, RA patients and normal controls (P<0.001). The percentage of DNT cells in SLE patients were higher compared with normal controls (2.45±1.45vs.1.08±0.62,P<0.001). The percentage of DNT cells in SLE patients were increased compared with RA patients (2.45±1.45vs.1.57±0.98,P=0.002). There is no statistically significant between RA patients and normal controls.1.2The percentages of DNT cells were significantly higher in onset SLE patients than review. Active SLE patients presented higher percentage of DNT cells than inactive patients did. Compared with normal complement group, DNT cells were increased in patients with low complement. Correlation of the percentages of DNT cells and autoantibodies was not found. Positive correlations were found between the percentages of DNT cells with SLEDAI (r=0.376, P=0.005).2. The expression rate of Fas receptor on the surface of DNT cells and the correlation with disease activity.2.1The percentages of Fas receptor on the surface of DNT cells were significantly different in SLE patients, RA patients and normal controls (P=0.015). The Fas expression of DNT cells in SLE patients were higher than normal controls.(1.44±0.91vs.0.91±0.55, P=0.004). There is no significantly difference between SLE patients and RA patients, as well as RA patients and normal controls.2.2The rate of Fas expression on the surface of DNT cells were significantly different in SLE patients, RA patients and normal controls (P<0.001). The Fas expression rate of DNT cells in SLE patients were lower than normal controls (61.39±19.20vs.75.95±8.88,P<0.001). Compared with RA patients, the Fas expression rate were markedly decreased in SLE patients(61.39± 19.20vs.74.11±12.82,P=0.004). There is no significantly difference between RA patients and normal controls.2.3Positive correlation between the percentages of DNT cells and Fas receptor on the surface of DNT cells was found (r=0.796, P<0.001). Compared with normal complement group, the Fas receptor expression of DNT cells were increased in SLE patients with low complement. Positive correlation were detected between Fas receptor expression of DNT cells with SLEDAI (r=0.332, P=0.014). Negative correlation between the Fas expression rate of DNT cells with the titers of anti-DNA and ANA antibodies in sera were detected. The relationship between the Fas expression rate of DNT cells and SLEDAI did not reach statistical significance.3. The levels of IL-17production of different T cell subsetsin condition of different stimulate and the relationship between percentages of DNT cells and IL-17with disease activity.3.1After5hours cultured, the percentages of TCRαβ+cells in SLE patients were similar with normal controls. TCRa(3+IL-17+cells were significantly increased in SLE patients compared with normal controls (1.22±0.74vs.0.42±0.29, P=0.030); the percentages of CD4+CD8-cells in SLE patients were lower than normal controls (37.22±7.02vs.45.80±6.99, P=0.019). CD4+CD8-IL-17+cells in SLE patients were similar with normal controls. There is no significantly difference between the percentages of CD8+CD4-cells, CD8+CD4-IL-17+cells of SLE patients with normal controls. The percentages of DNT cells in SLE patients were higher than normal controls (4.76±1.15vs.1.80±0.62, P<0.001). Compared with normal controls, DNT+IL-17+cells were significantly increased in patients with SLE (0.42±0.31vs.0.03±0.02, P<0.001).3.2After5days cultured without anti-CD3, the percentages of TCRαβ+cells, CD4+CD8-cells and CD8+CD4-cells in SLE patients were similar to normal controls. The percentages of DNT cells in SLE patients were significantly increased compared with normal controls (7.83±1.71vs.1.97±0.88, P<0.001). The percentages of IL-17-production by these four cell subsets were significantly higher than normal controls (P<0.05, respectively).3.3After5days stimulated with anti-CD3, the percentages of TCRαβ+cells and CD8+CD4-cells in SLE patients were similar to normal controls. The percentages of CD4+CD8-cells in SLE patients were lower than normal controls (44.11±9.21vs.57.14±9.97, P=0.014). The percentages of DNT cells were increased in SLE patients compared with normal controls (16.94±4.43vs.5.74±2.35, P<0.001). TCRαβ+IL-17+cells were significantly increased in SLE patients compared with normal controls (0.73±0.22vs.0.42±0.18, P=0.008).There is no significant difference of CD4+CD8-IL-17+cells between SLE patients and controls, as well as CD8+CD4-IL-17+cells.The percentages of DNT cells in SLE patients were significantly higher than controls (1.50±0.90vs.0.64±0.50, P=0.024).3.4Compared cells cultured with or without anti-CD3in SLE patients, the percentages of CD4+CD8-cells were lower with anti-CD3-stimulated than without stimulated (44.11±9.21vs.48.50±13.03, P=0.028). The percentages of DNT cells stimulated with anti-CD3were significantly increased in patients compared with non-stimulated (16.94±4.4vs.37.83±1.71, P=0.001). Compared with non-stimulated group, CD4+CD8-IL-17+cells decreased in stimulated group (0.68±0.31vs.0.46±0.17, P=0.028); DNT+IL-17+cells increased significantly in anti-CD3-stimulated group.(0.67±0.40vs.1.50±0.90, P=0.002).3.5The expression of IL-17by DNT cells increased in SLE patients with low complement or active stage after5-day cultured in the presence of plate-bound anti-CD3. The levels of IL-17which produced by DNT cells correlated neither with the titers of anti-DNA antibodies nor ANA in sera. There are markedly positive corelations between DNT+IL-17+cells and SLEDAI(r=0.573, P=0.041). Conclusion1. The percentages of DNT cells are different from SLE patients, RA patients and normal controls. DNT cells are significantly increased in SLE patients. The percentages of DNT cells are positively related to SLEDAI. These results indicate that the increased DNT cells in SLE are related to the activity of disease, and DNT cells may involve in the pathogenic of SLE.2. The significantly decreased Fas expression rate of DNT in SLE patients indicates the decreased sensitivity of DNT cells to Fas-mediated apoptosis. It maybe one of the reasons that lead to increased DNT cells in SLE patients. The expression of Fas receptor on the surface of DNT cells are closely related to disease activity in SLE patients. These results demonstrate that the Fas receptor expression of DNT cells may as a marker in the evaluation of disease activity.3. IL-17producing increased in SLE patients compared with normal controls. After stimulated with anti-CD3, DNT cells expanded and produced much more IL-17than other cell subsets. Morever, the percentages of DNT+IL-17+cells are closely related to disease activity. Our results imply that DNT cells may induce inflammatory response though the way of IL-17production, and DNT+IL-17+cells can be a candidate for evaluating activity of disease in patients with SLE. |
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