The Differences In BAFF, APRIL As Well As Their Receptors BR3, BCMA and TACI In SLE Patients

BackgroundSLE (systemic lupus erythematosus) is a chronic, multisystem-involved typical autoimmune disease in which abnormally activated and differentiated B cells act as one of the important characteristics of SLE. B cells not only function as effector cells by producing autoantibodies and secreting inflammatory mediators to trigger, sustain and expand immune response, but also act as antigen presenting cells to activate T cells, so B cells contribute a great deal to the pathogenesis of SLE.BAFF (B cell activating factor belonging to TNF family), also known as BLys (B-lymphocyte stimulator), was discovered in 1999 as a new member of TNF (tumor necrosis factor) superfamily of ligands. Through binding to its receptors, BAFF exerts potent stimulation to B cells, regulates the differentiation and maturation of B cells, promotes isotype switch, supports the survival of B cells and spares plasma cells from apoptosis. Over-expression of BAFF may help auto-reactive B effector cells overcome "death signal" triggered by autoantigen which may be involved in the onset and development of SLE. APRIL shares some functions with BAFF.Although several literatures have reported the status of BAFF expression in autoimmune disease, the results are divergent and controversial. One possible reason for the discrepancy might be that patients had received immunosuppressive treatment before being recruited, and this was not considered as an influential.factor.In this research, we tried to analyze the differences in BAFF, APRIL as well as their receptors BR3 (BAFF receptor, BAFF-R, BR3), BCMA(B cell maturation antigen) and TACI (transmembrane activater of and calcium modulator and cyclophilin ligand interactor) in different subgroups of SLE patients. We also analyzed the correlations of these items with clinical manifestations, hoping to expand our knowledge about BAFF and its role in the pathogenesis of SLE and provide some evidence for selecting candidates for BAFF-targeting therapy.MethodSeventy-three patients diagnosed of SLE were recuited. Twenty-eight sex and age matched health volunteers were accepted as health controls (HC). Anti-coagulated peripheral blood was collected and mononuclear cells were isolated.Expression of BR3, TACI and BCMA on peripheral B cells were detected by flow cytometer.Plasma BAFF levels and APRIL levels were detected by ELISA.BR3 mRNA, TACI mRNA and BCMA mRNA were measured and quantified by real-time polymerase chain reaction.Coculture of health PBMC with exogenous recombinant human BAFF was processed to investigate the influence of elevated BAFF on expressions of its recetpors.Clinical data was collected and disease activity was evaluated according to SLEDAI 2000 system. Patients were classified into subgroups according to organ involvement and/or status of medical treatment and/or SLEDAI scores。Results1. SLE patients had higher BAFF levels in their plasma than health controls. (2111.59±220.08pg/ml vs.914.84±43.66pg/ml, p<0.001). And in new-onse patients with active disease, BAFF was especially high (3403.89±640.65pg/ml, p<0.001). BAFF levels correlated positively with SLEDAI scores (p=0.018) and reduced after effective treatment (p=0.026).2. Detectable BR3 protein on peripheral B cells was significantly down-regulated in SLE patients compared to HC (p<0.001) and correlated negatively with plasma BAFF levels (p=0.022). The down-regulation of detectable BR3 was parallel among CD19+IgD+CD27- naive B cells, CD19+IgD+CD27+pre-switch memory B cells and CD19+IgD-CD27+ post-switch B cells and was more distinguished in patients with active disease than those with inactive disease (p=0.013). Post-therapeutic patients had less detectable BR3 than new-onset patient (p=0.018), so did patients with lupus nephritis (LN) than those without (p=0.002). For new-onset patients, expression of BR3 on B cells correlated negatively with SLEDAI (p=0.002) and positively with C3 level (p=0.028).3. Patients with LN had higher expression rate of TACI on peripheral B cells than HC and those patients without LN (p<0.05). TACI expression rate was expecially high in those new-onset patheints with LN (p=0.001) and reduced after immunosuppressive therapy (p=0.007).4. Only low levels of BCMA on developing B lymphocytes had been demonstrated in our study. But a trendency of up-regulation of BCMA expression on B cells in patients can be observed (p=0.032). And the percentage of CD 19+BCMA+ cells tended to correlate with the titers of anti-dsDNA and SLEDAI scores (p=0.035 and p=0.019). What's more, those without lupus nephritis had higher percentage of this group of cells.5. No significant differences of BR3 mRNA and BCMA mRNA level were found between SLE patients and HC. But SLE patients had higher TACI mRNA expression than HC (p=0.023).6. Eleavated BAFF may result more binding with BR3 which in turn caused down-regulation of detectable BR3 protein on B cell surface.ConclusionsHigher levels of BAFF and less expression of BR3 were prominent in SLE patients, and they could act as biomarkers for active disease. BR3 was the fundamental receptor of BAFF which was expressed exclusively on B cells. The actual BR3 expression may not differ between SLE patients and health controls and the down-regulation of detected BR3 may due to prior occupation by elevated BAFF. BAFF/BR3 axis may be over-activated in SLE patients since lower BR3 detection means more binding of BAFF with BR3 which was more distinguished in patients with active disease and patients with lupus nephritis. Up-regulation of TACI in SLE patients was significant in patients with LN. New-onset SLE patients with active disease especially those with nephritis may benefit the most from BAFF-targeting therapy. BackgroundMalfunction of T helper cells plays a central role in the immunological disorders in Systemic lupus erythematosus. Except for well-known Thl and Th2 cells, two new members have joined T helper family, which are T regulatory cell and T helper 17 cells. These four subgroups not only connect with each other on their origin, but also restrict with each other on their development and function. Cytokines secreted by them constitute a network:Thl can secret IFNγ,IL-2,TNF-α,IL-12, induce the activation of macrophage and stimulate the development and proliferation of cytotoxic T cells, thus participate in cellular immunity; Th2 can secret IL-,IL-5,IL-10,IL-13, stimulte B cells to proliferate and produce antibody, thus paticipate in humoral immuity; Treg possesses anergic property and is capable of suppressing the proliferation of T effector cells by cell contact. But Treg is also capable of secreting cytokines like TGF-βand IL-10 to enhance immunoregulation. Thl7 cells were named for its capability of secreting IL-17. IL-17 and other cytokines secreted by Th17 like IL-22 are pro-inflammatory cytokines that are involved in anti-infections immunity, tissue damage and inflammatory osteocalsia. Now the pathogenetic role of Th17 in rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis was recognized. But its role in SLE is rather ambiguous. In this research, through intracellure cytokines staining, T helper subgroups were identified and their correlations with clinical manifestations were analyzed.MethodForty-six patients diagnosed of SLE were recuited. Twenty-nine sex and age matched health volunteers were accepted as health controls (HC). Anti-coagulated peripheral blood was collected and peripheral blood mononuclear cells (PBMC) were isolated.Surface staining of CD4 and CD25 as well as intracellular Foxp3 were used to indentify Treg cells.PBMC was cultured in RPMI 1640 complete culture medium with PMA and Ionomycin to stimulate the expression of intracellular cytokines in the presence of protein transport inhibitor-Golgiplug. Thl, Th2 and Th17 cells were identified by the expression of surface CD4 and intracellular cytokine IFNy, IL-4 and IL-17 respectively.Clinical data was collected, organ involvement was assessed and SLEDAI score was evaluated according to SLEDAI 2000 system.Results1. SLE patients with active disease had higher percentage of Thl cells among CD4+T cells than HC (15.20±1.62%vs.10.85±1.05%, p=0.041). No significant difference was found between patients with organ involvement and those without.2. Percentage of Th2 cells among CD4+T cells increased in SLE patients (3.20±0.62% vs.1.01±0.19%, p=0.021), but did not correlated with the titters of anti-dsDNA antibody, C3 or IgG level. And no difference between patients with active disease and inactive disease was found. And patients with organ involvement did not differ from those without. Short-term follow-up (n=7) showed no significant change ocurred (p=0.398).3. Patients with active disease had higher percentage of Thl7 cells among CD4+T cells compared with HC and those with inactive disease (2.20±0.21 vs.1.53±0.16% and 1.32±0.19%, p=0.011and p=0.010); Thl7 percentage in CD4+ T cells was higher in new-onset patients than post-therapeutic patients (2.29±0.26% vs. 1.58±0.18%, p=0.019), and higher in those with lupus nephritis than those without (2.56±0.34% vs.1.59±0.14%, p=0.002). Although patients with NPSLE had higher percentage of Th17 cells than HC (p=0.026), they did not differ from those without NPSLE (p=0.122).4. Percentage of Th17 cells among CD4+T correlated positively with SLEDAI scores and titers of anti-dsDNA (p=0.008 and p<0.001) and correlated negatively with C3 level (p=0.005).5. No significant difference of Treg percentage was found between SLE patients and HC (p=0.058), although patients with NPSLE tended to have higher percentage of Treg than HC (5.42±1.14% vs.3.1±0.28%, p=0.02). CD4+CD25-Foxp3+ cells significantly increased in SLE patients no matter they were with active disease or not, or with organs involvement or not (P<0.001).6. The ratio of Th17/Treg correlated negatively with C3 level (p=0.038), while positively with titters of anti-dsDNA antibody (p=0.012).ConclusionImbalance of T helper subgroups exists in SLE patients. SLE patients had higher percentage of Th2 cells, which indicates one of the mechanisms of overactivation of B cells and continuous production of autoantigen. Patients with active disease had higher percentage of Thl and Th17 cells, and increased Th17 cells percentage correlating positively with disease activity and nephritis, indicating the possible pathogenetic role of Thl7 in SLE and organ involvement. Althogh patients with NPSLE tended to have a higher percentage of Treg cells than HC, no significant difference was found between HC and SLE patients as a whole. The increase of Treg in patients with NPSLE may be protective. The significantly increased CD4+CD25-Foxp3+T cells deserved more study to investigate its property. BackgroundCD200 is a type I membrane glycoprotein which is expressed on a number of cell types uniquely relevant to the inflammatory and immune cascade including dendritic cells, endothelial cells and activated T cells. Previous studies have shown that CD200 could regulate the activation threshold of inflammatory immune responses, polarize cytokine production and maintain the immune homeostasis. Therefore it plays an important role in prevention of graft rejection, autoimmune diseases and spontaneous abortion. Till now most researches about CD200 have been operated in animal models such as collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE). And the studies of CD200/CD200R1 axis have focused on regulating the function of macrophage and mast cells. The knowledge of CD200/CD200R axis in human especially in SLE patients was very limited and lack of report. In the previous study of our lab, CD200 mRNA and CD200R mRNA decreased in SLE patients, yet the percentage of cells in PBMC expressing CD200 increased, indicating an abnormality in CD200 signal. In this research we try to investigate the possible effect of CD200 signal on the proportion of Th cells and on the proliferation of PBMC.MethodPBMC from SLE patients and health controls were cocultured with CD200Fc,CD200R1 Fc and anti-CD200R1 separately for 48 hours. Intracellular staining was processed and flow cytometry detection was analyzed to determine the change of Thl, Th2, Treg and Th17 cells. The concentration of IL-17 in the supernatant was measured by ELISA.Certain amount of PBMC were also stained with CFSE under sterile conditions and cocultured with CD200 Fc, CD200R1 Fc or anti-CD200R1 in the presence or absence of stimulator (PHA or anti-CD3/CD28) for 96 hours. The status of proliferation was detected by flow cytometer.Results1. Soluble CD200 increased in SLE sera compared with HC (306.60±72.63pg/ml vs. 109.48±23.39pg/ml, p=0.001), but reduced to normal after immunosuppressive therapy (140.32±31.58pg/ml, p<0.001 as compared to before treatment).2. The effect of CD200 Fc on the balance of T helper cells in health controls was rather negligible. But in SLE patients, CD200R1 Fc could increase the percentage of Thl cells which CD200 Fc could reduce the percentage of Th17 cells.3. IL-17 levels in culture supernatant did not differ among the groups cocultured with or without CD200 Fc, CD200R1 Fc or anti-CD200R1.4. Both CD200 Fc and CD200R1 Fc have little influence on the auto-proliferation of PBMC, nor did they have an effect on the proliferation of PBMC under PHA or anti-CD3/CD28 stimulation. But SLE patients may respond to antiCD3/CD28 stimulation more actively in the presence of anti-CD200R1.ConclusionSLE patients had higher soluable CD200 in their sera which decreased to normal after immunosuppressive therapy. CD200 Fc, CD200R Fc and anti-CD200R1 had little effect on the proportion of T helper cell subgroups in HC. But in SLE patients, CD200R1 which blocks CD200 could increase the propotion of Thl cells while CD200 Fc could reduce the percentage of Th17 cells although IL-17 levels in culture supernatant were not altered. CD200 signal may take a part in the regulation of T helper cell subgroups. As shown by the proliferation experiment in vitro, anti-CD200R1 may promote the proliferation of PBMC under the stimulation of anti-CD3/CD28. CD200/CD200R1 axis may exert protective function in SLE patients.