| Background and objectivesSystemic lupus erythematosus (SLE), which develops predominantly in women of childbearing age, is a chronic autoimmune disease with a wide spectrum of clinical and immunological abnormalities. It is characterized by overactivation of autoreactive T and B lymphocytes, the presence of various autoantibodies, especially those directed to dsDNA,and circulating immune complexes deposit in tissues and organs. Both the accurate etiology and the pathogenesis of SLE are still unexplained, which might involve genetics, environment, infection and drugs and so on. In recent years, insight into the pathogenesis of SLE has deepened. In particular studies on apoptosis disorders in lupus have shed a new and intriguing light on the development and course of the disease.Increasing knowledge has considered that the defective regulation of apoptosis is the central defects of immune function of SLE. Under the circumstances of the genetic and environmental factors, excessive nuclear antigens released from apoptotic cells without being removed by impaired clearance result in overloaded self-antigens, abnormal activation of autoreactive lymphocytes, end-organ damage by immunocomplexes deposition on organs in SLE.Many studies have confirmed that an increased rate of peripheral blood lymphocytes apoptosis could be in part responsible for the lymphopenia of SLE patients and the skewed apoptotic signal pathways result in abnormal lymphocytes subsets apoptosis. The apoptosis of CD4+T and CD8+T cells increase but not B cells. CD4+ T cells are at a state of active apoptosis and the ratio of CD4+ / CD8+ T cells keep on decreasing. Especially natural CD4+ CD25+ regulatory T cell depletion and high apoptotic sensitivity of NK T cells result in autoreactive lymphocytes overactivation in SLE.CD4+ T cells are considered to play a pivotal role in the pathogenesis of SLE. A dysfunction in their regulatory action can account for the altered immunological homeostasis and the generation of losing self-tolerance. Although CD4+ T cells regulate multiple layers of autoimmunity, apoptotic signaling is an essential component of this balance. Increasing knowledge have confirmed that there is an altered expression of apoptosis-related molecules in CD4+ T cells of SLE. Extrinsic apoptotic pathways mediated by death receptor are evidently aberrant. Increased Fas, TRAIL, soluble Fas(sFas) and TNF-αexpression results in a higher apoptotic susceptibility of CD4+ T cells. Intrinsic apoptotic pathways mediated by the Bcl-2 families are also remarkably disordered in SLE with Bim, PUMA,Bcl-XL upregulated in SLE and so on. These unbalanced apoptotic signal result in the aberrant apoptosis of SLE CD4+ T cells. But, the complicated interaction mechanisms of apoptotic molecules and unclear signal regulation of SLE lymphocytes indicates that there must exist other unknown factors taking part in the disordered cell apoptosis. It is evidently important to investigate the aberrant apoptotic signal pathways in order to further elucidate the pathogenesis of SLE and find the effective methods to correct the imbalance of apoptosis.We have previously obtained genomewide gene expression profiles of CD4+ T cells from a female SLE patient at severe activity and nonactivity state of disease by using long serial analysis of gene expression (LongSAGE) and found a novel apoptosis-related gene, BclGL expression was significantly higher in peripheral blood CD4+ T cells. BclGL is a newly identified special pro-apoptotic members of Bcl-2 families. Besides its apoptotsis inducing effects on several tumor cell lines, it has been demonstrated to have the inhibiting effects on cell activation, which indicates that it may have different effects on different kinds of cells. But, up to now, its regulatory function on lymphocytes apoptosis and proliferation and its precise mechanisms are still unknown, and there is no the relative reports of BclGL in the pathogenesis of SLE. So, we conjecture that the increased BclGL expression in SLE CD4+ T cells may participate in the abnormal apoptosis of CD4+ T cells. In order to verify this presumption, in the present study, we first want to detect the expression distribution of BclGL in peripheral blood lymphocytes of SLE patients and analyze its relationships with cell apoptosis and clinical parameters, and further investigate its possible regulatory mechanisms in aberrant SLE CD4+T apoptosis. Our study will provide experimental clues and theoretical evidences for discovering molecular mechanisms of disordered lymphocytes apoptosis and screening diagnostic and therapeutic targets of SLE in the future.Methods and results1. Screening apoptosis-related differential genes in CD4+T cells of SLE patients by microarray analyses Peripheral CD4+T cells of 3 recent-onset SLE female patients and age-, area-,ethnically matched normal controls were isolated with immunomagnetic beads. The purity of isolated cells were identified by flow cytometry(FCM). The results showed that the expression of apoptosis-related genes and type I interferon signaling genes are evidently increased in SLE CD4+T cells compared to normal controls. Combining with LongSAGE profiles, we found the expression of a novel apoptosis-related gene BclGL was greatly increased in SLE CD4+T cells(p<0.01).2. BclGL gene expression in SLE lymphocytes and its relationships with cell apoptosis and clinical parameters 95 Chinese female SLE patients were consecutively recruited and the clinical parameters and disease activity determined with SLEDAI score were analyzed at time of blood acquisition. Seventy-five age-, gender-, course-matched and ethnically matched healthy female subjects, 43 active rheumatoid arthritis (RA) and 30 dermatomyositis (DM) patients were included from the same geographical area as controls. BclGL gene expression was detected by real-time PCR and wester blot in the peripheral blood CD4+T, CD8+ T and B cells. The percentage of apoptotic lymphocyte subsets were detected by FCM with Annexin V-FITC/PI double staining. The results showed that BclGL expression was significantly increased in SLE CD4+T cells (p<0.05) but not in CD8+T and B cells(p>0.05) compared to controls. The apoptotic rates of CD4+T and CD8+T cells in SLE patients were higher than other controls(p<0.01,p<0.05).The apoptosis of B cells could be seen but not reach significant levels(p>0.05). The increased BclGL expression was positively correlated with enhanced CD4+T cell apoptosis, antinuclear antibody (ANA) titer and proteinnuria(r=0.82,0.70 and 0.77,p<0.05).The SLE patients who have positive anti-dsDNA antibody, renal diseases, lymphopenia and serositis had relatively higher BclGL expression than those without these(p<0.05).3. Detection of BclGL expression and cell apoptosis after different sera stimulation BclGL expression and cell apoptotic rates of normal human CD4+T, CD8+ T and B cells (n=6) were detected after stimulation with different sera of SLE, RA, DM patients and normal controls(NHS) for 4h,16h and 24h. The results showed that BclGL expression and cell apoptosis in SLE sera stimulated CD4+T cells were significantly increased as compared with other sera(p<0.05). Increased BclGL expression was positively correlated with CD4+T cells apoptotic rates(r=0.73,p<0.05) with time-dependence. The apoptosis rates of CD8+T cells were also increased but the expression of BclGL were not increased in CD8+T and B cells by SLE sera stimulation(p>0.05).4. Construction of lentivirus FUGW-BclGL and detection of its apoptotic inducing effects A recombinant lentivirus vector Lenti-BclGL-GFP was constructed in vitro and transfected into 293FT cells to package lentivirus.The virus titer was detected after concentration.The virus was tranducted into Jurkat cell line and normal human CD4+T cells. Green fluorescent protein(GFP) expression was detected by immunofluorensence microscopy and FCM. BclGL expression was detected by real-time PCR and Western blot. Cell apoptosis was detected by FCM with Annexin V-APC/PI double staining. The results showed that Lenti-BclGL-GFP vector was successfully constructed by enzyme cutting and sequencing. The virus titer could reach 2.0×106/ml after packaging and concentration. The BclGL expression and cell apoptosis were both simultanously increased in transfected Jurkat and normal CD4+T cells(p<0.05),which indicated that BclGL had apoptosis-inducing effects on CD4+T cell apoptosis5. Construction of retrovirus pSilencer5.1-H1-BclGL and detection of BclGL silence efficiency and its effects on apoptosis and proliferation of T lymphocytes Three shRNA encoding sequences were synthesized by shRNA analysis software and retrovirus vectors pSilencer5.1-H1-BclGL (P1,P2, P3) were constructed and transfected into PT67 packaging cells. Stable cell clones were set up by puromycin selection. Three recombinant retrovirus and empty control retrovirus were packaged,concentrated and tranducted into Jurkat cells for 56h. Silence efficiency of BclGL was detected by real-time PCR and Western blot. The proliferation of cells was detected by CCK-8. The silence efficiency of normal CD4+T cells previously was detected by infection retrovirus into BclGL overexpressed CD4+T cells. After normal CD4+T cells were infected by shRNA retrovirus for 72h, the cell apoptosis by SLE sera stimulation was detected. The results showed that 3 retovirus vectors were all successfully constructed and the titers were all above 105CFU/mL. BclGL expression could be silenced by 3 siRNA retrovirus and P2 was most efficient. The proliferation of Jurkat cells was increased after BclGL silence. SLE serum-induced CD4+T cell apoptosis could be partially inhibited by BclGL silence.Conclusions1. BclGL expression was significantly increased in peripheral blood CD4+T cells of SLE patients compared with RA,DM patients and normal controls, but there was no significant differences of BclGL expression in CD8+T and B cells. Increased BclGL expression was positively correlated with enhanced CD4+T cell apoptosis. The patients with positive anti-dsDNA, renal diseases, lymphopenia and serisitis had relatively higher BclGL expression compared those without these.2. SLE sera could induce BclGL expression in normal CD4+T cells but not in CD8+T and B cells, and CD4+T cell apoptosis was also simultaneously enhanced by SLE sera stimulation. Increased BclGL expression was positively correlated with enhanced CD4+T cell apoptosis with time-dependence.3. BclGL gene overexpression could induce Jurkat and normal CD4+T cells apoptosis in vitro. The proliferation of Jurkat cells could be increased and the apoptosis-inducing effects of SLE serum could be inhibited after BclGL gene silencing, which indicated that BclGL might play important roles in regulating T lymphocyte apoptosis and proliferation, and its abnormal expression could participate in the intracellular apoptotic signal pathways induced by SLE serum stimulation and play important roles in the pathogenesis of SLE CD4+T cells apoptosis.4. Abnormal BclGL expression in SLE CD4+T cells could contribute to the aberrant SLE CD4+T cell apoptosis which results in immune disorder and imbalanced homeostasis in SLE. BclGL might be an important factor in regulating SLE CD4+T cell apoptosis but not the decisive factor. Deeply investigation of the BclGL signal pathways is significant and will provide worthy experimental clue and theoretical evidences for elucidating the pathogenesis and uncover useful and potential diagnostic and therapeutic targets of SLE in the future. |
PDF Full Text Request