The Effect Of Costimulatory Molecules 4-1BB/4-1BBL On T Lymphocyte Activation With Systemic Lupus Erythematosus

The Effect of Costimulatory Molecules 4-1BB/4-1BBL on T Lymphocyte Activation with Systemic Lupus ErythematosusIntroductionSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by hypergammaglobulinemia and the production of pathogenic autoantibodies, which results in multiple organ damage. The abnormal activation of T cell and T-B cell interaction plays a key role in the pathogenesis of SLE. T cell activation requires two signals: one is a signal transduced through the binding of Ag/MHC to the TCR, and the other is the costimulatory molecules without MHC restriction such as CD28/B7,4-1BB/4-1BBL and CD40/CD40L. Two signals altogether make T cell activate and proliferate to secrete cytokine and educe cytotoxicity T cell effect. Deficiency of the first or the second signals, T cell will display immunological anergy and immunological tolerance and Apoptosis. Recently researches have demonstrated that the abnormal expression of costimulatory molecules correlates with the development of autoimmune response.Interaction of 4-1BB expressed on active T cell and its ligand 4-1BBL expressed on APC provides the second independent transmit signal for T cell activation beside CD28. It has been think highly gradually that the contribution of 4-1BB/4-1BBL on inducing T cell activation and secreting cytokine and prolonging immunologic response. 4-1BB named ILA (receptor induced by lymphocyte activation) and CD137, was a kind of I type membrane glucoprotein of 30 KD, and expressed on active T cell by means of monomeric or dimeric form of 50KD. However, It no still clear whether the expression of 4-1BB on T cell of SLE is abnormal. Recently it has been showed in some research that Th1 and Th2 cells participate in autoimmune disease. It play a important role in immunology inflammation of SLE with IFN-γ,IL-12 produced by Thl cell and IL-4 produced by Th2 cell. However, It had never been reported whether 4-1BB/4-1BBL participates in T cell activation and cytokine secretion in SLE patients, p38 mitogen-activated protein kinase (MAPK) pathway is essential for T cells activation as well as the development of both Th1 and Th2 cells. NF-κB, as the member of Rel protein and indirect substrate of p38MAPK, is also a indispensable transcription factor for T cells activation. However, it need to be investigated how 4-1BB/4-1BBL affect p38MAPK pathway and activate transmit factor NF-κB. So we carried out series researches to detect the expression of 4-1BB on T cell in SLE patients in order to identify the effect of costimulatory molecules 4-1BB/4-1BBL on T cell activation with SLE patients and to approach the pathogenesis of SLE.Method1,Lymphocytes were isolated from heparinized whole blood of 40 SLE patients and 20 normal controls and cultured with RPMI-1640 for 48h. The expression of 4-1BB on CD4+T and CD8+T before and after T cell activation was detected by flow cytometry.2,Lymphocytes isolated from 30 SLE patients and 20 normal controls were cultured in RPMI-1640 and divided into five groups: normal control group, pure culture group, anti-CD3mAb activated group, anti-4-1BBmAb blocked group, Dexamethasone group. After cultured for 48h, the supernatants of every group were collected in order to detect the levels of IFN-γand IL-4 by ELISA. The T cell proliferation of every group was detected by MTT.3,T cell of every group was collected after cultured for 48h. The expression levels of p38MARKmRNA and NF-κB mRNA were detected by RT-PCR. The protein levels of p-p38MAPK and NF-κB were detected by Western blot.4,Statistics analysis: The data was indicated with (?)±s. The variances between two groups were compared by independent-samples T test and pair-samples T test. The correlation analysis was carried out between the different indexs. The software SPSS12.0 was used to manipulate the data.Result1,The expression of 4-1BB on CD4~+T and CDS~+T lymphocytes from SLE patients were significantly higher than that of normal controls (P＜0.01, P＜0.01). There were more expression of 4-1BB on CD4~+T and CDS~+T lymphocytes stimulated by anti-CD3 antibody from SLE patients (P＜0.01, P＜0.01). The expression of 4-1BB on CD4+T was higher than that of CD8+T before and after T cell activation (P＜0.01, P＜0.01). The expression of 4-1BB on CD4+T in SLE patients before and after T cell activation was positively correlated with the level of IgG and the amount of urinary micro-albumin (MA) (r=0.623, P＜0.01, r=0.407, P＜0.01, r=0.605, P＜0.01, r=0.463, P＜0.01).2,The proliferation of T cell with stimulated by antiCD3mAb was higher than that of unstimulated group (P＜0.01). The proliferation of T cell in anti-4-1BBmAb blockage group was inhibited by anti-4-1BBmAb, and there was statistic difference compared with stimulated group (P＜0.01). The proliferation of T cell treated with Dexamethasone was also inhibited (P＜0.01).3,The levels of IFN-γand IL-4 in supernatants oft cell with SLE patients were higher than that of normal controls (P＜0.05, P＜0.05). The levels of IFN-γand IL-4 in supernatants of stimulated group were significantly higher than that of unstimulated group (P＜0.01, P＜0.01). The levels of IFN-γand IL-4 in supernatants of T cell of SLE when 4-1BB/4-1BBL pathway was blocked by anti-4-1BBmAb were obviously decreased (P＜0.01, P＜0.01), especially, the degrade levels of IL-4 levels was the most significant, achieve to 50%.4,The expression of NF-κB mRNA and p38 MAPK mRNA oft cell with SLE patients were higher than that of normal controls (P＜0.01, P＜0.01). The expression levels of NF-κB mRNA and p38MAPK mRNA of T cell with SLE patients stimulated by anti-CD3mAb were higher than that of unstimulated group (P＜0.01, P＜0.01). In anti-4-1BbmAb blockage group, there was decreased expression of p38MAPK mRNA(P＜0.01), but not the decreased NF-κB mRNA expression.5,The protein expression of NF-κB and p-p38 MAPK of T cell with SLE patients were higher than that of normal controls (P＜0.01, P＜0.01). The expression levels of NF-κB protein and p-p38 MAPK protein of T cell with SLE patients stimulated by anti-CD3mAb were higher than that of unstimulated group (P＜0.01, P＜0.01). In anti-4-1BBmAb blockage group, there was decreased protein expression of p-p38 MAPK(P＜0.01), but not the decreased NF-κB protein expression.Conclusion1,The expression of 4-1BB on CD4~+T and CD8~+T lymphocytes from SLE patients before and after T cell activation increased significantly.2,When 4-1BB/4-1BBL pathway was blocked by Anti-4-1BBmAb, T cell activation and proliferation could be inhibited and the Th1 and Th2 cytokines secretion could be decreased obviously.3,Anti-4-1BBmAb inhibited T lymphocyte activation through blocking p38 MAPK signal transduction pathway, but not restraining NF-κB activation.4,These results suggest that costimulary molecule 4-1BB plays a critical role in T cell activation of SLE. Our investigation will provide an original view for pathogenesis of SLE and provide rationale and fresh target for autoimmune disease biotherapy.