| Rheumatoid arthritis(RA)is a chronic autoimmune inflammatory disease characterized by synovitis.Etanercept is the first biological agent approved for use in RA.It can relieve signs and symptoms of disease,improve body function,and inhibit the progression of structural damage in patients with moderate and severe active RA.TNF-αplays a very important role in the pathological mechanism of RA.TNF-αinduces the expression of receptor activator of nuclear factor-κB ligand(RANKL)and directly promotes osteoclast differentiation in collaboration with RANKL.TNF-αalso promotes bone loss by mobilizing bone marrow CD11b~+,promoting osteoclast precursors,and reducing bone formation by inhibiting osteoblast differentiation and function.In addition,TNF-αcan also induce the production of inflammatory chemokines,resulting in the accumulation of proinflammatory cells(including neutrophils,monocytes and activated T cells).First,TNF-αstimulates endothelial cells to express integrin and adhesion molecules on their surface,which leads to leukocyte accumulation.Secondly,TNF-αinduces the production and release of chemokines CXCL8,CCL2 and CCL5,which also play an important role in leukocyte production and angiogenesis.The third function of TNF-αis to induce the release of other pro-inflammatory cytokines,such as IL-1,.IL-6 and IL-18.In addition,TNF-αstimulated endothelial cells to produce osteoclast factors,such as macrophage colony stimulating factor(M-CSF).TNF-αalso promoted dendritic cell differentiation.The autoantigen in synovium of RA patients is presented to t cells,which stimulates the pathogenesis of RA.Etanercept is a fusion protein composed of the extracellular dimer of human TNF-αreceptor p75 and the Fc segment of human IgG1,which binds to soluble and transmembrane TNF-αon cell surface.The mechanism involves competitive inhibition of TNF-αand TNF-βbinding to tumor necrosis factor receptor on cell surface and regulation of multiple immune and inflammatory pathways.Although there have been a lot of reports on T cell mediated pathogenetic mechanism of RA,there is evidence of disease related autoantibodies such as rheumatoid factor(RF)and anti-cyclic citrullinated peptide(anti-CCP)in serum and synovial fluid of RA patients.These results suggest that B cells play an important role in the pathogenesis of RA.About 30%of RA patients had synovial infiltration of B cells and plasma cell in the ectopic lymphoid center.These germinal centers have been shown to be B-cell-dependent.The cell transfer test of severe combined immunodeficient mice showed that the germinal center failed to develop after rituximab depletion of B cells,suggesting the key role of B cells in germinal center activation.In 70-80%of RA patients,B cells differentiated into plasma cells producing RF and anti-CCP antibodies.Besides producing autoantibodies,B cells are effective antigen presenting cells,leading to T cell activation.Antigen-specific B cells with specific antigen membrane combined with immunoglobulin,can process and present antigens,and the efficiency is 1000times higher than that of other antigen-presenting cells(such as dendritic cells).In addition,activated B cells can express costimulatory molecules CD80/86 and CD40which are necessary for T cell activation.B cells also produce cytokines,such as lymphotoxins are crucial,for the development of tertiary lymphoid structures and IL-10,IL-4 and IL-6.Therefore,B cells play an important role in the pathogenesis of RA.However,little attention has been paid to the regulation of B cell group in RA by Etanercept,and there is no consensus in the literature on the effect of Etanercept on B cells in peripheral blood.Our previous study showed that there was abnormal activation of B cell subsets in peripheral blood of patients with RA.Whether there is a correlation between B cell subsets in peripheral blood and indexe of laboratory and clinical s in RA patients,and the effect of Etanercept on B cell subsets in peripheral blood,the effects on the differentiation of B cells and the signaling pathway that affect the differentiation of B cells are all problems to be solved in this study.Objective1.To explore the correlation between B cell subsets in RA patients with indicators of laboratory and clinical.2.To study the effect of Etanercept on B cell subsets(CD19~+total B cells,CD19~+CD27~+memory B cells,CD19~-CD27~+CD138~+plasma cells)in peripheral blood of RA patients.3.To explore the effect of Etanercept on the differentiation of B cells.Methods1.The peripheral blood and serum of RA patients who treated with Etanercept were collected and followed up for six months.2.Clinical indicators(swollen joint counts(SJC),tender joint counts(TJC),health assessment questionnaire score(HAQ),disease activity score in 28 joints(DAS28))and laboratory indicators(erythrocyte sedimentation rate(ESR),rheumatoid factor(RF),c-reactive protein(CRP))of RA patients were detected and analysis the correlation between B cell subsets(CD19~+total B cells,CD19~+CD27~+memory B cells,CD19~-CD27~+CD138~+plasma cells)and clinical indicators(SJC,TJC,HAQ,DAS28)and laboratory indicators(ESR,RF,CRP).3.The percentages of peripheral blood B cell subsets by flow cytometry in RA patients before and after threatment with Etanercept including:CD19~+total B cells,CD19~+CD27~+memory B cells,CD19~-CD27~+CD138~+plasma cells were detected by by flow cytometry.4.The changes of serum inflammatory cytokines TNF-αand BAFF in peripheral blood of patients with RA were detected by enzyme-linked immunosorbent assay(ELISA)5.In vitro experiments,the optimal concentration of TNF-αon the proliferation of mouse spleen B cells and the effect of Etanercept on the proliferation of mouse spleen B cells were detected by CCK-8.6.The expression of TNFRI,TNFRII and TRAF2 on Type I and Type II transitional B cells was detected by flow cytometry.7.The percentages of mature B cells of TNF-αand BAFF stimulated spleen B cells were detected by flow cytometry.Results1.Percentages of CD19~-CD27~+CD138~+plasma cell were correlated with laboratory indicators(ESR,RF)in RA patients,there was no correlation between CD19~+total B cell,CD19~+CD27~+memory B cell and laboratory indicators.The percentage of CD19~-CD27~+CD138~+plasma cells in peripheral blood of RA patients were detected by flow cytometry and the collected of laboratory indicators.There is a correlation between percentages of plasma cell with ESR(p=0.034)and RF(p=0.028).But there was no correlation between CD19~+total B cell,CD19~+CD27~+memory B cell and laboratory indicators.2.After treatment with Etanercept,the percentage of total B cells,memory cells and plasma cells in peripheral blood of RA patients decreased compared with before treatment with Etanercept.The percentages of B cell subsets in peripheral blood of RA patients were detected by flow cytometry.Compared with before,the percentage of CD19~+total B cell was decreased after three months and six months with treatment with Etanercept;Compared with before treatment with Etanercept,the percentage of CD19~+CD27~+memory B cell was decreased after three months and six months with treatment with Etanercept;Compared with before treatment with Etanercept,the percentage of CD19~-CD27~+CD138~+plasma cell was decreased after three months and six months with treatment with Etanercept.3.The changes of levels of inflammatory cytokines in serum of RA patients after treatment with Etanercept.The levels of inflammatory cytokines(TNF-αand BAFF)in serum of RA patients were detected by ELISA.The results showed that the levels of TNF-α,BAFF in the serum of RA patients were significantly higher than those in the normal group,and the level of TNF-α,BAFF was significantly decreased after treatment with etanercept.4.After treatment with Etanercept,the clinical and laboratory indicators of RA patients were decreased compared with before treatment with Etanercept.The HAQ was significantly decreased after three months and six months with treatment with Etanercept;after three months and six months of treatmentwith etanercept,SJC was significantly decreased;TJC was significantly decreased after three months and six months of treatment with Etanercept;compared with before,DAS28 was significantly decreased after three months and six months of treatment with Etanercept.5.After treatment with Etanercept,the laboratory indicators of RA patients were decreased compared with before treatment with Etanercept.The ESR was significantly decreased after three months and six months of treatment with Etanercept.CRP was significantly decreased after three months and six months of treatment with Etanercept.Compared with before,RF was significantly decreased after three months and six months of treatment with Etanercept.6.After treatment with Etanercept,some RA patients were improved to ACR20/50standard.Be treatmented with Etanercept,the percentage of RA patients who have improved to ACR20 standards is 60%and the percentage of RA patients who have improved to ACR50 standards is 20%after three months.The percentage of RA patients who have improved to ACR20 standards is 75%and the percentage of RA patients who have improved to ACR50 standards is 50%after six months.7.Etanercept inhibits TNF-α-stimulated B cell proliferation.The results showed that TNF-α(40ng/ml)could significantly stimulate the proliferation of B cells at 48h.Etanercept(40μg/ml)could significantly inhibit the proliferation of TNF-α-stimulated B cell.8.TNFRI,TNFRII and TRAF2 are expressed on type I and type II transitional B cells.B cells were isolated from the spleens of normal mice,labeled CD19-FITC,IgD-BV421,IgM-PE,TNFRI-APC,TNFRII-APC,TRAF2-AF64.Fow cytometry was used to detect,the result showed that TNFRI,TNFRII and TRAF2 were expressed on type I and type II transitional B cells.9.Etanercept inhibits the differentiation of transitional B cells into mature B cells.Flow cytometry was used to detect the percentage of B cell subsets in BAFF and TNF-αstimulated spleen B cells.The results showed that the percentage of mature B cell in the BAFF and TNF-αgroup is significantly increased compared with that in the control group.The percentage of mature B cell is significantly decreased with the effect of EtanerceptConlusions1.Plasma cells are correlated with laboratory indicators,suggesting that B cells play an important role in RA.2.It might be one of important mechanisms that Etanercept could improve clinical symptoms and laboratory indicators by down-regulating abnormally activated B cell subsets.3.Etanercept could inhibit the differentiation of B cells by regulating TNFRII/TRAF2/NF-κB signaling pathway. |
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