Clinical Significance Of Changes In Bruton Tyrosine Kinase In Patients With Lupus Erythematosus And Regulation Of Expression By Mesenchymal Stem Cells

Background:Systemic lupus erythematosus(SLE)is a multiple organ damage autoimmune disease.Glomerulonephritis,cutaneous lesions and arthritis are very common in SLE patients.B cells are reported to be involved in the onset of SLE.B cell antigen receptor(BCR)is essential for the proliferation,activation and differentiation of B cells.Bruton's tyrosine kinase(Btk)is a downstream molecule of B-cell receptor(BCR)signaling pathway.It is reported that transgenic mice overexpressing Btk specifically in B cells could produce antinuclear antibody and develop a lupus-like symptoms.Umbilical cord mesenchymal stem cells(UC-MSC),a kind of multipotent progenitor cells,have been used to treated autoimmue diseases now,for its properties of immunoregulation and repair.Objective: The aim of our study was to identify the specific role of Btk in lupus nephritis and detect whetherUC-MSC could regulate the expression of Btk in B cells.Methods: The peripheral blood mononuclear cells from SLE patients and healthy controls were collected,and isolated with Ficoll.ELISA was used to detect the serum anti-dsDNA antiody.And the percentages of Btk positive B cells were detected by flow cytometry.B cells of human and mice were purified by MicroBeads.Western Blot(WB)was used to measue the expression of Btk in B cells of human and mice.The correlation between the percentages of Btk positive B cells and some lupus related clinical indicators were analyzed.Immunohistochemistry was used to detect the Btk expression in kidney sections from 9 lupus nephritis(LN)patients and 8 controls.The levels of Btk expression in glomerulus were analyzed by image-pro plus analysis software,and represented by mean optical density(MOD).PBMCs were cocultured with UC-MSCs at a ratio of 10:1,2.5?g/ml CpG2395,3?g/ml anti-CD40,2?g/ml F(ab')2 anti-human IgM,50ng/ml IL-4 were added in the culture medium to stimulate the PBMCs for 72 hours.160?mol/L cyclophosphamide(CTX),1?mol/L dexamethasone(DEX),and 1?mol/L Btk inhibitor(RN486)were used to treat the PBMCs respectively.Then the percentage of B cells and percentage of Btkhigh B cells were detected by flow cytometry.Results: The percentage of Btkhigh cells in SLE patients is increased compared with the healthy controls(1.74% ± 0.15% vs 0.86% ± 0.06%,p<0.001).And the percentage of Btkhigh B cells from SLE patients is up-regulated compared with the healthy controls(3.89% ± 0.31% vs 2.61% ± 0.21%,p<0.01).The percentage of Btkhigh B cells significantly correlated with the SLE activity(SLEDAI)(r=0.53,p<0.01),levels of serum anti-dsDNA antibody(r=0.41,p<0.05),levels of serum C3(r=-0.41,p<0.05),and the amount of 24 hours urine protein(r=0.59,p<0.05).The results of Western blot showed that the expression of Btk in B cells significantly increased in MRL/lpr mice compared to the C57BL/6 mice.However,only one of the three SLE patients showed higher Btk expression compared with the healthy control.We also found that the frequency of Btkhigh B cells in the patients with lupus nephritis was significantly higher than the patients without lupus nephritis(4.71% ± 0.481% vs 3.19% ± 0.30%,p<0.05).The levels of Btk expression in glomerulus markedly increased in LN patients compared with controls,as the MOD value of Btk in glomerulus in LN was higher than controls(0.03 ± 0.003 vs 0.01 ± 0.002,p<0.001).The percentage of Btkhigh B cells was significantly increased compared with the controls when the CpG2395,anti-CD40,F(ab')2 anti-human IgM and IL-4 were all added into the coculture system(40.63% ± 10.84% vs 3.17% ± 0.25%,p<0.05).And the results of Btk MFI in B cells was increased consistently(12.53 ± 0.30 vs 9.58 ± 0.79,p<0.05).In addition,the percentage of Btkhigh B cells in SLE patients was markedly reduced when cocultured with UC-MSCs(29.00% ± 1.34% vs 50.03% ± 4.69%,p<0.05).What's more,similarly to the results in SLE patients,UC-MSC can also inhibit the Btkhigh B cells in healthy donors(16.90%±1.65% vs 39.48%±1.14%,p<0.001).It seems that DEX and CTX,which are the common drugs for SLE treatment,cannot reduce the Btkhigh B cells.Conclusion: The expression of Btk was significantly increased in SLE patients.UC-MSC could reduce the percentage of Btkhigh B cells.These findings suggested that Btk might be a promising therapeutic molecular target for SLE,and UC-MSCs might regulate the proliferation and differentiatin of B cells through Btk.