A Role For B Lymphocyte Stimulator In Graves Disease

Objective: B lymphocyte stimulator (BLyS, also known as TALL-1, BAFF, THANK, and zTNF4), is a recently identified 285–amino acid protein that is a member of the tumor necrosis factor (TNF) ligand superfamily. It is a type Ⅱmembrane protein that exists in both membrane-bound and soluble forms. The mRNA for BLyS was expressed at high levels by peripheral blood mononuclear cells, spleen, lymph node and bone marrow and at low levels by placenta, heart, lung, fetal liver, thymus and pancreas. BLyS has a crucial role in the humoral immune responds, it can induce B lymphocyte proliferation, differentiation and Ig secretion. Overexpression of BLyS directly leads to B cell hyperplasia and presence of autoantibody, thereby induces the development of various autoimmune diseases. BLyS transgenic mice develop a SLE-like clinical syndrome and elevated titers of multiple autoantibodies (including antinuclear antibodies, anti-double-stranded DNA antibodies [anti-dsDNA], and rheumatoid factor [RF]), circulating immune complexes, and Ig deposits in their kidneys. Serum levels of BLyS are significantly elevated in human patients with a variety of autoimmune disorders, such as systemic lupus erythematosus, rheumatoid arthritis, sjogren's syndrome and so on. Nowadays, the relationship between human BLyS and autoimmune disease has been a new hot spot in the study field of autoimmune disease. Graves disease is an organ-specific autoimmune disease with high levels of thyroid hormone, its pathogenesis hasn't been completely clarified until now. Recent studies have demonstrate that Graves disease is developed under the interaction of genetic susceptibility and environment. Reduced suppressor T lymphocyte function and relative increased help T lymphocyte function in vivo lead to the secretion of various autoantibodies by B cells. Among of them, TSH receptor antibody (TRAb) has a crucial role in the pathogenesis of autoimmune thyroid disease. TRAb includes thyroid stimulating antibody (TSAb) and TSH-binding antibody (TBAb). TSAb can compete for the binding of TSH to its specific receptor site in the cell membrane and is able to activate adenylate cyclase and to stimulate the synthesis and secretion of T3,T4, thus leads to a series of hyperthyroid symptoms. In this study, we want to examin the expression of BLyS mRNA in peripheral blood mononuclesr cells and the serum levels of BLyS and TSAb in patients with Graves disease, and to explore the probable action of BLyS in Graves disease. Thus we can provide a new experimental evidence for explaining the pathogenesis of human Graves disease. Methods: Thirty-one patients with Graves disease first diagnosed (untreated group), twenty-nine patients with Graves disease who were recovered to euthyroid after antithyroid drugtherapy (euthyroid group) and twenty-three healthy people (control group) were recruited for the study. Their morning fasting sitting 5ml blood samples were colleced from elbow vein. The blood was uncoagulated by heparin, and the peripheral blood mononuclear cells were isolated. Total RNA was prepaired from mononuclear cells by quanidinum thiocyanate. Then BLyS and ?-actin mRNA was respectively amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) procedure. The RT-PCR products were observed by agarose gel eletrophoresis. Then we took photo, the density of BLyS and ?-actin band were scanned. The relative expression quantity of BLyS was represented with the ratio of band densitometry unit of BLyS to that of ?-actin. Sera from those subjects were harvested at the same time and stored at -20℃until use. T3, T4 and TSH were examined by chemiluminescence, we also examined the serum levels of BLyS and thyroid stimulating antibody (TSAb) by enzyme linked immunosorbent assay (ELISA). All data were treated with SPSS 11.5. Continuous variables were presented as mean±s. We analysed data by one-way ANOVA and line correlation. The significance was indicated by P value, the difference which had the significance was indicated by P<0.05. Results 1 Serum levels of T3, T4 and TSH The serum levels of T3 and T4 were significantly higher in untreated group than those in euthyroid group and control group(T3: 5.06±1.54 vs 1.81±0.04, 1.77±0.30 nmol/ml, T4: 240.39±48.84 vs 118.30±24.25, 121.88±20.20 nmol/ml, respectively). The serum levels of TSH were lower in untreated group (0.12±0.07 mIU/ml) compared with euthyroid group (1.51±1.24 mIU/ml) and control group (1.41±0.83 mIU/ml), the differences were significant (P<0.001). there was no significant difference between the latter two groups (P>0.05). It demonstated that patients with graves disease in euthyroid group recovered to euthyroid after antithyuroid drug therapy. 2 Expression of BLyS mRNA in various groups The expression of BLyS mRNA in peripheral blood mononuclear cells was higher in untreated group (0.73±0.06) compared with euthyroid group (0.55±0.03) and control group (0.51±0.07) , the difference was significant (P<0.001). 3 Serum levels of BLyS in varoius groups The serum levels of BLyS in patients of untreated group (1.57±0.33 ng/ml) were higher than those of euthyroid group (1.30±0.17 ng/ml) and control group (1.25±0.23 ng/ml), the diffrence was significant (P<0.001). There was no significant difference between euthyroid group and control group (P>0.05). In untreated group, 28 of 31 cases (90%) serum BLyS densities were higher than 1.3 ng/ml, 4 case BLyS levels were higher than 2.0 ng/ml. In euthyroid group and control group, most people (>50%) serum BLyS densities were lower than 1.3 ng/ml. There was no one whose BLyS level was higher than 2.0 ng/ml. 4 Serum contents of TSAb in varoius groupsThe serum contents of TSAb in patients of untreated group (0.99±0.11) were higher than those of euthyroid group (0.92±0.07) and control group (0.91±0.05), the diffrence was significant (P<0.01). There was no significant difference between euthyroid group and control group (P>0.05). In untreated group, 12 of 29 cases (39%) serum TSAb contents were higher than 1.0, 5 cases TSAb contents were higher than 1.1. In euthyroid group and control group, most people (>80%) serum TSAb contents were lower than 1.0. There was no one whose TSAb content amounted to 1.1. 5 Correlation of serum BlyS, TSAb, T3, T4 and TSH The serum levels of BLyS correlated positively with T3 and T4 (r was 0.60 and 0.51, respectively, P<0.01) correlated negatively with TSH (r=-0.32, P<0.01). In contrast, the serum contents of TSAb didn't correlated with T3,T4 and TSH (r was 0.21, 0.20, -0.22, respectively, P>0.05). The serum levels of BLyS didn't correlated with the titers of TSAb neither (r=0.13, P>0.05). Conclusion: BLyS appears to be involved in the pathogenesis of Graves disease, although its mechanism was different with TSAb. The changes of serum BLyS levels can predict GD's progress and estimate its prognosis, thus to direct the treatment of Graves disease. Blockade of BLyS function with anti-BLyS antibody or with a soluble form of BLyS...