1. Subjects characteristics
Characteristics of the patients and controls were listed in Table 1.
2. Elevated serum BAFF levels in patients affected with AITD
Serum BAFF levels were elevated in autoimmune diseases including GD in previous studies. To confirm these findings, we measured serum BAFF concentrations in both AITD patients and healthy controls by ELISA. As shown in Figure 1, serum BAFF levels were significantly higher in patients with either GD (1.18 ± 0.33 ng/ml, P=0.0027) or HT (1.02±0.24, P=0.0331) than those in healthy controls (0.93 ± 0.24 ng/ml, Figure1). BAFF concentrations did not differ between euthyroid patients and hypothyroid patients in HT group (data were not shown).
Despite previous studies showed serum BAFF levels were associated with activity of autoimmune, there were inconsistent results on the correlations between BAFF expression levels and autoantibodies titers. We thus investigated whether serum BAFF levels correlated with either thyroid function or thyroid autoantibodies titers in AITD patients. As shown in Table 2, serum BAFF levels positively correlated with serum free thyroxine concentrations (Spearman r = 0.2460, P = 0.0394), but not the TRAb titers (P = 0.4734) in GD patients. However, serum BAFF concentrations were associated with TPOAb titers in both GD (R=0.2451, P=0.0399, Table 2) and HT (R=0.2983, P=0.0325, Table 2). There was no correlation between sera BAFF levels and free thyroxine concentrations in HT (P=0.2655, Table 2).
3. Altered expression profile of receptors for BAFF on peripheral blood B lymphocytes in Graves’ disease
BAFF receptors include BR3, TACI and BCMA. Because BCMA expression is restricted on plasma cells and plasmablasts, both of them exist at extremely low frequencies in peripheral blood, we only measured BR3 and TACI expression in this study. Most of CD19+ cells express BR3 while only part of B lymphocytes express TACI. The percentages of TACI-expressing B lymphocytes were comparable between healthy controls and GD patients (data were not shown). Expression levels of BR3 on peripheral blood B lymphocytes in GD patients were higher than that in healthy controls (mean MFI: 4.52 ± 2.06 in GD vs 3.00 ± 0.87 in HC, P=0.0015, Figure 2A). However, expression of TACI was decreased in GD patients without statistical significance (mean MFI: 7.96 ± 4.06 in GD vs 9.10 ± 3.37 in HC, P =0.1285, figure 2B.). No difference of BR3 and TACI expression between HT patients and controls was observed (figure 2B). These data suggested altered expression of BAFF receptors on peripheral blood B lymphocytes in GD patients.
4. Altered expression of BAFF receptors was corrected by steroids in GO
Steroids have been shown to suppress serum BAFF concentrations in GO patients, however, their effects on expression of BAFF receptors have not been studied. We therefore analyzed serum BAFF concentrations, expression levels of BR3 and TACI on peripheral blood B lymphocytes before and at the end of steroids therapy in 10 GO patients. At baseline, serum BAFF levels and BAFF receptors expression did not differ between GO group and non-GO group (data were not shown). Mean serum BAFF concentrations were decreased from 1.18 ± 0.27 ng/ml at baseline to 0.97 ± 0.10 ng/ml (P=0.0364, Table 3) at the end of methylprednisolone therapy. BR3 expression on peripheral blood B lymphocytes in GO patients was reduced (MFI was decreased from 6.26 ± 4.91 at baseline to 4.05 ± 1.58 at the end of therapy, P=0.0083, Table 3). TACI expression in B lymphocytes after steroids therapy was increased without statistical significance (MFI 6.67 ± 1.96 at baseline vs 7.05 ± 2.32 after steroids therapy, P=0.1974, Table 3). These results indicated immunosuppressive effect of steroids on B lymphocytes was not only on the B cell surviving factor BAFF, but also on its receptors, mainly BR3.