Serum BAFF and thyroid autoantibodies in autoimmune thyroid disease

Serum BAFF and thyroid autoantibodies in autoimmune thyroid disease

Clinica Chimica Acta 462 (2016) 96–102 Contents lists available at ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clin...

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Clinica Chimica Acta 462 (2016) 96–102

Contents lists available at ScienceDirect

Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim

Serum BAFF and thyroid autoantibodies in autoimmune thyroid disease Jiunn-Diann Lin MD a,b,c, Yuan-Hung Wang PhD a,d, Wen-Fang Fang MD e, Chia-Jung Hsiao MS a, Amarzaya Chagnaadorj MD a, Yuh-Feng Lin MD a,f, Kam-Tsun Tang MD g, Chao-Wen Cheng PhD a,⁎ a

Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC Division of Endocrinology, Department of Internal Medicine, Shuang-Ho Hospital, Taipei Medical University, New Taipei City, Taiwan, ROC Division of Endocrinology and Metabolism, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC d Department of Medical Research, Shuang-Ho Hospital, Taipei Medical University, New Taipei City, Taiwan, ROC e Department of Family Medicine, Shuang-Ho Hospital, Taipei Medical University, New Taipei City, Taiwan, ROC f Division of Nephrology Department of Internal Medicine, Shuang-Ho Hospital, Taipei Medical University, New Taipei City, Taiwan, ROC g Division of Endocrinology and Metabolism, Department of Internal Medicine, Veterans General Hospital, Taipei, Taiwan, ROC b c

a r t i c l e

i n f o

Article history: Received 30 June 2016 Received in revised form 6 September 2016 Accepted 6 September 2016 Available online 8 September 2016 Keywords: B-lymphocyte activating factor Graves' disease Hashimoto's thyroiditis Autoimmune thyroid disease Autoantibody

a b s t r a c t Background: This study investigated the association of serum B-lymphocyte activating factor (BAFF) levels with autoimmune thyroid disease (AITD) in a Chinese population. Materials and methods: We enrolled 221 patients with AITD [170 patients with Graves' disease (GD), 51 patients with Hashimoto's thyroiditis (HT)], and 124 healthy controls. Serum BAFF levels, thyroid function and thyroid autoantibody (TAb) levels, including of thyroid-stimulating hormone receptor antibody (TSHRAb), anti-thyroid peroxidase antibody (Anti-TPO Ab), and antithyroglobulin antibody (ATA), were measured at baseline. Results: Serum BAFF levels were higher in the GD, HT, and AITD groups than in the control group. Significant correlations were observed between BAFF and TSHRAb levels (r = 0.238, p = 0.018), between BAFF and Anti-TPO Ab levels (p = 0.038), and between BAFF and ATA titers (p = 0.025) in women but not in men. In addition, serum BAFF levels were significantly associated with free thyroxine (r = 0.430, p = 0.004) and TSHRAb (r = 0.495, p = 0.001) levels in women with active GD but not in those with inactive GD. Conclusions: Serum BAFF levels are increased in GD, HT, and AITD. The correlation between serum BAFF and TAb levels exhibits a dimorphic pattern, particularly in active GD. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Autoimmune thyroid disease (AITD) is the most common autoimmune disease (AID) in the general population [1,2] and includes Graves' disease (GD) and Hashimoto's thyroiditis (HT). GD is characterized by the generation of a B-cell immune response resulting in the formation of thyroid-stimulating hormone (TSH) receptor antibody (TSHRAb), which causes thyroid follicular cell hyperplasia and increases thyroid hormone production. By contrast, HT is mainly triggered by a T-cell-mediated immune reaction, followed by the destruction of thyroid follicular cells and finally the occurrence of overt hypothyroidism [3]. In addition, HT development is associated with B lymphocyte activation and autoantibody formation; thus, thyroid autoantibodies (TAbs) can be detected in most patients with HT [1]. Abbreviations: AITD, autoimmune thyroid disease; Anti-TPO Ab, anti-thyroid peroxidase antibody; ATA, antithyroglobulin antibody; BAFF, B-lymphocyte activating factor; GD, Graves' disease; HT, Hashimoto's thyroiditis; TAb, thyroid autoantibody; TSHRAb, thyroid-stimulating hormone receptor antibody. ⁎ Corresponding author at: Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, 250 Wuxing St., Taipei 110, Taiwan, ROC. E-mail address: [email protected] (C.-W. Cheng).

http://dx.doi.org/10.1016/j.cca.2016.09.004 0009-8981/© 2016 Elsevier B.V. All rights reserved.

B lymphocytes are essential for maintaining a normal humoral immune reaction through the promotion of antibody formation [4]. However, because of the dysregulation of polyclonal activation, production of autoantibodies, and costimulation of autoreactive T cells, B cells have a pathogenic role in the development of AIDs [5]. B-lymphocyte activating factor (BAFF) has a critical role in regulating the maturation, proliferation, and differentiation of B cells and prolonging the survival of B cells [6,7]. BAFF transgenic mice develop hypergammaglobulinemia and have phenotypes similar to those of autoimmune lupus-like disease, including the presence of autoantibodies to nuclear antigens and immune complex deposits in the kidney [8]. This evidence indicates that BAFF acts as a stimulator of immunoglobulin production in AIDs. A high serum BAFF level has been correlated with several human AIDs including systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren's syndrome [9,10,11]. In addition, serum BAFF levels have been associated with serum autoantibody levels in AIDs [10,12]. Several studies have reported a relationship between AITD and BAFF expression. Fabris et al. reported that the plasma BAFF level was higher in patients with GD and HT than in healthy controls [13]. Moreover, Vannucchi et al. reported that serum BAFF level was increased in

J.-D. Lin et al. / Clinica Chimica Acta 462 (2016) 96–102 Table 1 Demographic characteristics of the Graves' disease (GD), Hashimoto's thyroiditis (HT), and control groups.

Age (y) Women/Men (women %) Smoking (%) Family history of thyroid disease (%)

whether serum BAFF level is associated with the clinical manifestations of GD and HT remains unclear.

Control (124)

GD (170)

HT (51)

AITD (221)

2. Materials and methods

46 ± 12 76/48 (61.3)c,d

45 ± 13c 121/49 (71.2)c

51 ± 14b 45/6 (88.2)a,b

46 ± 13 166/55 (75.1)a

2.1. Patients

15.1d 4.0b,c,d

24.6c 29.4a

10.0b 19.1a

21.2a 27.0a

Age is expressed as the mean ± SD. AITD, autoimmune thyroid disease (GD + HT). a p b 0.05 vs. the control group. b p b 0.05 vs. GD. c p b 0.05 vs. HT. d p b 0.05 vs. AITD.

patients with GD; however, the level decreased after immunosuppressive therapy [14]. Campil et al. demonstrated that BAFF protein expression in infiltrating lymphocytes was higher in AITD than in multinodular goiter tissues [15]. In our recent study, we reported that rs2893321, a BAFF single-nucleotide polymorphism variant, affected susceptibility to the development of GD and AITD [16]. All these studies have suggested the involvement of BAFF in the pathogenesis of AITD. However,

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The serum samples of 221 patients with AITD, including 170 patients with GD and 51 patients with HT, aged N 20 y, were collected by the Division of Endocrinology, Internal Department, Shuang-Ho Hospital (New Taipei, Taiwan) from January 2013 to September 2014. The blood samples of 124 patients without AITD or other AIDs and aged N20 y were obtained by the Health Screening Center of Shuang-Ho Hospital from May to August 2014. Patients with AITD and healthy controls were excluded if they were aged b20 y, pregnant, alcoholic, or had a history of drug intoxication. The study protocol was approved by the Joint Institutional Review Board of Taipei Medical University, and all the patients provided written informed consent prior to participation. GD was diagnosed if one of the following criteria was met: (1) presence of a low TSH level, normal or high free thyroxine (FT4) level, and TSHRAbs; (2) presence of thyrotoxicosis without TSHRAbs but increased or normal diffuse thyroid uptake of I131; or (3) a proven diagnosis by another hospital, as indicated by medical records. HT was diagnosed on the basis of the presence of a Hashimoto autoantibody (either an anti-thyroid peroxidase antibody [Anti-TPO Ab], antithyroglobulin antibody [ATA], or both) and hypoechogenic thyroid parenchyma on a thyroid sonogram.

Fig. 1. Comparison of serum B-lymphocyte activating factor (BAFF) levels in each group. GD, Graves' disease; HT, Hashimoto's thyroiditis; AITD, autoimmune thyroid disease (GD + HT). The number in the column indicates the number of patients. Data are presented as box plots (median, 25th–75th percentile). ***p b 0.001 compared with the controls.

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Table 2 Association of demographic data and thyroid function at baseline with serum B-lymphocyte activating factor (BAFF) protein levels in the Graves' disease (GD) and Hashimoto's thyroiditis (HT) groups. HT (n = 51)

Age Women/Men (women %) Smoking (%) FH of thyroid disease Free T4 (ng/dl) TSH (μIU/mL)

GD (n = 170)

Low BAFF

High BAFF

p value

Low BAFF

High BAFF

p value

47.09 ± 12.11 21/2 (91.3%) 9.1% 23.8% 1.22 ± 1.11 17.85 ± 34.49

53.67 ± 15.07 24/4 (85.7%) 10.7% 15.4% 0.78 ± 0.21 15.10 ± 20.54

NS NS NS NS NS NS

44.39 ± 12.20 62/25 (71.3%) 24.4% 30.8% 4.40 ± 1.68 b0.03

45.89 ± 13.08 59/24 (71.1%) 24.7% 28.0% 4.70 ± 2.02 b0.03

NS NS NS NS NS –

FH, family history; free T4 and thyroid-stimulating hormone (TSH) values are expressed as the mean ± SD; p b 0.05 was considered significant.

2.2. Laboratory analyses Serum FT4 and TSH levels were determined through the electrochemiluminescence immunoassay method by using commercial Roche Elecsys reagent kits (Roche Diagnostica). The normal range of FT4 is 0.93–1.7 ng/dl (with an intra-assay coefficient of variation [CV] of b2.0% and an interassay CV of b4.8%) and that of TSH is 0.27–4.20 μIU/mL (with an intra-assay CV of b3.0% and an interassay CV of b7.2%). Serum Anti-TPO Ab and ATA titers were determined through the particle agglutination method by using available commercial kits (Fujirebio). A reciprocal titer of ≥ 1:100 was considered positive. Serum TSHRAb levels were quantified through the radioimmunoassay method by using a commercial TSHRAb-coated tube kit (R.S.R., Ltd.). Data were expressed as the percentage of blocking of I125-labeled TSH binding to the TSH receptor coated onto the test tube [17]. A value of N15% was considered positive. Serum BAFF levels were determined using aj enzyme-linked immunosorbent assay kit (R&D Systems) according to the manufacturer's

protocol. The serum samples were diluted 1:3-fold. The results are expressed as picograms per milliliter. The patients with AITD were divided into low and high BAFF groups according to the median value of the serum BAFF protein level. 2.3. Statistical analysis All statistical analyses were performed using the SPSS software, ver 13.0. Quantitative values are presented as mean ± SD. The independent t-test was used to compare differences in demographic data and FT4, TSH, and TSHRAb levels between 2 groups. The χ2 test was used to assess differences between the GD, HT, and AITD groups and the control group. Pearson correlation was performed to assess the relationship between either FT4 or TSHRAb and BAFF levels. One-way analysis of variance was used to compare differences in clinical parameters among the GD, HT, and control groups. The Bonferroni test was used for post hoc examinations. Furthermore, the χ2 test or Fisher's exact test was used to assess differences in categorical data between two groups. Because

Fig. 2. Association of serum B-lymphocyte activating factor (BAFF) levels with thyroid-stimulating hormone receptor antibody (TSHRAb) titers in (A) all patients, (B) women, (C) men.

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Fig. 3. Association of serum B-lymphocyte activating factor (BAFF) levels with anti-thyroid peroxidase antibody titers in (A) all patients, (B) women, and (C) men.

Anti-TPO Ab and ATA titers derived using the immunoagglutination method were b1:100, 1:100, 1:400, 1:1600, 1:6400, 1:25,600, 1:102,400, and N1:102,400, comparing differences between 2 groups was difficult. Thus, we divided the patients with AITD into three subgroups according to their Anti-TPO Ab levels: low (≤1:1600), medium (1:6400–1:25,600), and high (N1:25,600). In addition, we divided the patients with AITD into three subgroups according to their ATA levels: low (≤1:100), medium (1:400–1:6400), and high (N 1:6400). All statistical tests were 2-sided, and a p b 0.05 was considered significant. 3. Results 3.1. Demographic data of the GD, HT, AITD, and control groups The demographic data of the HT, GD, AITD, and control groups are listed in Table 1. The patients in the HT group were older than those in the GD group, and the percentage of women was higher in the HT group than in the GD and control groups. The proportion of smokers was higher in the GD group than in the HT group. The number of patients with a family history (FH) of thyroid disease was higher in both the GD and HT groups than in the control group. The proportions of women, smokers, and patients with a FH of thyroid disease were higher in the AITD group than in the control group. 3.2. Serum BAFF level comparison BAFF protein levels were significantly higher in the GD, HT, and AITD groups than in the control group (p b 0.001, Fig. 1A). The serum BAFF levels did not significantly differ between the GD and HT groups. We determined serum BAFF levels in both sexes at baseline and observed that BAFF protein levels were higher in the GD, HT, and AITD groups than in

the control group in both sexes (Fig. 1B). Furthermore, the serum BAFF levels did not significantly differ between the GD and HT groups in either sex. Although the serum BAFF levels were higher in women than in men in the control group (p = 0.01, data not shown), they did not significantly differ between men and women in the GD, HT, and AITD groups (data not shown). 3.3. Association of BAFF protein levels with thyroid function at baseline We determined the association of thyroid function in HT and GD at baseline with serum BAFF protein levels. No significant association of HT severity with serum BAFF protein levels was observed (Table 2). Furthermore, we determined the association of serum BAFF levels with thyroid function in both sexes. We did not observe a significant association of serum BAFF protein levels with thyroid function at baseline in either sex. The results are listed in Supplementary Table 1. 3.4. Association of BAFF protein levels with TSHRAb, Anti-TPO Ab, and ATA titers There was a significant, although weak, correlation between serum BAFF levels and TSHRAb titers at the baseline in the patients with GD (r = 0.185, p = 0.029, Fig. 2A). In addition, the association of TSHRAb levels with BAFF levels was evaluated in both sexes. A significant association of serum BAFF levels with TSHRAb levels was remain observed in women (r = 0.238, p = 0.018, Fig. 2B) but not in men (r = 0.041, p = 0.800, Fig. 2C). Serum Anti-TPO Ab levels significantly increased with a baseline increase in serum BAFF levels in the patients with AITD (p = 0.032, Fig. 3A). Moreover, a significant correlation was observed between serum BAFF levels and Anti-TPO Ab titers in women (p = 0.038, Fig. 3B) but not in men (p = 0.587, Fig. 3C).

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Similarly, we found there was a significant correlation between serum BAFF and TSHRAb levels in the women (r = 0.495, p = 0.001, Fig.5C) with active GD but not in those with inactive GD (r = −0.025, p = 0.852, Fig.5D). However, there was no association of serum BAFF and TSHRAb levels in the men with active or inactive GD (r = −0.145, p = 0.478 and r = 0.168, p = 0.506, respectively). 4. Discussion

Fig. 4. Association of serum B-lymphocyte activating factor (BAFF) levels with antithyroglobulin antibody titers in (A) all patients and (B) women.

A significant association of serum BAFF levels with serum ATA levels was observed in the patients with AITD (p = 0.035, Fig. 4A). In addition, a correlation was observed between serum BAFF levels and ATA titers in women (p = 0.025, Fig. 4B). However, the relationship between serum BAFF levels and ATA titers could not be analyzed because the ATA data of only three men were available.

3.5. Association of BAFF protein levels with TSHRAb and FT4 levels in active and inactive GD To further understand the impact of BAFF on thyroid function and TSHRAb levels in different stages of GD, we classified GD patients into two groups, active GD (patients with new onset or recurrence prior to medication or those receiving treatment with high thyroid function), and inactive GD (patients in remission status or those receiving medication with normal thyroid function) according to the status in which we collected the blood samples. The women with active GD had higher serum BAFF levels than those with inactive GD (p = 0.005; Supplementary Table 2). In addition, there was a significant correlation between serum BAFF and FT4 levels in the women with active GD (r = 0.430, p = 0.004, Fig.5A) but not in those with inactive GD (r = − 0.059, p = 0.668, Fig.5B). In the meanwhile, serum BAFF levels were not associated with FT4 levels in the men with either active or inactive GD (r = −0.175, p = 0.437 and r = 0.145, p = 0.554, respectively).

We observed that serum BAFF levels were higher in the patients with GD, HT, and AITD than in the healthy controls in an ethnic Chinese population. In addition, serum BAFF levels were associated with TAb levels. These results suggest that BAFF is linked to the occurrence of GD, HT, and AITD and the production of TAbs. In addition, we observed that the association of serum BAFF levels with TSHRAb and Anti-TPO Ab levels exhibited a sexual dimorphism, which is consistent with the results of our previous genetic study [16]. Serum BAFF levels were significantly associated with TSHRAb and Anti-TPO Ab levels only in women but not in men. Finally, we observed that serum BAFF levels were significantly associated with TSHRAb and FT4 levels in active GD but not in inactive GD. Moreover, in active GD, serum BAFF levels were also correlated with FT4 and TSHRAb levels only in women but not in men, which further supported the concept that the BAFF could play an important role in the activation of GD and its function was affected by sex. Studies have reported a correlation between circulating BAFF levels and autoantibody levels in several AIDs [9,10,12,18]. Mariette et al. reported a correlation between serum BAFF levels and anti-SSA and rheumatoid factor levels [10]. In addition, Zhang et al. and Chemma et al. demonstrated that anti-dsDNA titers were higher in patients with SLE who had higher serum BAFF titers than in those who had lower serum BAFF titers [9,12]. Consistent with previous findings for other AIDs, we observed a significant correlation between serum BAFF levels and three TAb types in AITD. However, our observation of a significant association between serum BAFF levels and TAbs was inconsistent with the results of two other AITD studies [13,14]. Fabris et al. reported no significant correlation between serum BAFF levels and TAbs in GD, HT, and AITD [13]. In addition, Vannucchi et al. reported that serum BAFF levels were associated with ATA levels but not with TSHRAb and Anti-TPO Ab levels in patients with AITD [14]. The inconsistency between our results and those of these two previous studies might be attributable to differences in the ethnicity of the population, sample size, methods used for quantifying TAbs, and disease statuses. Additional studies are required to clarify this inconsistency. In GD, the TSHRAb level was weakly associated with the serum BAFF level (r = 0.185, p = 0.029), and the association was mildly weaker than that of the serum BAFF level with the anti-dsDNA level in patients with SLE [12,18]. This finding might be attributable to the fact that not all serum BAFF levels in GD were quantified in active stages in our study. Carter et al. reported that the association of serum BAFF levels with anti-dsDNA levels in the remission stage is weaker than that in the relapse stage [18]. This may be also the reason for our observation of no association between the serum BAFF level and thyroid function in GD. To further confirm our hypothesis, we divided the GD patients into two groups, active GD and inactive GD in the present study. Interestingly, we observed serum BAFF levels were associated with TSHRAb (r = 0.299, p = 0.017, data not shown) and FT4 levels (r = 0.270, p = 0.031, data not shown) in the active GD but not in the inactive GD. which further suggests BAFF plays a significant role in the production of TSHRAb, enhancement of thyroid hormone synthesis and the activation of GD. In the meanwhile, HT is mainly driven by T-cellmediated immunity, and TAbs have a limited role in the occurrence of HT, implicating a decreased effect of BAFF on thyroid function in HT, despite the significant association of BAFF with ATA and Anti-TPO Ab in AITD [19]. In the present study, we observed that the associations of BAFF with the TSHRAb and Anti-TPO Ab in both GD and AITD differed between

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Fig. 5. Association of serum B-lymphocyte activating factor (BAFF) levels with free thyroxine (FT4) levels in women with (A) active, (B) inactive Graves' disease (GD) and thyroidstimulating hormone receptor antibody (TSHRAb) titers in women with (C) active, (D) inactive GD.

men and women. In brief, the association of BAFF with either TSHRAb or Anti-TPO Ab is stronger in women than in men. In addition, the associations of serum BAFF with TSHRAb and FT4 levels in active GD also were in a sex-deviation pattern, which further suggests the BAFF function in the activation of GD is influenced by sexes. This dimorphic pattern might be mainly attributable to the difference in the primary immune reaction between men and women [20]. Because of the immunoregulatory effect of estrogen, women have a stronger immune response, greater immunocyte activation, and higher immunoglobulin and antibody formation than men do [20]. In addition, estrogen has a role in triggering and promoting the onset of AIDs, leading to a sex bias [20,21]. Moreover, a recent study reported that the administration of estrogen to both cultured immunocytes from lupus mice and a macrophage cell line could upregulate BAFF mRNA levels, strongly indicating the immunomodulatory role of estrogen in BAFF gene expression [22]. In this study, although no significant differences were observed in serum BAFF levels between women and men with GD, HT, and AITD, a correlation between estrogen and BAFF may be present, contributing to the stronger association of TAb levels with BAFF levels in women than in men. However, additional studies are required to clarify this correlation. Our study has several limitations that should be addressed. First, our sample size for HT was relatively small; an additional study with a large sample size can make our results more reliable. Second, most serum samples of patients with AITD were collected in the euthyroid status or under medication, in which the alteration of BAFF levels compared with those prior to medications is expected. Collecting and analyzing all serum samples in the active stage prior to antithyroid drug and eltroxin administration can make our observations more convincing. Finally, serum BAFF levels in GD can significantly change after medical treatment [14]. The present study is only a cross-sectional study and future well-design longitudinal studies analyzing the association of serum

BAFF with TAbs and thyroid function in different stages, including fresh diagnosis prior medication, under treatment, remission and relapse, would be more valuable and can clarify the role of BAFF in the pathogenesis and clinical manifestations of AITD. Acknowledgments We thank all the participants in this study. This work was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 104-2314-B-038 -048; MOST 105-2314-B-038-035) and was partly supported by a grant from Taipei Medical University and Shuang-Ho Hospital (104TMU-SHH-09). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2016.09.004. References [1] A. Hasham, Y. Tomer, Genetic and epigenetic mechanisms in thyroid autoimmunity, Immunol. Res. 54 (2012) 204–213. [2] D.S. McLeod, P. Caturegli, D.S. Cooper, P.G. Matos, S. Hutfless, Variation in rates of autoimmune thyroid disease by race/ethnicity in US military personnel, JAMA 311 (2014) 1563–1565. [3] Tomer Y, Huber A The etiology of autoimmune thyroid disease: a story of genes and environment. J. Autoimmun. 32 (2009) 231–239. [4] K. Yanaba, J.D. Bouaziz, T. Matsushita, C.M. Magro, E.W. St Clair, T.F. Tedder, B-lymphocyte contributions to human autoimmune disease, Immunol. Rev. 223 (2008) 284–299. [5] J.A. Gross, J. Johnston, S. Mudri, R. Enselman, S.R. Dillon, K. Madden, W. Xu, J. ParrishNovak, D. Foster, C. Lofton-Day, M. Moore, A. Littau, A. Grossman, H. Haugen, K. Foley, H. Blumberg, K. Harrison, W. Kindsvogel, Clegg CH TACI and BCMA are

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