Journal of Neuroimmunology 199 (2008) 151–154www.elsevier.com/locate/jneuroim

Short communication

Serum BAFF expression in patients with myasthenia gravis

Jee Young Kim a, Young Yang b, Joon-Shik Moon c, Eun Young Lee d, Sun Hwha So d,Hong-Soo Lee e, Kee Duk Park a, Young-Chul Choi d,⁎

a Department of Neurology, Ewha Womans University College of Medicine, Republic of Koreab Department of Life Science, Sookmyung Women's University, Republic of Korea

c Department of Neurology, Mayo Clinic, Rochester, MN USAd Department of Neurology, Brain Korea 21 Project for medicine, Yonsei University College of Medicine, Republic of Korea

e Department of Family Medicine, Ewha Womans University College of Medicine, Republic of Korea

Received 12 February 2008; received in revised form 14 April 2008; accepted 20 May 2008

Abstract

A B-cell activating factor of the tumor necrosis factor (TNF) family (BAFF) is an essential B-cell survival factor. Myasthenia gravis (MG) isone of the most typical antibody-mediated autoimmune disorders. To test whether serum BAFF levels are increased in MG patients, we comparedthe serum BAFF levels of 40 MG patients with those of 30 healthy controls. Serum BAFF levels of MG group were significantly higher thanthose of healthy group. These results suggested that the BAFF might play a role in the immunopathogenesis of MG and further study on B-cell andT-cell activation may be needed.© 2008 Elsevier B.V. All rights reserved.

Keywords: BAFF; B lymphocyte stimulator; Myasthenia gravis (MG)

1. Introduction

A B lymphocyte stimulator (BLys or BAFF) is a B-cellactivating factor of the tumor necrosis factor (TNF) family thatis secreted by various myeloid cells, such as macrophages,dendritic cells, and neutrophils (Thangarajh et al., 2006). BAFFoccurs as both a membrane-bound ligand and a biologicallyactive soluble protein, and is essential for B-cell survival anddevelopment (Thangarajh et al., 2004). BAFF binds to 3 cell-surface receptors: BAFF-receptor (BAFF-R), B-cell maturationantigen (BCMA), and transmembrane activator and calcium-modulator and cyclophilin ligand (TACI) (Thangarajh et al.,2004, 2006). These receptors are expressed at different stages ofB-cell development and differentiation (Tangye et al., 2006). Atthese stages, T-cell derived signals, such as CD40L and IL-10,

⁎ Corresponding author. Department of Neurology, Yongdong SeveranceHospital Yonsei University College of Medicine, 146-92 Dogok-dong, Kang-nam-gu, Seoul, Republic of Korea. Tel.: +82 2 20193323, 3320; fax: +82 2 34625904.

E-mail address: ycchoi@yuhs.ac (Y.-C. Choi).

0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.jneuroim.2008.05.010

are required to increase BAFF production by myeloid cells(Tangye et al., 2006). BAFF maintains B-cell survival bymodifying expression of pro- and anti-apoptotic molecules, andalso induces class switch recombination of B cells (Tangyeet al., 2006). And according to recent studies, BAFF enhancesproliferation and cytokine secretion of T cells and the in vitroresponse of CD4+ and CD8+T cells as well as naïve and effectorT cells (Tangye et al., 2006).

In recent years, there have been many studies on elevatedBAFF levels in serum or synovial tissue of patients with severalautoimmune disorders, including systemic lupus erythematosus(SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SjS), andWegener's granulomatosis (Cheema et al., 2001; Mackay et al.,2007; Mariette et al., 2003; Zhang et al., 2001). BAFF has alsobeen found to be correlated with titers of some antibodies, such asanti-Ro/La, rheumatoid factor, and anti-dsDNA (Cheema et al.,2001; Mackay et al., 2007; Mariette et al., 2003; Zhang et al.,2001). Recently a human monoclonal antibody (Ab) against thehuman B lymphocyte stimulator (BLys) (LymphoStat-B®,Belimumab) has been attempted to treat patients with certainautoimmune disorders (Ding and Jones, 2006).

Table 2Serum BAFF levels between ocular type MG patients and generalized type MGpatients

Ocular type Generalized type p-value

Number 20 19BAFF level (pg/mL) 794.99 (593.97) 720.11 (507.64) 0.90

Values are presented as median (standard deviation).

Table 1Serum BAFF levels between healthy controls and MG patients

Controls(n=30)

Totalpatients(n=40)

p-value MG patients onanti-cholinesteraseonly (n=28)

p-value

Age (years) 43.50(11.05)

44.25(12.96)

0.34 45.25(12.46)

0.67

F:M (%) 60:40 55:45 0.81BAFF level(pg/mL)

562.03(269.30)

747.59(544.96)

0.04 771.43(623.93)

0.02

Values are presented as median (standard deviation).*p-value between control and MG patients (either total MG patients or MGpatients on anti-cholinesterase only) by Student's t-test.F; female.M; male.

152 J.Y. Kim et al. / Journal of Neuroimmunology 199 (2008) 151–154

Myasthenia gravis (MG) characterized by fatigue andfluctuating weakness of certain voluntary muscles, is one ofthe most typical antibody-mediated autoimmune disordersamong numerous neurologic diseases (Ropper and Brown,2005). In MG, antibodies against the nicotinic acetylcholinereceptor (AChR) reduce the number of functional AChR on thepostsynaptic membrane of the neuromuscular junction (Ropperand Brown, 2005; Thangarajh et al., 2006). There are a fewstudies on the relationship between MG and BAFF levels(Thangarajh et al., 2006, 2007). Thangarajh et al. (2006) re-ported that plasma BAFF levels did not significantly differbetween healthy controls and MG patients, but they demon-strated that macrophages in hyperplastic thymi of MG patientsexpressed BAFF (Thangarajh et al., 2006).

Our study was aimed at testing whether serum BAFF levelsare increased in patients with MG. If the relationship betweenBAFF and immunopathogenesis of MG is established, targetingBAFF might be a potential therapy of MG.

2. Materials and methods

2.1. Subjects

Forty patients with MG were enrolled in this study. Diagnosisof MG was made on the clinical, electromyographic, andpharmacological findings at the Department of Neurology atEwha Womans University Medical Center and YongdongSeverance Hospital. MG patients were diagnosed based on thefollowing criteria: 1) a fluctuating weakness of certain voluntarymuscles and fatigability; 2) electrophysiological assessments,including repetitive nerve stimulation; 3) positive responseto anti-cholinesterase drugs; and 4) serum levels of anti-AChRbinding or blockingAb. There were 18males and 22 females withages ranging from 20–60 years (yr). Thirty healthy controlsubjects who visited the Health Promotion Center of EwhaWomans University Medical Center for a health examinationconsisted of volunteers who had no prior history or family historyof any medical diseases (12 males, 18 females; age 20–60 yr).

MG patients were classified by disease types (ocular, bulbar,limb, or generalized), presence or absence of thymic abnorm-alities on chest computer tomography (CT), presence or ab-sence of anti-AChR blocking or binding Ab, treatment methods

(anti-cholinesterases, anti-cholinesterases and steroids, immu-nosuppressants, or thymectomy). All subjects provided in-formed consent. This study was approved by Ewha WomansUniversity Medical Center's Ethics Committee.

2.2. Detection of serum BAFF proteins

Sera from healthy controls and MG patients were quantifiedby quantitative sandwich enzyme immunoassay using commer-cial Quantikine® kit (R&D Systems, USA) according to themanufacturer's instructions.

2.3. Statistical analysis

The data were analyzed using SPSS 13.0 program. SerumBAFF levels of healthy controls and MG patients werecompared using Student's t-test. Serum BAFF levels of MGpatient subgroups were compared by Mann–Whitney orKruskal–Wallis test. Values are presented as mean±standarddeviation and described as significant if pb0.05.

3. Results

The mean ages of healthy controls and MG patients were43.5±11.1 years and 44.3±13.0 years, respectively, and the ratioof females to males was 60:40 in the healthy group and 55:45 inMG patients. There was no statistically significant differencein mean age (p=0.34) and sex ratio (p=0.81). Serum BAFFlevels were 562.03±269.30 pg/mL in the healthy groupand 747.59±544.96 pg/mL in MG patients, which were sig-nificantly higher in the MG patients (p=0.04). Serum BAFFlevels in MG patients taking anti-cholinesterase only were771.43±623.93 pg/mL, which were significantly higher thanthose in healthy controls (p=0.02) (Table 1). Among 20 ocularand 19 generalized MG patients, serum BAFF levels were794.99±593.97 pg/mL and 720.11±507.64 pg/mL, respec-tively, and did not differ significantly (p=0.90) (Table 2).

Patients with anti-AChR binding Ab (+) were 67.5% (27/40)and BAFF levels of anti-AChR binding Ab (+) and anti-AChR binding Ab (−) were 849.77±617.38 pg/mL and 553.17±262.03 pg/mL, respectively, which tended to be higher in anti-AChR binding Ab (+) group, but did not show statisticalsignificance (p=0.13) (Table 3A). Patients with anti-AChRblocking Ab (+) were 27.5% (11/40), and BAFF levels of anti-AChR blocking Ab (+) and anti-AChR blocking Ab (−) were919.88±638.26 pg/mL and 691.62±425.43 pg/mL, respectively,which tended to be higher in the anti-AChR blocking Ab (+) groupbut did not show statistical significance (p=0.35) (Table 3A).Serum BAFF levels tended to be higher in MG patients with both

Table 3Serum BAFF levels among MG patients with and without anti-AChR bindingAb or anti-AChR blocking Ab

(A) Anti-AchRbindingAb (+)

Anti-AChRbindingAb (−)

p-value Anti-AChRblockingAb (+)

Anti-AChRblockingAb (−)

p-value

Number 27 12 11 17BAFF

level(pg/mL)

849.77(617.38)

553.17(262.03)

0.13 919.88(638.26)

691.62(425.43)

0.35

(B) BothAb (+)

Anti-AChRBindingAb only (+)

Anti-AchRBlockingAb only (+)

BothAb (−)

p-value

Number 9 18 2 10BAFF

level(pg/mL)

995.34(686.89)

776.99(586.60)

580.85(134.56)

547.63(285.84)

0.25(0.07⁎)

Values are presented as median (standard deviation).*p-value between MG patients with both antibodies positive and those with bothantibodies negative by Mann–Whitney test.

Table 5Serum BAFF levels among MG subgroups classified by treatment methods

Anti-cholinesterase

Anti-cholinesteraseand steroids

Azathioprine Nomedication

p-value⁎

Number 28 7 1 2BAFF(pg/mL)

771.43(623.93)

784.60(313.74)

794.54 397.07(58.50)

0.25

Values are presented as median (standard deviation).*p-value between patients taking only anti-cholinesterase and patients takinganti-cholinesterase and steroids by Mann–Whitney test.

153J.Y. Kim et al. / Journal of Neuroimmunology 199 (2008) 151–154

anti-AChR binding and blocking antibodies than in those withouteither antibodies, which showed a marginal statistical significance(995.34±686.89 pg/mL vs 547.63±285.84 pg/mL, p=0.07)(Table 3B).

Fifteen out of 30 MG patients had thymoma or thymichyperplasia on chest CT and their BAFF levels were 939.47±792.43 pg/mL while those without chest CT abnormalitieswere 689.78±281.40 pg/mL, which did not show statisticallysignificant difference (p=0.91) (Table 4). Seventeen patientsunderwent thymectomy, and their BAFF levels did not differsignificantly from those who did not undergo a thymusoperation (819.33 ±609.40 pg/mL vs 607.38±283.61 pg/mL,p=0.35). Among MG patients, twenty-eight were taking anti-cholinesterases only, seven were taking anti-cholinesterasesand steroids, one was taking azathioprine, and two were nottaking any medication. BAFF levels of patients taking anti-cholinesterases only and those taking anti-cholinesterases andsteroids were 771.43±623.93 pg/mL and 784.60±313.74 pg/mL(p=0.25), respectively (Table 5).

4. Discussion

BAFF is essential for B-cell homeostasis and acts as acostimulator to T cells (Thangarajh et al., 2004). In transgenic

Table 4Serum BAFF levels between MG patients with normal findings on chest CT andthose with thymoma or thymic hyperplasia

Normal findingson chest CT

Thymoma or thymichyperplasia on chest CT

p-value

Number 14 15Age (years) 43.00 (13.04) 50.33 (11.77) 0.09BAFF level (pg/mL) 689.78 (281.40) 939.47 (792.43) 0.91

Values are presented as median (standard deviation).

mice that overexpress BAFF, greatly increased levels ofautoantibody production and associated autoimmune manifes-tations were observed (Mackay et al., 2007). In many studies onpatients with autoimmune disorders, BAFF levels were elevatedin their serum or synovial fluid (Cheema et al., 2001; Mackayet al., 2007; Mariette et al., 2003; Zhang et al., 2001).

Unlike the previous study of Thangarajh et al., our studyshowed that serum BAFF levels were significantly higher inMG patients than in healthy controls. Especially serum BAFFlevels were statistically higher in MG patients taking anti-cholinesterases only without any other immunotherapycompared with healthy controls. This different result betweentwo studies might be affected by clinical status of subjects.Even though there was no statistical significant difference,serum BAFF levels of patients with antibodies against AChRtended to be higher than those without these antibodies. Takinginto account these results, we presumed that there might besome association between serum BAFF and the production ofautoantibodies directed against the AChR in MG patients.

Autoantibody production in MG is a T-cell-dependent processand CD4+Tcells regulate the production of anti-AChR antibodies(Katirji et al., 2002). But it is clear that B cells and autoantibodiesare crucial to MG pathogenesis. At present, it is difficult to saywhether BAFF plays a critical role in the pathogenesis of MG, butT cells have an effect on the action of BAFF and vice versa(Tangye et al., 2006). Therefore, the result of our research sug-gests the possibility of a link between BAFF and the immuno-pathogenesis of MG. However, it is not yet unknown whether theserum BAFF expression is constant or variable during course ofautoimmune diseases. We plan to monitor changes in serumBAFF levels with disease course for MG patients. When therelationship between BAFF and the immunopathogenesis of MGbecomes clear and accurate, targeting the B-cell stimulator mightbe a potential therapy of MG.

References

Cheema, G.S., Roschke, V., Hilber, D.M., Stohl, W., 2001. Elevated serum Blymphocyte stimulator levels in patients with systemic immune-basedrheumatic diseases. Arthritis Rheum. 44, 1313–1319.

Ding, C., Jones, G., 2006. Belimumab Human Genome Sciences/CambridgeAntibody Technology/GlaxoSmithKline. Curr. Opin. Investig. Drugs 7,464–472.

Katirji, B., Kaminski, H.J., Preston, D.C., Ruff, R.L., Shapiro, B.E., 2002.Neuromuscular Disorders in Clinical Practice. Butterworth-Heinemann,Boston, pp. 916–930.

154 J.Y. Kim et al. / Journal of Neuroimmunology 199 (2008) 151–154

Mackay, F., Silveira, P.A., Brink, R., 2007. B cells and the BAFF/APRIL axis:fast-forward on autoimmunity and signaling. Curr. Opin. Immunol. 19,327–336.

Mariette, X., Roux, S., Zhang, J., Bengoufa, D., Lavie, F., Zhou, T., Kimberly, R.,2003. The level of BLys (BAFF) correlates with the titre of autoantibodies inhuman Sjögren's syndrome. Ann. Rheum. Dis. 62, 168–171.

Ropper, A.H., Brown, R.H., 2005. Adams and Victor's Principles of Neurology,8th ed. McGraw-Hill, New York, pp. 1250–1264.

Tangye, S.G., Bryant, V.L., Cuss, A.K., Good, K.L., 2006. BAFF, APRIL andhuman B cell disorders. Semin. Immunol. 18, 305–317.

Thangarajh, M., Gomes, A., Masterman, T., Hillert, J., Hjelmström, P., 2004.Expression of B-cell-activating factor of the TNF family (BAFF) and itsreceptors in multiple sclerosis. J. Neuroimmunol. 152, 183–190.

Thangarajh, M., Masterman, T., Helgeland, L., Rot, U., Jonsson, M.V., Eide, G.E.,Pirskanen, R., Hillert, J., Jonsson, R., 2006. The thymus is a source of B-cell-survival factors-APRIL and BAFF-inmyasthenia gravis. J. Neuroimmunol. 178,161–166.

Thangarajh, M., Kisiswa, L., Pirskanen, R., Hillert, J., 2007. The expression ofBAFF-binding receptors in not altered in multiple sclerosis or myastheniagravis. Scand. J. Immunol. 65, 461–466.

Zhang, J., Roschke, V., Baker, K.P.,Wang, Z., Alarcon, G.S., Fessler, B.J., Bastian,H., Kimberly, R.P., Zhou, T., 2001. Cutting edge: a role for B lymphocytestimulator in systemic lupus erythematosus. J. Immunol. 166, 6–10.