International Congress Series 1240 (2003) 1163 1167

Antibodies against inner ear proteins in the sera of patients with inner ear diseases
M. Suzuki a, M.S. Krug b, K.C. Cheng b, Y. Yazawa a, J. Bernstein c, S.S. Kwon b, R. Mora d, T.J. Yoo b,*
b a Department of Otolaryngology, Shiga University of Medical Science, Seta, Ostu 520-21, Japan Division of Allergy and Immunology, Department of Medicine, University of Tennessee, Memphis, 956 Court Avenue, Room H300, Memphis, TN 38163, USA c Department of Otolaryngology, State University of New York, Buffalo, NY, USA d Department of Otolaryngology, University of Genoa, Genoa, Italy

Abstract Sera from patients with various inner ear diseases, especially Menieres disease, were investigated by Western blot against guinea pig inner ear preparations. Of 45 patients, 24 (53%) with various inner ear diseases had antibodies against inner ear proteins, compared with none of 10 (0%) in control subjects without inner ear diseases. Of the 10 proteins that showed a positive reaction with patient sera, the 28-kD band was unique in that it appeared only in the membranous fraction of the inner ear and was highly positive (28%) in reaction with Menieres disease patient sera. The results in the present study with proteins extracted from guinea pig inner ears were consistent with our previous study with proteins from human inner ear. It suggests that the 28-kD protein may be a candidate for detecting autoimmune inner ear disease (AIED). D 2003 International Federation of Otorhinolaryngological Societies (IFOS). All rights reserved.
Keywords: Autoantibody; Inner ear disease; Menieres disease; Western blot assay

1. Introduction Since McCabe [1] first described autoimmune sensorineural hearing loss, now accepted as autoimmune inner ear disease (AIED) [2], a number of experimental and clinical studies were reported in the field of auto immunology [3 7]. These reports presented evidence

* Corresponding author. Tel.: +1-901-448-6663; fax: +1-901-448-8284. E-mail address: tyoo@utmem.edu (T.J. Yoo). 0531-5131/ D 2003 International Federation of Otorhinolaryngological Societies (IFOS). All rights reserved. doi:10.1016/S0531-5131(03)00857-4

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that the etiology and pathogenesis of certain inner ear diseases, for example, Menieres disease and progressive sensorineural hearing loss (PSNHL), might be related to autoimmunity, but have not yet fully confirmed the autoimmune pathological mechanisms. In a previous investigation, we also found that three of six patients with Menieres disease had antibodies directed against the 30-kD protein extracted from human inner ear in Western blotting [5]. In this report, we focus on Menieres disease and attempt to define the localization of antigenic epitope and molecular size on Western blotting with the proteins extracted from guinea pig inner ear.

2. Materials and methods 2.1. Preparation of patients sera Sera from 45 patients with various inner ear diseases were supplied courtesy of Drs. J. Bernstein and Y. Yazawa. The diagnosis of Menieres disease was based on the AAO-HNS criteria [8]. All of the patients with Menieres disease were symptomatic. The sera from the patients with other inner ear diseases and the control subjects without inner ear diseases were subjected to the present study. 2.2. Preparation of cochlear tissue from guinea pigs The cochlea tissues were obtained from 20 Hartley strain male guinea pigs. Guinea pigs were sacrificed painlessly under deep anesthesia. Immediately both temporal bones were removed. The inner ear was divided into two parts under a dissecting microscope. The first part is the membranous labyrinth containing basement membrane, organ of Corti, stria vascularis, spiral ligament and vestibular epithelium (the membranous part of the inner ear). The other part contained the spiral ganglion and cochlear nerve in the modiolus, and vestibular nerve in the temporal bone (the neural part of the inner ear). The facial nerve, brain, and heart were also obtained from the animals. The tissues in lysis buffer were sonicated for 20 s. The supernatant was filtered through a 0.22-Am filter and boiled for 5 min in a boiling water bath. The resultant antigen preparations were frozen at 80 jC. 2.3. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer of proteins to polyvinylidene difluoride (PVDF) membrane Electrophoresis was performed in vertical electrophoresis apparatus utilizing a 12% SDS-PAGE. Separated proteins were then stained with Coomassie brilliant blue. For Western blotting, the gel was transferred immediately to polyvinylidene difluoride (PVDF; BIO-RAD, USA) membrane without gel staining. The separated proteins were transferred to PVDF membrane using a semi-dry type transblot cell (BIO-RAD) with a transfer buffer. The PVDF membrane was dried and kept at 80 jC until the Western blotting. One piece was stained with protein detection kit (BIO-RAD) to visualize the transferred proteins.

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2.4. Western blotting The membrane was incubated with patient serum that was diluted to 1:50. The paper was rocked overnight at 4 jC. The paper was then reacted with a peroxidase-conjugated goat anti-human polyvalent immunoglobulin (Accurate Chemical and Scientific, USA) for 2 h at room temperature. Finally, the paper was stained with 0.05 M Tris HCl (pH 7.6) containing 0.02% 3, 3V-diaminobenzidine (Chemicon International, USA) and 0.01% H2O2.

3. Results The proteins extracted from the membranous part of the inner ear showed five major bands (79, 52, 46, 30 and 28 kD) and eight distinguishable minor bands (67, 44, 42, 40, 35, 32, 25 and 22 kD). The proteins from the neural part of the inner ear showed four major bands (79, 52, 46 and 30 kD) and eight distinguishable minor bands (71, 67, 42, 40, 35, 32, 25 and 22 kD). Of these bands, the distinguishable bands with the 44- and 28-kD molecular weight were found only in membranous part, and the band at a 71-kD apparent molecular weight was found only in the neural part. Although a strong 30-kD band was found in the neural part, two bands with 30- and 28-kD molecular weights were found in the membranous part. Western blotting was carried out with the above-extracted proteins from guinea pig inner ear, and patient and control sera. In 24 (53%) of 45 patients with various inner ear diseases, Western blot analysis with the membranous part and/or the neural part of guinea pig inner ear showed at least one positive band (Table 1). In contrast, normal subjects (n = 10) did not show any positive reaction to these inner ear antigens at all. Of 25 patients with Menieres disease, 15 (60%) showed positive Western blots. The most common positive bands were located at 28-, 52- and 67-kD molecular weights. Of these 10 positively reactive bands, the 28-kD protein was the most common band found in Menieres disease sera with 7 out of 25 sera reactive ( p = 0.06; chi-square test compared with control subjects). The marked difference in the Western blotting results between the two parts of the inner ear was the
Table 1 Distribution of positive bands in Western blot assay 25 kD Menieres disease Otosclerosis HL and Tinnitus Progressive SNHL Cogans syndrome Sudden deafness Strial atrophy Hereditary HL Syphilitic labyrintitis Total 28 kD 30 kD 32 kD 40 kD 42 kD 46 kD 52 kD 5 1 0 1 0 0 0 0 0 7 67 kD 79 kD (4%) (17%)

2 (8%) 7 (28%) 2 (8%) 2 (33%) 0 0 0 2 (33%) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 (9%) 9 (20%) 2 (4%)

0 0 0 0 0 1 (17%) 1 (50%) 0 0 0 0 0 0 0 0 0 0 0 1 (2%) 1 (2%)

0 1 (4%) 0 0 1 (17%) 0 1 (50%) 0 0 0 0 0 0 0 0 0 0 0 2 (4%) 1 (2%)

(20%) 4 (17%) 0 0 (50%) 1 0 0 0 0 1 (16%) 6

(16%) 1 1 0 (50%) 0 0 0 0 0 (100%) 1 (13%) 3

(100%) (7%)

HL: Hearing loss; ( ): (%).

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28-kD protein. This protein was detected only in the membranous part of the guinea pig inner ear. To investigate organ specificity of the reactivity against the 28-kD protein, Western blot analysis was carried out between the sera of nine patients with the positive reaction against the 28-kD protein and the tissues of brain and heart. Of 10 control subjects, the four sera selected at random were also examined on the immunoreactivities against brain and heart. Of nine patients, five patients showed the positive blots against brain and three against heart. However, the positive band with the 28-kD molecular weight was not detected in the fractions of brain and heart. No positive blots were found between the sera of four controls and the tissues of brain and heart.

4. Discussion Of 45 patients, 24 (53%) with various inner ear diseases had the antibodies against guinea pig inner ear in the present study, compared with 0% in the control group. In spite of the limited number of patients examined, this result suggests that certain cases of inner ear diseases might have autoimmune involvement. The positive bands in the guinea pig inner ear fraction were widely distributed in size, but the most common bands were at 28-, 52- and 67-kD molecular weight. The significant presence of inner ear proteins as antigenic epitopes in AIED except for the 68-kD protein remains obscure [5]. Cao et al. [7] have reported 30- and 58-kD protein bands reacted with sera from patients with various inner ear diseases in Western blotting using guinea pig. In this study, we found 10 proteins with different molecular weights reacted with sera of patients with inner ear disease. Of these proteins, the 28-kD protein was only found in the membranous part of guinea pig inner ear and antibodies to it were also frequently found in Menieres disease patient sera. However, in a bovine inner ear protein study, the inner ear specific, but disease nonspecific antigen described by Gottschlich et al. [9] migrated as a doublet at 33 35 kD and thus is unlikely to bear any relationship to the relatively sharp single antigens at 28 or 30 kD reported in this paper. Although the importance of this 28- and 30-kD protein is unclear at the present time, the results in this study suggest that it may have a key role as an autoimmune factor in inner ear diseases, especially Menieres disease. Further studies, for example, the correlation between the positive blots and disease activity or immunosuppressive therapy and protein sequence, to address these issues are under way.

Acknowledgements This study is supported by the UTMG Allergy support fund.

References
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