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lpr mouse spleen cells
Hearing Research 117 (1998) 173^177
Induction of immune-mediated hearing loss in SCID mice by injection of MRL/lpr mouse spleen cells Hiroshi Iwai a; *, Koichi Tomoda b , Naoki Hosaka c , Shigeo Miyashima c , Yuko Suzuka b , Hiroki Ikeda a , Shinryu Lee a , Muneo Inaba c , Susumu Ikehara c , Toshio Yamashita a a
Department of Otorhinolaryngology, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570, Japan b Department of Otorhinolaryngology, Kanazawa Medical University, Ishikawa, Japan c First Department of Pathology, Kansai Medical University, Osaka, Japan Received 28 July 1997; revised 24 December 1997; accepted 31 December 1997
Abstract The MRL/lpr mouse, which has a mutation in the Fas gene encoding a cell-surface receptor for apoptosis, shows an accumulation of abnormal immunocompetent cells and SLE-like disease. It has recently been reported that this mouse also manifests sensorineural hearing loss (SHL) with cochlear pathology at 20 weeks of age. We examined the effects of injecting MRL/lpr spleen cells on the development of SHL in severe combined immunodeficient (SCID) mice, which originally develop neither SHL nor cochlear pathology. Immune-mediated SHL and cochlear pathology were, indeed, transferred to the SCID mice by the injection of spleen cells from the MRL/lpr mice. These findings suggest that cell-mediated immunity is involved in the development of SHL and cochlear pathology. z 1998 Published by Elsevier Science B.V. Key words: Autoimmune disease; Stria vascularis; Immunocompetent cell; Systemic lupus erythematosus
1. Introduction McCabe (1979) described the relationship between inner ear disease and immunological disorders. Clinical observations suggest a correlation between inner ear disorders and autoimmune diseases such as systemic lupus erythematosus (SLE) (Macrae and O'Reilly, 1975), relapsing polychondritis (Cody and Tones, 1971), rheumatoid arthritis (Goodwill et al., 1972), and Behc°et's disease (Brama and Fainaru, 1980). This has prompted investigators to carry out experimental studies on autoimmune mechanisms in inner ear diseases including Meniere's disease (Nonaka et al., 1992 ; Iwai et al., 1995) and sensorineural hearing loss (SHL) (Kusakari et al., 1992; Ruckenstein et al., 1993 ; McMenomey et al., 1992; Trune et al., 1996) in animal models. In the present study, we used two strains of mice : * Corresponding author. Tel.: +81 (6) 992-1001; Fax: +81 (6) 992-1055.
MRL/Mp-lpr/lpr (MRL/lpr) mice established by Murphy and Roths (1979) and severe combined immunode¢cient (SCID) mice established by Bosma et al. (1983). The MRL/lpr mouse, a murine model of SLE, has a mutation in the Fas gene that encodes a cell-surface receptor for apoptosis. The mice show not only the accumulation of abnormal immunocompetent cells due to the escape from Fas-mediated apoptosis but also SLE-like disease accompanied by the production of autoantibodies (Andrews et al., 1978 ; Theo¢lopoulos and Dixon, 1985 ; Watanabe-Fukunaga et al., 1992). Recent works indicate that these mice manifest SHL as a consequence of immune complex deposits containing IgG on the stria vascularis at 20 weeks of age but not at 4 weeks of age (Kusakari et al., 1992). The SCID mouse is known as an ideal recipient for examining the transfer of autoimmune diseases, since it is de¢cient in active B and T cells (Bosma et al., 1983). The SCID defect has been ascribed to the abnormalities in the process of gene rearrangement for immunoglobulin and T cell receptor (Schuler et al., 1986; Lieber et
0378-5955 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 0 1 5 - X
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al., 1988). Recently, it has been demonstrated that even xenogenic tissue or cells including cells from patients with autoimmune diseases can be successfully transplanted into this mouse (Mosier et al., 1988; Krams et al., 1989 ; Duchosal et al., 1990; Macht et al., 1991). In the present study, we attempt to transfer SHL and cochlear pathology to SCID mice by injecting them with spleen cells from MRL/lpr mice, and show that SHL and cochlear pathology can indeed be transferred to SCID mice. 2. Materials and methods 2.1. Animals Inbred SCID (FOX CHASE C.B17/Icr-scid) and C57BL/6 mice were obtained from CLEA Japan, Inc. (Osaka, Japan). MRL/lpr mice were purchased from SLC Japan, Inc. (Shizuoka, Japan). These mice were maintained under speci¢c pathogen-free conditions in our facilities. 2.2. Transfer of donor spleen cells SCID (H-2d ) mice (7 weeks) were intraperitoneally injected with 4U107 of spleen cells from either MRL/ lpr mice (H-2k ) or C57BL/6 mice (H-2b ) at the age of 20 weeks. For several assays, mice were killed 20 weeks after injection. SCID, C57BL/6 and MRL/lpr mice without injection of spleen cells were also used as controls. 2.3. Auditory brainstem response The auditory function of mice was evaluated by their auditory brainstem response (ABR). Prior to testing, the mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and atropine sulfate (0.5 mg/kg, i.p.). Otoscopic and histopathological examinations revealed that the tympanic membranes or tympanic cavities were normal in all the mice. Needle electrodes were inserted into the occipital, vertex and hindpaw (ground). Click stimuli (0.1 ms) were produced using a sound stimulator (MEM-4104, Nihon Kohden, Tokyo, Japan) and a closed tube sound delivery system sealed into the left ear canal. The frequency and intensity (dBpeSPL) of the stimuli at the ear drum of each mouse were con¢rmed using an oscilloscope and a calibration microphone in a preliminary study. The stimulus was set at 20 stimulations per second. The signal was band-pass ¢ltered at 50 to 3000 Hz and averaged 1000 times. ABRs were obtained by decreasing the stimuli in 4or 6-dB steps from a maximum intensity of 106 dBpeSPL. The threshold was de¢ned as the lowest intensity producing a clearly visible ¢rst peak of wave.
2.4. Statistical analyses Data presented means þ S.D. The statistically signi¢cant di¡erences in the ABR were determined using Student's paired t-test. 2.5. Flow cytometry Spleen cells (1U106 ) were suspended in phosphatebu¡ered saline (PBS) containing 2% fetal calf serum (FCS) plus 0.05% sodium azide, then incubated on ice with FITC-coupled anti-H-2d , anti-H-2k , or anti-H-2b antibody (Ab) (Meiji Institute of Health Science, Odawara, Kanagawa, Japan). The stained cells were analyzed on a FACScan (Beckton Dickinson). 2.6. Immunohistochemistry After splenectomy for £ow cytometry, the mice were subjected to intracardiac perfusion with saline, followed by periodate-lysin-paraformaldehyde (PLP) ¢xation at 4³C and decalci¢cation at 4³C in 5% bu¡ered ethylene diamine-tetra-acetate (EDTA) for 7 days. The temporal bones were frozen in OCT compound (Tissue-Tek II, Lab-Tek Products, Naperville, IL), sectioned into 5 Wm sections horizontally to the long axis of the cochlea, then air-dried at room temperature. The sections were stained with hematoxylin and eosin (H and E). Histopathological examinations revealed that the tympanic cavities are normal in all mice, and showed no sign of cell in¢ltration. Rabbit anti-mouse IgG Ab (Cappel, Organon Teknika Corporation, NC) was also applied to the sections immunohistochemically using a modi¢ed indirect immunoperoxidase technique (Takahashi and Harris, 1988) to detect IgG-bearing cells. The avidin-biotin complex immunostaining method was applied using anti-rabbit ABC kits (Vectastain, Vector Lab, Burlingame, CA). The sections were counterstained with hematoxylin. The care and use of the mice reported on this study were approved by a grant application agency (grants-inaid for scienti¢c research 08671994 from the Ministry of Education, Science and Culture). 3. Results 3.1. Analyses of chimerism Flow cytometric analyses were carried out to examine the chimerism using the spleen cells of [MRL/ lprCSCID] mice. Although SCID mice carry the H2d antigen of the major histocompatibility complex on the lymphocyte cell surface, more than 95% of the spleen cells from the [MRL/lprCSCID] mice were of
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[MRL/lprCSCID] mice (42.8 dBpeSPL) were signi¢cantly higher than in the SCID, C57BL/6 and [C57BL/6CSCID] mice, and reached the level of MRL/lpr mice (40.4 dBpeSPL).
Fig. 1. H-2 typing of spleen cells in MRL/lpr and [MRL/ lprCSCID] mice. Spleen cells of A: MRL/lpr mice (20 weeks old) and B: [MRL/lprCSCID] mice (20 weeks after injection of MRL/ lpr spleen cells) were examined for H-2 type spleen cells. The merge graphs show the £uorescence intensity of spleen cells in the unstained control group (line) or stained groups with either anti-H-2d FITC (dots) or anti-H-2k -FITC (dotted line) monoclonal Ab. Note: More than 95% of the spleen cells from the [MRL/lprCSCID] mice were of donor (MRL/lpr) origin (H-2k ) and were similar to the cells of the MRL/lpr mice.
donor (MRL/lpr) origin (H-2k ) (Fig. 1). SCID mice which had been injected with spleen cells from C57BL/6 also showed more than 90% of spleen cells of donor (C57BL/6) origin (H-2b ) (data not shown). 3.2. Thresholds Similar mean thresholds of ABR were obtained in 20-week-old SCID (20.5 dBpeSPL), 20-week-old C57BL/6 (19.7 dBpeSPL), and [C57BL/6CSCID] mice (20 weeks after injection) (20.5 dBpeSPL), as shown in Table 1. In contrast, the thresholds in the
Fig. 2. Transfer of pathology in stria vascularis of MRL/lpr mice to SCID mice. Sections from the temporal bones of A: MRL/lpr mice, B: [MRL/lprCSCID] chimeras, and C: [C57BL/6CSCID] chimeras were stained with rabbit anti-mouse IgG Ab using an ABC method (U400). The arrows indicate IgG deposits on the endothelial cells in capillaries of stria vascularis (A, B). The sections were counterstained with hematoxylin.
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Table 1 Transfer of MRL/lpr mouse hearing loss to SCID mice Groups
Animals
n
dBpeSPL (mean þ S.D.)
A
SCID C57BL/6 [C57BL/6CSCID] MRL/lpr [MRL/lprCSCID]
5 5 7 10 10
20.5 þ 8.7 19.7 þ 8.3 20.5 þ 9.1 40.4 þ 8.3 42.8 þ 9.6
B*
*P 6 0.01 compared to the A group using a t-test. There was no signi¢cant di¡erence between thresholds in group A or B.
3.3. Immunohistochemistry Our pilot studies using H and E staining revealed that MRL/lpr mice and [MRL/lprCSCID] mice show proliferation of endothelial cells in the capillaries and edema in the stria vascularis, while no histopathologic change was found in the cochleas of the SCID, C57BL/ 6, or [C57BL/6CSCID] mice. Based on these ¢ndings, we carried out immunohistochemical analyses to examine the pathological ¢ndings of the stria vascularis. Endothelial cells in the capillaries were positively stained with anti-IgG Ab from the basal turn to the apical turn in the stria vascularis of all 10 of [MRL/lprCSCID] chimeras as well as in all 10 of the non-treated MRL/lpr mice (Fig. 2A, B). In contrast, no IgG deposits were observed in the cochleas of SCID or C57BL/6 mice (data not shown), or the [C57BL/ 6CSCID] mice (Fig. 2C). 4. Discussion In the present study, we have demonstrated that donor cells are repopulated in [MRL/lprCSCID] and [C57BL/6CSCID] mice, and that both hearing loss and IgG deposits on the endothelial cells of capillaries in the stria vascularis develop in [MRL/lprCSCID] but not [C57BL/6CSCID] mice. Ashany et al. (1992) have reported that the donor (MRL/lpr) spleen cells in [MRL/lprCSCID] mice can continuously produce autoantibodies such as anti-DNA and anti-Sm antibodies. Trune et al. (1997) have recently hypothesized that cell-surface DNA receptors of 30 and 70 kDa size in the cochlea could be masked or down regulated by circulating antibodies in the autoimmune SHLs of both human and MRL/lpr mice. Since no lymphocyte in¢ltration was found in the stria vascularis of [MRL/ lprCSCID] mice and no hearing loss was observed in [C57BL/6CSCID] mice, graft-versus-host (GvH) disease can be excluded ; autoantibodies are likely responsible for the development of the inner ear disease. Although C57BL/6 mice exhibit high frequency (over 4000 Hz) presbycusis with the degeneration of outer
hair cells and spiral ganglion cells in the basal turn of the cochlea from the age of 28 weeks (Li and Borg, 1991 ; Willott, 1991), we used the mice at the age of 20 weeks and applied a clicking stimulus with the signal band-pass ¢ltered at 50 to 3000 Hz in ABR. The [C57BL/6CSCID] mice showed neither the elevated thresholds nor the cochlear alteration observed in both MRL/lpr mice and [MRL/lprCSCID] mice. No abnormal ¢ndings in tympanic membranes or tympanic cavities were found in any mice used in this study. It has been reported that the atrophy of the stria vascularis causes sensorineural hearing loss through impaired ion transport and a decline in the endocochlear potential (Schulte and Schmiedt, 1992; Gratton and Schulte, 1995 ; Gratton et al., 1996). Kusakari et al. (1992) have shown that IgG deposition and degeneration of the stria vascularis in 20-week-old MRL/lpr mice are responsible for dysfunction of the stria vascularis and SHL. In conclusion, SHL and the pathology of the stria vascularis in MRL/lpr mice can be transferred by the injection of MRL/lpr spleen cells to normal mice. It is likely that inner ear diseases due to autoantibodies produced by donor spleen cells carrying the lpr gene develop while recipient mice bear no lpr gene. Acknowledgments This study was supported by grants-in-aid for scienti¢c research 08671994 from the Ministry of Education, Science and Culture, by a research grant for Intractable Diseases (Vestibular Disorders) from the Ministry of Health and Welfare, and by the Katano prize of Kansai Medical University in Japan.
References Andrews, B.S., Eisenberg, R.A., Theo¢lopoulos, A.N., Izui, S., Wilson, C.B., McConahey, P.J., Murphy, E.D., Roths, J.B., Dixon, F.J., 1978. Spontaneous murine lupus-like syndromes. J. Exp. Med. 148, 1198^1215. Ashany, D., Hines, J.J., Gharavi, A.E., Mouradian, J., Drappa, J., Elkon, K.B., 1992. MRL/lprCsevere combined immunode¢ciency mouse allografts produce autoantibodies, acute graft-versus-host disease or a wasting syndrome depending on the source of cells. Clin. Exp. Immunol. 90, 466^475. Bosma, G.C., Custer, R.P., Bosma, M.J., 1983. A severe combined immunode¢ciency mutation in the mouse. Nature (Lond.) 301, 527^530. Brama, I., Fainaru, M., 1980. Inner ear involvement in Behc°et's disease. Arch. Otolaryngol. 106, 215^217. Cody, D., Tones, D.A., 1971. Relapsing polychondritis; Audiovestibular manifestations. Laryngoscope 81, 1208^1222. Duchosal, M.A., McConahey, P.J., Robinson, C.A., Dixon, F.J., 1990. Transfer of human systemic lupus erythematosus in severe combined immunode¢cient (SCID) mice. J. Exp. Med. 172, 985^ 988.
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H. Iwai et al. / Hearing Research 117 (1998) 173^177 Goodwill, C.J., Lord, I.J., Jones, R.P.K., 1972. Hearing in rheumatoid arthritis. Ann. Rheum. Dis. 31, 170^173. Gratton, M.A., Schmiedt, R.A., Schulte, B.A., 1996. Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis. Hear. Res. 94, 116^124. Gratton, M.A., Schulte, B.A., 1995. Alterations in microvasculature are associated with atrophy of the stria vascularis in quiet-aged gerbils. Hear. Res. 82, 44^52. Iwai, H., Tomoda, K., Inaba, M., Kubo, N., Tsujikawa, S., Ikehara, S., Yamashita, T., 1995. Evidence of cellular supplies to the endolymphatic sac from the systemic circulation. Acta Otolaryngol. (Stockh.) 115, 509^511. Krams, S.M., Dorschkind, L., Gershwin, M.E., 1989. Generation of biliary lesions after transfer of human lymphocytes into severe combined immunode¢cient (SCID) mice. J. Exp. Med. 170, 1919^1930. Kusakari, C., Hozawa, K., Koike, S., Kyogoku, M., Takasaka, T., 1992. MRL/Mp-lpr/lpr mouse as a model of immune-induced sensorineural hearing loss. Ann. Otol. Rhinol. Laryngol. 101, 82^ 86. Li, H., Borg, E., 1991. Age-related loss of auditory sensitivity in two mouse genotypes. Acta Otolaryngol. (Stockh.) 111, 827^834. Lieber, M.R., Hesse, J.E., Lewis, S., Bosma, G.C., Rosenberg, N., Mizuuchi, K., Bosma, M.J., Gellert, M., 1988. The defect in murine severe combined immune de¢ciency: joining of signal sequence but not coding segments in V(D)J recombination. Cell 55, 7^16. Macht, L., Fukuma, N., Leader, K., Sarsero, D., Pegg, C.A.S., Pillips, D.I.W., Yates, P., McLachlan, S.M., Elson, C., Rees Smith, B., 1991. Severe combined immunode¢cient (SCID) mice: a model for investigating human thyroid autoantibody synthesis. Clin. Exp. Immunol. 84, 34^42. Macrae, D., O'Reilly, S., 1975. On some neuro-oto-ophthalmological manifestations of systemic lupus erythematosus and polyarteritis nodosa. Eye Ear Nose Throat Mon. 36, 721^726. McCabe, B.F., 1979. Autoimmune sensorineural hearing loss. Ann. Otol. Rhinol. Laryngol. 88, 585^589. McMenomey, S.O., Russell, N.J., Morton, J.I., Turne, D.R., 1992. Stria vascularis ultrastructural pathology in the C3H/lpr autoimmune strain mouse: a potential mechanism for immune-related hearing loss. Otolaryngol. Head Neck Surg. 106, 288^295.
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Mosier, D.E., Gulizia, R.J., Baird, S.M., Wilsom, D.B., 1988. Transfer of a functional human immune system to mice with severe combined immunode¢ciency. Nature (Lond.) 335, 256^259. Murphy, E.D. and Roths, J.B. (1979) Autoimmunity and lymphoproliferation: Induction by mutant gene lpr and acceleration by a male-associated factor in strain BXSB mice. In: Rose, N.R., Bibazzi, P.E. and Warner, N.L. (Eds.), Genetic Control of Autoimmune Disease. Elsevier, Amsterdam, pp. 207^220. Nonaka, M., Tomiyama, S., Gotoh, Y., Ikezono, T., Yagi, T., 1992. Vestibular disorders following immune response of the endolymphatic sac in the guinea pig. Ann. Otol. Rhinol. Laryngol. 101, 54^ 57. Ruckenstein, M.J., Mount, R.J., Harrison, R.V., 1993. The MRL-lpr/ lpr mouse: a potential model of autoimmune inner ear disease. Acta Otolaryngol. (Stockh.) 113, 160^165. Schuler, W., Weiler, I.J., Schuler, A., Phillips, R.A., Rosenberg, N., Mak, T.W., Kearney, J.F., Perry, R.P., Bosma, M.J., 1986. Rearrangement of antigen receptor genes is defective in mice with severe combined immune de¢ciency. Cell 46, 963^972. Schulte, B.A., Schmiedt, R.A., 1992. Lateral wall Na, K-ATPase and endocochlear patients decline with age in quiet-reared animals. Hear. Res. 61, 35^46. Takahashi, M., Harris, J.P., 1988. Anatomic distribution and localization of immunocompetent cells in normal mouse endolymphatic sac. Acta Otolaryngol. (Stockh.) 106, 409^416. Theo¢lopoulos, A.N., Dixon, F.J., 1985. Murine models of systemic lupus erythematosus. Adv. Immunol. 37, 269^390. Trune, D.R., Kempton, J.B., Mitchell, C., 1996. Decreased auditory function in the C3H/lpr autoimmune disease mouse. Hear. Res. 95, 57^62. Trune, D.R., Kempton, J.B., Hefeneider, S.H., Bennett, R.M., 1997. Inner ear DNA receptors in MRL/lpr autoimmune mice: potential 30 and 70 kDa link between autoimmune disease and hearing loss. Hear. Res. 105, 57^64. Watanabe-Fukunaga, R., Brannan, C.I., Copeland, N.G., Jenkins, N.A., Nagata, S., 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature (Lond.) 356, 314^317. Willott, J.F. (1991) Aging and the Auditory System: Anatomy, Physiology, and Psychophysics. Singular Press, San Diego, CA.
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Induction of immune-mediated hearing loss in SCID mice by injection of MRL/lpr mouse spleen cells Hiroshi Iwai a; *, Koichi Tomoda b , Naoki Hosaka c , Shigeo Miyashima c , Yuko Suzuka b , Hiroki Ikeda a , Shinryu Lee a , Muneo Inaba c , Susumu Ikehara c , Toshio Yamashita a a
Department of Otorhinolaryngology, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570, Japan b Department of Otorhinolaryngology, Kanazawa Medical University, Ishikawa, Japan c First Department of Pathology, Kansai Medical University, Osaka, Japan Received 28 July 1997; revised 24 December 1997; accepted 31 December 1997
Abstract The MRL/lpr mouse, which has a mutation in the Fas gene encoding a cell-surface receptor for apoptosis, shows an accumulation of abnormal immunocompetent cells and SLE-like disease. It has recently been reported that this mouse also manifests sensorineural hearing loss (SHL) with cochlear pathology at 20 weeks of age. We examined the effects of injecting MRL/lpr spleen cells on the development of SHL in severe combined immunodeficient (SCID) mice, which originally develop neither SHL nor cochlear pathology. Immune-mediated SHL and cochlear pathology were, indeed, transferred to the SCID mice by the injection of spleen cells from the MRL/lpr mice. These findings suggest that cell-mediated immunity is involved in the development of SHL and cochlear pathology. z 1998 Published by Elsevier Science B.V. Key words: Autoimmune disease; Stria vascularis; Immunocompetent cell; Systemic lupus erythematosus
1. Introduction McCabe (1979) described the relationship between inner ear disease and immunological disorders. Clinical observations suggest a correlation between inner ear disorders and autoimmune diseases such as systemic lupus erythematosus (SLE) (Macrae and O'Reilly, 1975), relapsing polychondritis (Cody and Tones, 1971), rheumatoid arthritis (Goodwill et al., 1972), and Behc°et's disease (Brama and Fainaru, 1980). This has prompted investigators to carry out experimental studies on autoimmune mechanisms in inner ear diseases including Meniere's disease (Nonaka et al., 1992 ; Iwai et al., 1995) and sensorineural hearing loss (SHL) (Kusakari et al., 1992; Ruckenstein et al., 1993 ; McMenomey et al., 1992; Trune et al., 1996) in animal models. In the present study, we used two strains of mice : * Corresponding author. Tel.: +81 (6) 992-1001; Fax: +81 (6) 992-1055.
MRL/Mp-lpr/lpr (MRL/lpr) mice established by Murphy and Roths (1979) and severe combined immunode¢cient (SCID) mice established by Bosma et al. (1983). The MRL/lpr mouse, a murine model of SLE, has a mutation in the Fas gene that encodes a cell-surface receptor for apoptosis. The mice show not only the accumulation of abnormal immunocompetent cells due to the escape from Fas-mediated apoptosis but also SLE-like disease accompanied by the production of autoantibodies (Andrews et al., 1978 ; Theo¢lopoulos and Dixon, 1985 ; Watanabe-Fukunaga et al., 1992). Recent works indicate that these mice manifest SHL as a consequence of immune complex deposits containing IgG on the stria vascularis at 20 weeks of age but not at 4 weeks of age (Kusakari et al., 1992). The SCID mouse is known as an ideal recipient for examining the transfer of autoimmune diseases, since it is de¢cient in active B and T cells (Bosma et al., 1983). The SCID defect has been ascribed to the abnormalities in the process of gene rearrangement for immunoglobulin and T cell receptor (Schuler et al., 1986; Lieber et
0378-5955 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 0 1 5 - X
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H. Iwai et al. / Hearing Research 117 (1998) 173^177
al., 1988). Recently, it has been demonstrated that even xenogenic tissue or cells including cells from patients with autoimmune diseases can be successfully transplanted into this mouse (Mosier et al., 1988; Krams et al., 1989 ; Duchosal et al., 1990; Macht et al., 1991). In the present study, we attempt to transfer SHL and cochlear pathology to SCID mice by injecting them with spleen cells from MRL/lpr mice, and show that SHL and cochlear pathology can indeed be transferred to SCID mice. 2. Materials and methods 2.1. Animals Inbred SCID (FOX CHASE C.B17/Icr-scid) and C57BL/6 mice were obtained from CLEA Japan, Inc. (Osaka, Japan). MRL/lpr mice were purchased from SLC Japan, Inc. (Shizuoka, Japan). These mice were maintained under speci¢c pathogen-free conditions in our facilities. 2.2. Transfer of donor spleen cells SCID (H-2d ) mice (7 weeks) were intraperitoneally injected with 4U107 of spleen cells from either MRL/ lpr mice (H-2k ) or C57BL/6 mice (H-2b ) at the age of 20 weeks. For several assays, mice were killed 20 weeks after injection. SCID, C57BL/6 and MRL/lpr mice without injection of spleen cells were also used as controls. 2.3. Auditory brainstem response The auditory function of mice was evaluated by their auditory brainstem response (ABR). Prior to testing, the mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and atropine sulfate (0.5 mg/kg, i.p.). Otoscopic and histopathological examinations revealed that the tympanic membranes or tympanic cavities were normal in all the mice. Needle electrodes were inserted into the occipital, vertex and hindpaw (ground). Click stimuli (0.1 ms) were produced using a sound stimulator (MEM-4104, Nihon Kohden, Tokyo, Japan) and a closed tube sound delivery system sealed into the left ear canal. The frequency and intensity (dBpeSPL) of the stimuli at the ear drum of each mouse were con¢rmed using an oscilloscope and a calibration microphone in a preliminary study. The stimulus was set at 20 stimulations per second. The signal was band-pass ¢ltered at 50 to 3000 Hz and averaged 1000 times. ABRs were obtained by decreasing the stimuli in 4or 6-dB steps from a maximum intensity of 106 dBpeSPL. The threshold was de¢ned as the lowest intensity producing a clearly visible ¢rst peak of wave.
2.4. Statistical analyses Data presented means þ S.D. The statistically signi¢cant di¡erences in the ABR were determined using Student's paired t-test. 2.5. Flow cytometry Spleen cells (1U106 ) were suspended in phosphatebu¡ered saline (PBS) containing 2% fetal calf serum (FCS) plus 0.05% sodium azide, then incubated on ice with FITC-coupled anti-H-2d , anti-H-2k , or anti-H-2b antibody (Ab) (Meiji Institute of Health Science, Odawara, Kanagawa, Japan). The stained cells were analyzed on a FACScan (Beckton Dickinson). 2.6. Immunohistochemistry After splenectomy for £ow cytometry, the mice were subjected to intracardiac perfusion with saline, followed by periodate-lysin-paraformaldehyde (PLP) ¢xation at 4³C and decalci¢cation at 4³C in 5% bu¡ered ethylene diamine-tetra-acetate (EDTA) for 7 days. The temporal bones were frozen in OCT compound (Tissue-Tek II, Lab-Tek Products, Naperville, IL), sectioned into 5 Wm sections horizontally to the long axis of the cochlea, then air-dried at room temperature. The sections were stained with hematoxylin and eosin (H and E). Histopathological examinations revealed that the tympanic cavities are normal in all mice, and showed no sign of cell in¢ltration. Rabbit anti-mouse IgG Ab (Cappel, Organon Teknika Corporation, NC) was also applied to the sections immunohistochemically using a modi¢ed indirect immunoperoxidase technique (Takahashi and Harris, 1988) to detect IgG-bearing cells. The avidin-biotin complex immunostaining method was applied using anti-rabbit ABC kits (Vectastain, Vector Lab, Burlingame, CA). The sections were counterstained with hematoxylin. The care and use of the mice reported on this study were approved by a grant application agency (grants-inaid for scienti¢c research 08671994 from the Ministry of Education, Science and Culture). 3. Results 3.1. Analyses of chimerism Flow cytometric analyses were carried out to examine the chimerism using the spleen cells of [MRL/ lprCSCID] mice. Although SCID mice carry the H2d antigen of the major histocompatibility complex on the lymphocyte cell surface, more than 95% of the spleen cells from the [MRL/lprCSCID] mice were of
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[MRL/lprCSCID] mice (42.8 dBpeSPL) were signi¢cantly higher than in the SCID, C57BL/6 and [C57BL/6CSCID] mice, and reached the level of MRL/lpr mice (40.4 dBpeSPL).
Fig. 1. H-2 typing of spleen cells in MRL/lpr and [MRL/ lprCSCID] mice. Spleen cells of A: MRL/lpr mice (20 weeks old) and B: [MRL/lprCSCID] mice (20 weeks after injection of MRL/ lpr spleen cells) were examined for H-2 type spleen cells. The merge graphs show the £uorescence intensity of spleen cells in the unstained control group (line) or stained groups with either anti-H-2d FITC (dots) or anti-H-2k -FITC (dotted line) monoclonal Ab. Note: More than 95% of the spleen cells from the [MRL/lprCSCID] mice were of donor (MRL/lpr) origin (H-2k ) and were similar to the cells of the MRL/lpr mice.
donor (MRL/lpr) origin (H-2k ) (Fig. 1). SCID mice which had been injected with spleen cells from C57BL/6 also showed more than 90% of spleen cells of donor (C57BL/6) origin (H-2b ) (data not shown). 3.2. Thresholds Similar mean thresholds of ABR were obtained in 20-week-old SCID (20.5 dBpeSPL), 20-week-old C57BL/6 (19.7 dBpeSPL), and [C57BL/6CSCID] mice (20 weeks after injection) (20.5 dBpeSPL), as shown in Table 1. In contrast, the thresholds in the
Fig. 2. Transfer of pathology in stria vascularis of MRL/lpr mice to SCID mice. Sections from the temporal bones of A: MRL/lpr mice, B: [MRL/lprCSCID] chimeras, and C: [C57BL/6CSCID] chimeras were stained with rabbit anti-mouse IgG Ab using an ABC method (U400). The arrows indicate IgG deposits on the endothelial cells in capillaries of stria vascularis (A, B). The sections were counterstained with hematoxylin.
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H. Iwai et al. / Hearing Research 117 (1998) 173^177
176
Table 1 Transfer of MRL/lpr mouse hearing loss to SCID mice Groups
Animals
n
dBpeSPL (mean þ S.D.)
A
SCID C57BL/6 [C57BL/6CSCID] MRL/lpr [MRL/lprCSCID]
5 5 7 10 10
20.5 þ 8.7 19.7 þ 8.3 20.5 þ 9.1 40.4 þ 8.3 42.8 þ 9.6
B*
*P 6 0.01 compared to the A group using a t-test. There was no signi¢cant di¡erence between thresholds in group A or B.
3.3. Immunohistochemistry Our pilot studies using H and E staining revealed that MRL/lpr mice and [MRL/lprCSCID] mice show proliferation of endothelial cells in the capillaries and edema in the stria vascularis, while no histopathologic change was found in the cochleas of the SCID, C57BL/ 6, or [C57BL/6CSCID] mice. Based on these ¢ndings, we carried out immunohistochemical analyses to examine the pathological ¢ndings of the stria vascularis. Endothelial cells in the capillaries were positively stained with anti-IgG Ab from the basal turn to the apical turn in the stria vascularis of all 10 of [MRL/lprCSCID] chimeras as well as in all 10 of the non-treated MRL/lpr mice (Fig. 2A, B). In contrast, no IgG deposits were observed in the cochleas of SCID or C57BL/6 mice (data not shown), or the [C57BL/ 6CSCID] mice (Fig. 2C). 4. Discussion In the present study, we have demonstrated that donor cells are repopulated in [MRL/lprCSCID] and [C57BL/6CSCID] mice, and that both hearing loss and IgG deposits on the endothelial cells of capillaries in the stria vascularis develop in [MRL/lprCSCID] but not [C57BL/6CSCID] mice. Ashany et al. (1992) have reported that the donor (MRL/lpr) spleen cells in [MRL/lprCSCID] mice can continuously produce autoantibodies such as anti-DNA and anti-Sm antibodies. Trune et al. (1997) have recently hypothesized that cell-surface DNA receptors of 30 and 70 kDa size in the cochlea could be masked or down regulated by circulating antibodies in the autoimmune SHLs of both human and MRL/lpr mice. Since no lymphocyte in¢ltration was found in the stria vascularis of [MRL/ lprCSCID] mice and no hearing loss was observed in [C57BL/6CSCID] mice, graft-versus-host (GvH) disease can be excluded ; autoantibodies are likely responsible for the development of the inner ear disease. Although C57BL/6 mice exhibit high frequency (over 4000 Hz) presbycusis with the degeneration of outer
hair cells and spiral ganglion cells in the basal turn of the cochlea from the age of 28 weeks (Li and Borg, 1991 ; Willott, 1991), we used the mice at the age of 20 weeks and applied a clicking stimulus with the signal band-pass ¢ltered at 50 to 3000 Hz in ABR. The [C57BL/6CSCID] mice showed neither the elevated thresholds nor the cochlear alteration observed in both MRL/lpr mice and [MRL/lprCSCID] mice. No abnormal ¢ndings in tympanic membranes or tympanic cavities were found in any mice used in this study. It has been reported that the atrophy of the stria vascularis causes sensorineural hearing loss through impaired ion transport and a decline in the endocochlear potential (Schulte and Schmiedt, 1992; Gratton and Schulte, 1995 ; Gratton et al., 1996). Kusakari et al. (1992) have shown that IgG deposition and degeneration of the stria vascularis in 20-week-old MRL/lpr mice are responsible for dysfunction of the stria vascularis and SHL. In conclusion, SHL and the pathology of the stria vascularis in MRL/lpr mice can be transferred by the injection of MRL/lpr spleen cells to normal mice. It is likely that inner ear diseases due to autoantibodies produced by donor spleen cells carrying the lpr gene develop while recipient mice bear no lpr gene. Acknowledgments This study was supported by grants-in-aid for scienti¢c research 08671994 from the Ministry of Education, Science and Culture, by a research grant for Intractable Diseases (Vestibular Disorders) from the Ministry of Health and Welfare, and by the Katano prize of Kansai Medical University in Japan.
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