J mice

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The Possible Role of Immunologic Injury in the Dysplastic Bony Lesion in LP/J Mice HILARY A. BRODIE, MD, AND RICHARDA. CI-IOLE, MD, PHD An immunohistologic study was performed on temporal bones from 30 LP/J mice and 17 CBA/ J mice to assess the role of immunologic injury in the pathogenesis of dysplastic bony lesions in LP/J mice. Temporal bones were harvested from animals aged 2 to 31 months to evaluate the progression of the disease. As early as 2 months of age, before the onset of bony lesions, the tympanic cavities frequently contained small effusions coating the ossicles and otic capsules that were demonstrated to contain immunoglobulins and pockets of macrophages. Later in the course of the disease, bony lesions grossly and histologically similar to human otosclerosis developed, which stained for immunoglobulins. No similar bony lesions, effusions, cellular infiltrates, or staining for immunoglobulins was detected in the control animals, even in the presence of acute otitis media. This study suggests a role of immunologic injury in the pathogenesis of dysplastic bony lesions in LP/J mice.

Otosclerosis is a disease of bone limited to the stapes and otic capsule that was first described by Valsalva in 1741. Over the ensuing two and one half centuries, the pathogenesis has remained obscure. Although the histopathology has been well described by a number of authors, 1-~ the relationship of the different lesions to each other and to clinical manifestations is still controversial. Numerous pathogenic mechanisms have been suggested as to the etiology of otosclerosis. Two widely held hypotheses include lysosomal enzymatic destruction and repair, a,~,7 and autoimmune injury directed at type II collagen. 8-m These theories are clearly not mutually exclusive. LyReceived from the Department of Otolaryngology-Head and Neck Surgery, University of California at Davis, Davis, California. Presented at the Workshop on Recent Advances in etasclerosis Research, Tenth Midwinter Research Meeting, Association for Research in Otolaryngology, February 4, 1987, Clearwater Beach, Florida. Accepted for publication at that time. Supported by a Medical Scholar Research Grant from the University at" California at Davis School of Medicine and by NIH grant R01 NS 21573-03 A1. Address reprint requests to Dr. Brodie: Otelogy Laboratory, 1159 Surge III, University of California at Davis, Davis, CA 95616. 0196-0709/87 $0,00 + ,25 342

sosomal release of hydrolytic enzymes resulting from unknown stimuli may expose a sequestered antigen (e,g., type II collagen), causing the production of an autoimmune response. Conversely, an autoimmune process may initiate the bone destruction. Stimulation of B cells and T cells could trigger a variety of responses, including the production of autoantibodies with subsequent antigen-antibody complex formation with activation of the complement cascade, This series, in turn, would initiate an inflammatory response with subsequent histiocytic release of hydrolytic enzymes. The absence of a clear understanding of the pathogenesis and natural progression of the dysplastic bony lesions of otosclerosis is partially due to an inability to study the ongoing disease process. The tissue available for study has been limited to postmortem temporal bones and minute bone fragments obtained from stapedectomies for essentially end-stage disease. However, recently the spontaneous development of abnormal bony lesions in the ossicles and otic capsule that are morphologically and histologically similar to the lesions in otosclerosis has been observed in LP/J mice. 11'12 By understanding the sequence of events and pathogenic mechanisms involved in the dysplastic bony lesions in LP/J

BRODIE A N D C H O L E

mice, we might better understand other diseases that result in bony lesions limited to the middle ear and otic capsule. The present study was designed to characterize the dysplastic bony lesions observed in LP/J mice and to assess the role of immunologic injury throughout the progression of the disease. MATERIALS AND METHODS

Tissue Processing Thirty inbred LP/J mice of both sexes and 17 CBA/J mice as control animals were included in the study. Three generations of LP/J mice were used, including the parental stock from Jackson Laboratory (Bar Harbor, Maine) and first and second generation offspring raised in our colony. The mice ranged in age from 2 to 31 months. They were fed a diet of Simonson Lab Chow and given tap water ad libitum. The ambient noise levels in the colony were not more than 40 dB. The mice were killed by intraperitoneal injection of a lethal dose of pentobarbital. The temporal bones were harvested and the ventral bullae wails partially excised. The bullae were filled with B5 fixative (6% mercuric chloride wilh 1.25% sodium acetate in distilled water mixed 9:1 with 37% formalin), carefully avoiding irrigating out the contents of the bullae. The temporal bones were then fixed in B5 fixative for 2 to 4 hours to retain maximal antigenicity, rinsed in phosphate-buffered saline (PBS), and decalcified overnight in Cal x (1.35 N HCI + 0.003 M chelating agent; Fisher Scientific Products, Santa Clara, California). Spleens from six CBA/J mice were also obtained to serve as positive controls for the immunohistologic staining and were exposed to the same fixation and decalcification procedure. The specimens were washed in PBS, dehydrated in graded alcohol, cleared in xylene, and embedded in paraffin using Ames VIP tissue processor. Care was taken to insure that the tissue was not exposed to temperatures greater than 60~ The tissue was serially sectioned at 4 p.m on a rotary microtome, and every fifth section was mounted on a slide coated with white glue. Every fifth slide was then stained with hematoxylin and eosin, coverslipped, and examined under a Olympus BH microscoi3e. The slides were evaluated for middle ear cellular infiltrates, effusions, concretions and dysplastic bony lesions. The bony lesions were assessed for cellularity, vascularity, size, and location. Unstained slides were selected that demonstrated both the peril-

nent findings for each animal as well as representative normal examples to be used for immunohistologic staining.

Immunohistologic Staining An avidin-biotin immunohistologic staining technique was used to assess for the presence of immunoglobulins. The tissue was deparaffinized in xylene and rehydrated through graded alcohols. The slides were placed in 0.5% alcoholic iodine for 3 minutes to remove the mercury crystal precipitates from B5 fixative. The tissue was

rinsed in running water and bleached in 5% sodium thiosulfate for 2 minutes. Following another rinse, the slides were transferred to a solution of 5.6 ml of 30% hydrogen peroxide in 250 ml of methanol for 30 minutes to remove endogenous peroxidase activity. The tissue was rinsed with PBS and placed in a buffer bath for 5 minutes. Four drops of normal rabbit sera diluted 1:5 was applied to the slides to bind the highly charged connective tissue elements to prevent nonspecific binding of the following antibody. The slides were then incubated for 20 minutes. The excess serum was tapped off and four drops of goat antimouse immunoglobulin either IgG, IgM or IgA (Zymed, San Francisco, California; Cooper Biomedical, Malvern, Pennsylvania) diluted 1:200 was applied. These primary antibodies will bind immunoglobulins intracellularly and extracellularly. The tissue was then incubated for 30 minutes. This incubation, as well as the following incubations, was followed by bathing in PBS for 10 minutes. Four drops of biotin-conjugated rabbit anti-goat IgG (Zymed; Vector Laboratories, Burlington, California) diluted 1:500 was applied and incubated for 30 minutes. This antibody preparation binds to any primary antibody remaining on the tissue. Next, four drops of horseradish peroxidase-conjugated biotin-avidin complexes (Zymed; Vector) were applied and incubated for 20 minutes. This solution was prepared 30 minutes before use using equal amounts of avidin and biotinylated peroxidase diluted 1:100. Avidin is an eggwhite glycoprotein that binds with high affinity and specificity to the vitamin biotin, thus linking the peroxidase-containing complex to the secondary biotinylated antibody. A freshly prepared solution of 10 mg of 3,3 diaminobenzidine [DAB) in 16.7 ml of Tris Volume 8 buffer (pH 7.6) mixed with 0.17 ml of 3% hydroNumber 5 gen peroxide was applied to the tissue for 5 minutes. DAB acts as a chromagen resulting in a red- September 1987 343

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BRODIEAND CHOLE brown precipitate at the sites tagged with peroxidase. Finally, the tissue was rinsed in running water, counterstained with hematoxylin, and coverslipped.

Immunohistologic Controls Both positive and negative controls are a critical aspect of immunohistologic research to verify that the technique is working and that results are specific for the antigens under study. Therefore, the following controls were used. The first control was to apply normal goat sera in place of the goat anti-mouse immunoglobulins to delineate the presence of nonspecific binding of goat immunoglobulins. The second control was to omit the primary antibody in the staining procedure and replace it with phosphate-buffered saline to assess the nonspecific background staining of the secondary antibody, the avidin-biotin, and endogenous peroxidase not eliminated during the staining procedure. The third control, spleen tissue, was used as a positive control to assess the ability of the technique to stain for immunoglobulins. And fourth, immunohistologic staining was also performed on temporal bones from CBA/I mice, which are not known to develop dysplastic bony lesions.

RESULTS One of the initial changes observed in the middle ears of LP/I mice, which began at approximately eight weeks of age, was a slight fluid effusion that coated the ossicles, middle ear mucosa, and round window (Fig. 1). Occasional monocytic cells accompanied the fluid layer. With progression of the disease process, a collagenous matrix was formed in the submucosa, and increasing effusions were present with small pockets of monocytes and macrophages. In mice as young as 4 months of age, foci of calcifying osteoid were noted to occur in the collagenous lesions (Fig. 2). The lesions progressed from eosinophilic, relatively acellular osteoid to increasingly cellular lesions with subsequent calcification and basophilic staining (Fig. 3). The lesions were located most frequently on the incus, with 70% of animals over 6 months old displaying lesions on this

ossicle. Twenty-one of the 30 LP/J mice examined developed at least one bony lesion, and only one LPIJ mouse over the age of 4 months was free of bony lesions. With aging, the cellular infiltrate of lymphocytes, monocytes, macrophages, and multinucleated giant cells increased in the middle ear. Concomitant with the increasing infiltrate was the development of concretions in the submucosa of the middle ear that occasionally appeared to be free within the tympanic cavity itself (Fig. 4). All of the LP/J mice developed either middle ear effusions, monocytic infiltrates, or both by 6 months of age. Pelymorphonuclear cells were absent in ears from mice less than 6 months old and were only a small component of the infiltrates in the older animals. In none of the LP/J mice in this series was there ever any evidence of acute otitis media. Cholesterol granulomas were present in some of the older mice. In control CBA/J mice, similar effusions or bony lesions were never encountered. Four of the CBA/J mice had middle ear exudates packed with polymorphonuclear cells and macrephages consistent with acute etitis media.

Immunohistologic Results When the immunohistologic techniques described earlier were applied, the earliest demonstration of staining for immunoglobulins was in the early fluid layer, coating the ossicles, which preceded the bony lesions in young LP/J mice (Fig. 5). The staining was positive for IgG and IgM but not IgA. Staining for IgG and IgM also occurred in the effusions and newly forming lesions (Fig. 6). Immunoglobulins were not detected in the older lesions or normal bone. The temporal bones tested for the presence of IgA had only scattered plasma cells that stained for this immunoglebulin (Fig. 7). The intensity of the immunohistologic staining varied with the immunoglobulin concentration in the tissue and the dilution of the primary staining antibody. Optimal staining to background ratio was obtained using the primary antibody diluted 1:200 and the secondary antibody diluted 1:500.

Figure 1 (top). Earlyfluid exudate(arrows] coveringthe incudomalleolarjointin an 8-week-oldLP/)mouse[paraffinsection, Volume 8 hematoxylinand eosin, x 60). ME, middle ear; T, tympanicmembrane; M, malleus,I, incus. Number 5 Figure 2 (bottom). 'Early focus of osteoid production (arrows) within a collagenouslesion associatedwith the malleus (M) in a LP/Jmouse (paraffinsection, hematoxylinand eosin, x 120). ME, middle ear; S, submucosa;E, middle ear effusion. September 1987 345

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Immunohistologlc Controls When the primary antibody was replaced with normal goat sera or phosphate-buffered saline, no significant staining was observed. Spleen tissue, used as a positive control, demonstrated dark staining for all three immunoglobulins (Fig. 8). The CBA/J middle ears had rare plasma ceils that stained for immunoglobulins; otherwise, uo significant staining was present in any of the normal bony tissue. In the temporal bones with acute otitis media, the exudate did not stain for any of the immunoglobulins. However, plasma cells in the submucosal connective tissue adjacent to the purulent effusion stained for IgG, IgM, and IgA (Fig. 9).

DISCUSSION Based on these results, we believe that immunologicaIly mediated injury may play a significant role in the pathogenesis of the dysplastic bony lesions in LP/I mice. This belief is supported by the observation of an immunoglobulincontaining fluid layer in the middle ear coating the middle ear mucosa and ossicles preceding the development of the bony lesions. This development is followed by increasing infiltrates of monocytes, macrophages, and lymphocytes, which would be predicted with antigen-antibody complex formation and activation of the complement cascade. In addition, prominent staining for IgG and IgM was observed in the early osteoid lesions, and subperiosteal immunoglobulin deposition was seen in areas of maturing lesions. In older calcified lesions, no further staining occurred. Whether these events are secondary to an initial destructive agent or inflammatory process or whether this is the primary pathogenic mechanism is still unknown. What actually initiates the fibroblasts, osteoblasts, or possibly the resting mesenchymai ceils to begin producing the collagenous matrix and osteoid in the developing lesions also has yet to be investigated. In attempting to understand the mediators of dysplastic bony lesions in the middle ear, this

disease process can be compared to other diseases that result in ectopic bone formation in this region. The more prevalent of these disorders include otosclerosis, tympanosclerosis, acute and chronic purulent infection, Paget's disease, and osteogenesis imperfecta, as well as any of the disorders on bone metabolism. No evidence of purulent infection was seen in the LP/J mice, thus eliminating this as a possible etiologic factor. Of these diseases, only otosclerosis and tympanosclerosis are limited to the middle ear and otic capsule. This detail is also true of the disease in LP/] mice. A third disorder, Cogan's syndrome, is reported to have caused dysplastic bone formation limited to the bony labyrinthl:L All three of these diseases have been hypothesized to involve immunologically mediated injury. Cogan's syndrome is manifested by nonsyphilitic interstitial keratitis and vestibule-auditory symptoms. This disease is believed to be a variant of polyarteritis nodosa, which is an autoimmune disease causing systemic necrotizing vasculitis involving small and medium-sized arteries. TM Ectopic bone formation occurs in the semicircular canals and modiolus. Interestingly, bony lesions have also been observed in the modiolus in LP/J mice. ~s The scarcity of temporal bones from patients with Cogan's syndrome and the endstage nature of the lesions reported make direct comparisons of histopathologic findings impossible. The relationship of tympanosclerosis to chronic inflamation has been long established. Recent evidence suggests the possibility of an autoimmnne etiology. White plaques were observed in traumatized tympanic membranes in guinea pigs passively immunized with anti-tympanic memhrane antisera and not in the non immunized animals, l,.17 The dysplastic bony lesions of tympanosclerosis, which occur infrequently on the ossicles of the middle ear, begin in the submucosa, as do the lesions in LP/J mice. The lesions tend to be exophytic, as are those in LP/] mice. Human otosclerosis and tiffs murine disease share many characteristics. ~'~2 Both are inherited disorders producing progressive toss of auditory function and bony lesions with gross and

Figure 3 [top). Light micrograph demonstratingthe three types of dysplasticbony lesions seen in the LP/J mouse: early acellular collagenous lesion (urrawheadl, intermediate lesion consisting of dense uncalcifiedosteoid (arrowheads, (note increasing cellularityJ, and mature, calcified lesion contiguouswith the incus (I} paraffinsection, hematoxylinand eosin, x 90). M, malleus; E, middle ear effusion;EAC,external ear canal. Volume 8 Figure 4 (bottom). Concretions(C) and cellular infiltrate in the middle ear of mature LP/J mouse. Note monocytes(Me), Number 5 lymphocytes[L},and multimmleatedgiantcell (arrow)(paraffinsection,hematoxylinand eosin, x 240].E,middleear effusion. September 1987 347

DYSPLASTIC BONY LESIONS tN LP/J MICE histologic similarity that result in ossicular fixation. The disease in LP/J m i c e differs f r o m otosclerosis in several respects. The diseases differ in the i n c i d e n c e of location of the lesions. Middle ear effusions are a c o m m o n finding in LP/J m i c e and are not k n o w n to be related to the h u m a n disease. H a i r cell loss is m u c h more p r o n o u n c e d in LP/J m i c e t h a n in otosclerosis. Also, the sequence of events on the f o r m a t i o n of lesions in LP/J m i c e differs from the c o m m o n l y h e l d theory in otosclerosis. Murine lesions a p p e a r to pass through three stages: 1) a m o r p h o u s collagenous matrix deposition, 2) osteoid f o r m a t i o n with increased cellularity, and 3} calcification a n d bone remodelling. Otosclerosis has b e e n frequently categorized into two groups: 1) vascular, highly cellular, eosinophilic lesions t e r m e d o t o s p o n giotic; a n d 2} relatively avascular h y p o c e l l u l a r lesions retaining the n a m e otosclerotic. Otospongiotic lesions are h y p o t h e s i z e d to precede otosclerotic lesions~e; h o w e v e r , this has not been clearly established. Recent e v i d e n c e suggests that the two types of lesion m a y not be sequential at all. S c h u k n e c h t and Barber ~9 exarnined 117 temporal bones containing otosclerotic lesions anterior to the oval w i n d o w . They d i v i d e d the lesions by size into three groups: 1~ less t h a n 2 m m ; 2) 2 to 4 ram; a n d 3) greater t h a n 4 mm. T h e lesions then were assessed for vascularity. T h e researchers f o u n d that v a s c u l a r i t y correlated directly with size a n d that n o n e of the 28 lesions less t h a n 2 m m w e r e highly vascular. Their findings are incongruous with the h y p o t h esis that the spongiotic lesions are the p r e c u r s o r lesions, If the spongiotic lesions p r e c e d e the sclerotic lesions, it would be unlikely that none of the small lesions were highly v a s c u l a r for "spongiotic" and that o n l y one of 33 lesions were

avascular or "sclerotic." Another argument against the traditional c o n c e p t of spongiotic lesions preceding sclerotic lesions w a s p r e s e n t e d b y P a r a h y a n d L i n t h i c u m . ~'2u O b s e r v i n g t h a t h y a l i n i z a t i o n occurred adjacent to o t o s p o n g i o t i c lesions but not to otosclerotic lesions, they argued it w o u l d be unlikely that the h y a l i n i z a t i o n w o u l d disappear but rather that the t w o lesions are i n d e p e n dent of one another. Due to the lack of u n d e r s t a n d i n g of the progression of the lesions in otosclerosis, a c o m p a r ison of d e v e l o p m e n t a l sequence w i t h the m u r i n e disease is p r e m a t u r e . A possible role of i m m u n o l o g i c injury in otosclerosis has b e e n p r o p o s e d but r e m a i n s u n s u b stantiated. Yoo et al. 8-~0 p r o p o s e d an interesting animal m o d e l in w h i c h rats were i m m u n i z e d to type II bovine collagen. T h e y r e p o r t that the animals d e v e l o p e d s p o n g i a t i c changes in the labyrinthine bone. R e - e x a m i n a t i o n of the a n i m a l m o d e l has not s u p p o r t e d the earlier observations. 2~ W h e n the m e c h a n i s m s r e s p o n s i b l e for the production of d y s p l a s t i c b o n y lesions in the m i d d l e ear of the LP/J m o u s e and the reasons this process is limited to t h e temporal bone are better u n d e r stood, p e r h a p s we c a n better u n d e r s t a n d diseases s u c h as otosclerosis, t y m p a n o s c l e r o s i s , a n d Cogan's s y n d r o m e , which, a l t h o u g h different diseases, m a y share c o m m o n u n d e r l y i n g p a t h o g e n i c mechanisms.

References 1. Guild S. Histologic ot0sclerosis. Ann Otol Rhinol Laryngol 1944;53:246-266 2. Kelemen G. Lithicum F: Labyrinthine otosclerosis. Acta Otolaryngol [Suppl] (Stockh) 1969;253-68

Figure 5 (top left). Immunobistologic staining for lgM in the early fluid exudate {arrowheads) covering the malleus (M) and atic capsule (OC) (paraffin section, avidin-biotin technique, hematoxy]in counterstain; original magnification • 60), Figure 6 Itop right). Photomicrograph demonstrating multiple dysplastic bony lesions surrounding the incudomalleolar joint. The middle ear effusion (E), the early collagenous lesion (EL), and the subperiosteal margin (arrowheads) of the intermediate lesion (IL) all are stained for IgG. Note the absence of staining in the mature calcified lesion (CL) (paraffin section, avidin-biotin technique, hematoxylin, counterstain; original magnification • 60). EAC, external auditory canal; M, malleus; l, incus. Figure 7 (second row). A, middle ear with effusion and cellular infiltrate in the LP/J mouse stained for IgA. Note lack of staining in the middle ear effusion (E) {paraffin section, avidin-biotin technique, hematoxylin, counterstain, x 60). M, mal!eus; I, incus. B, higher magnification of the submuscosal connective tissue in A. Note plasma cell which stains for the presence of lgA {original magnification • 240).

American Journal of Otoia ryngoJogy 348

Figure 8 [third row, left). Spleen from a CBA/J mouse stained for IgG. Note the presence of stained lymphocytes (paraffin section, avidin-biotin technique hematoxylin counterstain; original magnification x 240). Figure 9 (third row, right, and battom). A, middle ear of a CBA/J mouse with acute otitis media. Observe the absence of staining in the purulent exudate (PE)(paraffin section, avidin-biotin technique, hematoxylin counterstain; original magnification x 60). M, malleus; I, incus. B, higher magnification photomicrograph of the purulent exudate next to the middle ear mucosa seen in A. Note the stained plasma cells {arrows) in the middle ear mucosa (MM) adjacent to the nonstaining purulent exudate (PE) (paraffin section, avidin-biotin technique, hematoxylin eounterstain; original magnification x 240).

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DYSPLASTIC BONY LESIONS IN LP/J MICE 3. Bretluu P: Otosclerosis: Electron microscopic studies of biopsies from the labyrinthine capsule. Arch Otolaryn~nl 1971;93:551-551 4. Bretlau P, Chevance LG, Causse J, et el: Bane resorption in otospongiosis. Am J Otol 1982;3:284-289 5. Chavance LG, Bretlau P, ]orgensen MB, at ah Otosclerosis: An electron microscopic and cytochemical study. Acta Otolaryngo[ (Stockhl 1970;(suppl 272):1--44 6. Parahy C, Linthicum FH: Otasclerosis and otospongIosis: Clinical and histological comparisons. Laryngoscope 1984;94:508-512 7. Lhn DJ, Saunders WH: Otosclerotic stapes: Morphological and microchemical correlates--An Blectron microscopic and x-ray analytical investigation, Ann Oto[ Rhinol l,aryngol 1977;86:525-541 t]. 5'00 TJ, Townes AS, Stuart JM, et ah Type II collagen autoimmunity in otosclerosis and Meniere's disease. Sciance 1982;217:1153-1155 9. u TI, Tomoda K, Kang AH, e t a h Type I[ collageninduced autoimmune otospongiosis: A preliminary report. AIll'l Otol Rhinol Laryngol 1983;92:103-108 10. Yoo IT: Etlopathogenesis o[ otoscterasis: A hypothesis, Amx Otol Rhinol Laryngol 1984;93:28-33 1I, Chole RA, Henry If.R: Otasclerotic Lesions in the LP/J Mouse, Science 1983;221:881-882 12. Chela RA, Henry KR: Ossicular and otic capsular lesions in I.P/J mice, Ant10tol Rhinol Laryngol 1985;94:366372

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13, Rarey KE, Bicknell JM, Davis LE; Intra[abyrinthine osteogenesis in Cogan's syndrome. Am J Otolaryngal 1985;7:387-390 14. Chosen BD, Bluming AZ, Alroy J: Cogan's syndrome: A systemic vasculitis. Am J Med 1976;60:549-555 15, Chole RA, Tinling SP: Fine morphology of bony dysplasia of the murine ear: Comparisons with otosclerosis. Am ]" Otolaryngol 1987;8:325-331 16. Schiff M, Catanzaro A, Ryan AF, et ah Tympanosclerosis: A theory of pathogenesis. Ann Oral Rhinol Laryngol 1980;70(Suppl 89):1-16 17. Poliquin J, Catanzaro A, Robb ], at ah Adaptive immunity of the tympanic membrane. Am J Otolaryngo11981;2:9498 18. Nager GT: Histopathology of otasclerosis. Arch Otolaryngol 1969;89:341-363 19. Schuknecht HF, Barber W: Histologic variants in otosclerosis. Laryngoscope 1985;95:1307-1317 2(1, Parahy C, Linthicum FH: Otoscleros[s: Relationship of spiral ligament hyalinization to sensorineural hearing loss. Laryngoscope 1983;93:717-720 21. Harris ]P, Woolf NK, Ryan AF; A reexamination of experimental type II collagen autaimmunity: Middle and inner ear morphology and function, Ann Otol Rhinol Laryngo] 1986;95:176-180