ObjectiveslHypothesis: Sudden hearing loss (HL) can be caused by autoimmune disorders localized to the inner ear or secondary to systemic immune dis- eases. Studies in autoimmune animal strains showing HL have reported changes in the cochlear stria vas- cularis. The authors investigated the presence of an- tiendothelial cell antibodies (AECA) to see if immune- mediated vasculitis may play a role in human sudden HL. Study Design: A prospective study in patients with sudden HL. Methods: Fifteen  consecutive pa- tients (mean age, 32 y) affected by sudden HL and 14 normal subjects were included. Patients with familial deafness and metabolic diseases were excluded. Ex- tensive audiovestibular,  imaging,  microbiological, immunological, and routine examinations were per- formed. AECA were detected on rat kidney tissue sec- tions on the sera collected at -20°C. Results: AECA were positive in 8 of 15 patients (53%) (2 of 5 men and 6 of 10 women), thus differing significantly from the normal control  population, in which  only 2 of 14 tested AECA positive (P = .023). Conclusions: In pa- tients with  sudden HL, immune-mediated vascular damage can have  a  pathogenetic  role and AECA might represent a  serological marker of vasculitis. Key  Words: Sudden hearing loss, immune-mediated vascular damage, anti-endothelial cell antibodies

Cadoni, Gabriella (ORCID:0000-0001-6702-2616)

De Santis A.

Fetoni Ar

Marinelli L

Ottaviani, Fabrizio

English

Cadoni, Gabriella (ORCID:0000-0001-8244-784X)

PubliCatt

Thv Laryngoscope Lippincott Williams & Wilkins, Inc., Philadelphia 0 1999 The American Laryngological, Rhinological and Otological Society, Inc. Anti-Endothelial Autoantibodies in Patients With Sudden Hearing Loss Fabrizio Ottaviani, MD; Gabriella Cadoni, MD; Luca Marinelli, MD; Anna Rita Fetoni, MD; Antonella De Santis, MD; Anna Romito, MD; Paola Vulpiani, MD; Raffaele Manna, MD ObjectiveslHypothesis: Sudden hearing loss (HL) can be caused by autoimmune disorders localized to the inner ear or secondary to systemic immune dis- eases. Studies in autoimmune animal strains showing HL have reported changes in the cochlear stria vas- cularis. The authors investigated the presence of an- tiendothelial cell antibodies (AECA) to see if immune- mediated vasculitis may play a role in human sudden HL. Study Design: A prospective study in patients with sudden HL. Methods: Fifteen consecutive pa- tients (mean age, 32 y) affected by sudden HL and 14 normal subjects were included. Patients with familial deafness and metabolic diseases were excluded. Ex- tensive audiovestibular, imaging, microbiological, immunological, and routine examinations were per- formed. AECA were detected on rat kidney tissue sec- tions on the sera collected at -20°C. Results: AECA were positive in 8 of 15 patients (53%) (2 of 5 men and 6 of 10 women), thus differing significantly from the normal control population, in which only 2 of 14 tested AECA positive (P = .023). Conclusions: In pa- tients with sudden HL, immune-mediated vascular damage can have a pathogenetic role and AECA might represent a serological marker of vasculitis. Key Words: Sudden hearing loss, immune-mediated vascular damage, anti-endothelial cell antibodies. Laryngoscope, 1091084-1087,1999 INTRODUCTION Sudden hearing loss (HL) is defined as a sensorineu- ral HL of 30 dB or more over at least three contiguous audiometric frequencies that develops over a period of a few hours to  3 days' and whose etiology can be found only in 10% to 15% of patients.2 McCabe3 reported in 1979 that a group of patients with HL responded well to  immuno- suppressive treatment with steroids and cyclophospha- mide and described autoimmune inner ear disease as a clinical entity. Recently there has been considerable in- From the Departments of Otorhinolaryngology (F.o., c.c., L.M., A.H.F.) and Internal Medicine (A.D.s., AX. ,  r.v., R . M . )  of the Catholic University of the Sacred Heart, Rome, Italy. Editor's Note: This Manuscript was accepted for publication March 31, 1999. Send Reprint Requests to Fabrizio Ottaviani, MD, Institute of Oto- rhinolaryngology of the Catholic University of the Sacred Heart, Largo Agostino Gernelli, 8. 1-00168 Rome, Italy. E-mail: iclot@rm.unicatt.it vestigation into the relationship between immune system and sensorineural HL. Harris4 demonstrated the inner ear's capacity to  respond to local antigenic challenge and to produce systemic immunization. Local and systemic immunization was documented experimentally after ad- ministration of nonspecific4-6 or inner-ear-specific anti- gens.7~~ Several authors have described a significant increase of specific HLA class I1 genes,9 elevated levels of circulat- ing immune complexesl0.l' and the detection of inner ear autoantibodies in patients with progressive or sudden sen- sorineural HL.12-14 These data are consistent with an immune-mediated hypothesis of sudden HL. Neverthe- less, the existence of autoimmune sudden HL has not been demonstrated yet and the response to immunosuppressive treatment cannot be used as a diagnostic criterion. Evidence also indicates that sensorineural HL can be a manifestation of systemic autoimmune diseases15 such as Cogan's syndrome,lG temporal arteritis,'7 Wegener's granulomatosis,lS polyarteritis nodosa,'g and systemic lu- pus erythematosus.2O Although the vascular damage fre- quently observed in lupus erythematosus and other vas- culitides is probably mediated by immune autoantibodies against endothelial cells (AECA) have also been implicated in this process and could have a pathoge- netic role.22 Indeed AECA may damage endothelial cells via a complement-mediated or antibody-dependent cellu- lar cytotoxic mechanism, or by upregulation of adhesion molecules and increased secretion of proinflammatory and chemoattractant cytokines.22 The aim of this paper is to verify if a vascular damage could have a pathogenetic role and if AECA might represent a serological marker of vas- culitis in patients affected by idiopathic sudden HL. PATIENTS AND RlETHODS Fifteen consecutive patients (mean age, 32 y; age range, 14-50 y; 10 females, 5 males) affected by sudden HL were in- cluded. Patients with familial deafness and metabolic diseases were excluded. Table I shows the various types of HL. All patients underwent a routine general physical examination. Extensive audiovestibular (pure-tone average, speech discrimination, im- pedance audiometry, auditory brainstem response, electronystag- mogram, computed dynamic posturography), imaging (magnrtic resonance imaging, epi-aortic-vessels doppler ultrasound), micro- Laryngoscope 109: July 1999 Ottaviani et al.: Autoantibodies in Sudden Hearing Loss 1084 TABLE 1. Clinical Characteristics and Laboratory Data of 15 Patients With Sudden Hearing Loss Patient No Flat Loss High-Tone Loss U-Shaped Loss Low-Tone Loss ESR CRP CIC ACA ANA Anti-DNA d s  AECA ~ ___  - __ ~ _____ ___ + 1 Severe - - - High High Normal - - - (bilateral) + - - - - Normal Normal Normal - 2 Severe - 3 Severe - - - Normal Normal Normal - + - - 4 - Severe - - Normal Normal Normal - NA - 5 - Severe - - Normal Normal Elevated - + - 6 - Moderate - - Normal Normal Elevated - + - 7 Moderate - - - Normal Normal Normal - - a - - - Moderate Normal Normal Normal - - - 9 - - - Moderate Normal Normal Normal - - - - 11 - - Moderate - Normal Normal Elevated - + - - 12 _. - Moderate - Normal Normal Normal - 13 Mild - - - Normal Normal Elevated - - - 14 - - - Mild Normal Normal Elevated - + + 15 Severe - - - Normal Normal Normal - - - + + + - - + 10 - - - Moderate Normal High Elevated - + - - - - - + - + ~ ____ -~ CPR C reactive protein, ESR = erythrosedimentation rate, CIC = circulating irnmunocomplexes, ANA = antinuclear antibodies. AECA = antiendothelial cell antibodies, ACA = anticardiolipin antibodies, NA = not available biological (herpesvirus, cytomegalovirus, influenza and parain- fluenza virus, Epstein-Barr virus, coxsackie virus, hepatitis B and C virus, Venereal Disease Research Laboratory), immunolog- ical (antinuclear, anti-DNA double stranded, anticardiolipin, an- tiendothelial cell antibodies, immunocomplexes), and routine ex- amination (blood hemoglobin and leukocyte count, erythrocyte sedimentation rate, serum gamma-globulin, C reactive protein) work-ups were performed. Median follow-up was 4 months (range, 2-7 mo). A 1-year follow-up for AECA detection has been established. Fourteen normal subjects (7 men and 7 women; mean age, 29 y; age range, 17-45 y )  without history of HL o r  autoimmune or metabolic diseases were included as controls. Scsra were drawn from 15 patients and from 14 control subjects and collected at  -20°C. All samples of blood were ob- tained 1 day after the date of hospitalization and 3 days or less after the onset of sudden HL. The AECA detection was performed as described by Tan and Pearsoil':' and is based on the principle of indirect fluorescent antibody technique. The specific antibodies were detected on rat kidney tissue sections, which were coated onto the slides as monolager (Biogenetics, Padua, Italy). In the first step, the hu- man serum, diluted at 1:20 with PBS buffer solution, was brought into contact with the antigen substrate. Negative and positive human controls were provided with the kit (Biogenetics). The antibody, present in the test serum, attached to the antigen to form an antigen-antibody complex. Unbound material was re- moved by washing. In the next step, the antigen-antibody com- plexes were marked by specific antihuman polyvalent globulin conjugated to fluorescein (FITC). The positive reaction was shown by a grren fluorescence peritubular vessel in kidney sections and observed in inverted fluorescence microscope. 2 test (Statview 4.0-Macintosh) was performed to compare the patient and control groups. RESULTS Sudden HL was unilateral in 14 cases; only one case came to our observation with a bilateral HL, which was flat and severe. In unilateral cases, HL was severe in five cases, moderate in seven, and mild in two. No character- istic shape was detected, but U-shaped and low-tone losses were more frequently associated with less severe HL (Table I). Audiological tests showed the presence of recruitment and the absence of pathological adaptation in all cases. A retrocochlear lesion was excluded by means of impedance tests and auditory brainstem response when possible (auditory threshold) and by imaging techniques. Normal vestibular reflectivity was present in all patients. Anticardiolipin autoantibodies were negative in all patients. Antinuclear autoantibodies were positive in 6 of 14 patients (one missing) (42.8%). AECA were positive in 8 of 15 patients (53%) (2 of 5 males and 6 of 10 females), thus significantly differing from the normal population, in which only 2 of 14 tested AECA positive ( P  = .023). Al- though the number of patients was small, we did observe that severe hearing losses tended to be associated with positive AECA ( 5  of 61, whereas mild and moderate hear- ing losses were more frequently associated with negative AECA (6 of 9). Titers examined for IgM antivirus were negative in all patients. The relationship among AECA and other parameters (immunological or otherwise) is showed in Table I. After corticosteroid immunosuppressive therapy (methylprednisone 1 mgkg per day for 1 mo) no AECA- positive patients had a significant recovery, but they did show a reduction of inflammatory parameters (erythro- cyte sedimentation rate, C reactive protein) without wors- ening of hearing or new symptoms. DISCUSSION The evidence in humans of coexisting sensorineural HL and systemic autoimmune disease has suggested the Laryngoscope 109: July 1999 Ottaviani et al.: Autoantibodies in Sudden Hearing Loss 1085 possibility of isolated forms of immune-mediated deaf- ness. The results of treatment of individual cases with immunosuppressive agents also support the hypothesis that some cases of so-called “idiopathic” sudden HL are immune-mediated.12~24 Response to immunosuppressive treatment can be demonstrated by a reduction of inflam- matory parameters and by the absence of other systemic or local manifestations and not only by a recovery of hear- ing. Moreover, long-term studies are necessary to deter- mine if autoimmune sudden HL is an initial local mani- festation of a systemic autoimmune disease or if it is an isolated entity. This is the first report of AECA in sudden HL. The pathogenetic potential of AECA has been demonstrated in vascular diseases, especially for immune-mediated vascu- litis.26 For this reason, and because AECA were present in 53% of our patients (8/15), we suspect that AECA induced vascular damage of the inner ear in these cases. In fact, AECA, reacting against available surface an- tigens, may damage endothelial cells via a complement- mediated or antibody-dependent cellular cytotoxic mech- anism, or by upregulation of adhesion molecules. Although AECA do not display any disease specificity, their absence in diseases such as mixed essential cryoglob- ulinemia, in which vascular damage is clearly mediated by other immune effectors, suggests that these antibodies represent a primary event rather than merely a secondary immune response against antigens exposed in the course of the vascular inflammatory process.22 The hypothesis that vascular damage might have a pathogenetic role in immune-mediated sudden HL is sup- ported by labyrinthine fibrosis and cochlear ossification found in patients affected by autoimmune diseases and sudden HL,26 and by the observation of the breakdown of stria vascularis blood-labyrinth barrier in C3H/lpr auto- immune disease mice that have elevated auditory brain- stem response thresholds during the active phase of the disease. Some researchers suggest that either a humoral mechanism of injury or a direct injury to the endothelial cells is involved and that the breakdown of tight junctions between stria vascularis endothelial cells may be one eti- ologic mechanism of sensorineural HL in autoimmune disease.27 In our AECA-positive patients, the absence of HL recovery after steroid therapy might be explained by irre- versible vascular damage of the inner ear, as described in patients with sudden HL and systemic vasculitis.26 There- fore, the immunosuppressive treatment cannot be utilized as the only diagnostic criterion. The finding in our patients of antinuclear antibodies (42.8%) and circulating immunocomplexes (40%) also sup- ports the involvement of immune mechanisms in this dis- ease and is consistent with the experimental model of autoimmune inner ear disease of C3H/lpr mouse where significantly elevated threshold shifts correlated with de- velopment of antinuclear antibodies and circulating im- munocomplexes.28 A systemic disease should produce bilateral rather than unilateral ear symptoms over time. In fact, unilat- eral symptoms more often result from primary ear disease (e.g., eighth nerve schwannoma and chronic otitis media), but, as in ocular autoimmune diseases,zl”x30 the microvas- cular damage may be unilateral. Moreover, the finding of AECA supports a vascular hypothesis and should encour- age investigation about the existence of an immune sys- temic disease. The preponderance of women in our group of patients with sudden HL is noted in other autoimmune diseases. This link to the female sex may be influenced by endocrine effects on the immune system, but the cause remains unclear. CONCLUSION The present data suggest an immunological mecha- nism in the pathogenesis of some cases of idiopathic sud- den HL. Detection of serological nonspecific inner-ear au- toantibodies, such as AECA, in patients with sudden HL may be useful in clinical practice to identify a subset of sudden HL patients whose inner ear damage may be caused by immune-mediated vasculitis. The diagnosis of AECA-related autoimmune sudden HL should support an aggressive immunosuppressive treatment to avoid the worsening of HL or the onset of new symptoms. Further studies on a larger population are needed to support this hypothesis and to demonstrate the possible role of auto- antibodies such as AECA in the development of immune- mediated sudden HL. BIBLIOGRAPHY 1. Wilson WR, By1 FM, Laird N. The efficacy of steroids in the treatment of idiopathic sudden hearing loss. Arch Otolar- yngol 1980;106:772-776. 2. Mattox DE, Lyles CA. Idiopathic sudden sensorineural hear- ing loss. Am J Otol 1989;10:242-247. 3. McCabe BF. Autoimmune sensorineural hearing loss. Ann Otol Rhinol Laryngol 1979;88:585-589. 4. Harris JP.  Immunology of the inner ear: response of the inner ear to antigen challenge. Otolaryngol Head Neck Surg 5. Yo0 TJ, Yazawa Y, Floyd R, Tomoda K. Antibody activity in perilymph from rats with type I1 collagen-induced autoim- mune ear disease. Ann Otol Rhinol Laryngol 1984;13:1-2. 6. Tomoda K, Maeda N, Yamawaki T. Immunologically induced experimental hydrops. In: J B  Nadol, ed. Meniere’s Diseasc~. Amsterdam: Kugler & Ghedini; 1989: 165-172. 7. Harris JP. Experimental autoimmune hearing loss. Laryngo- scope 1987;97:63-76. 8. Soliman AM. Experimental autoimmune ear disease. Laryn- goscope 1989;99:188-193. 9. Cao MV,Thonnard J, Deggouj N, et al. HLA class II- associated genetic susceptibility in idiopathic progressive sensorineural hearing loss. 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Anti-endothelial autoantibodies in patients with sudden hearing loss.

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Anti-endothelial autoantibodies in patients with sudden hearing loss.

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