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Is there a relationship between myeloperoxidase activity and conductive hearing loss in chronic otitis media complicated by cholesteatoma?

Abstract

We conducted a prospective, controlled study of patients with chronic otitis media and cholesteatoma (1) to examine the expression of myeloperoxidase (MPO) using immunohistochemical staining techniques and (2) to investigate the relationship between MPO activity and the degree of conductive hearing loss in these patients. Our study population included 51 adults--26 men and 25 women, aged 18 to 58years (mean: 37.5)--who had been diagnosed with chronic otitis media and cholesteatoma by physical examination and computed tomography (study group). Another 30 patients--13 men and 17 women, aged 18 to 52 years (mean: 32.7)--who had chronic otitis media without cholesteatoma served as the control group. Following audiometric evaluations, all patients underwent appropriate surgery. Postoperatively, cholesteatoma samples were analyzed by immunostaining-for MPO positivity as a marker for acute inflammation. We found that MPO activity was present in all 51 study patients (100%) but in only 10 controls (33.3%); the difference was statistically significant (p < 0.01). In the study group, the degree of MPO activity was slight in 6 patients (11.8%), moderate in 24patients (47.1%), and intense in 21 patients (41.2%), while in the control group, all 10 MPO-positive cases showed only a slight degree of activity. We also found a statistically significant association in the study group between the degree of MPO activity and the degree of conductive hearing loss (ff = 13.518; p < 0.001). We encourage further study of all steps in the process of cholesteatoma formation.

Introduction

Chronic otitis media is generally associated with some degree of hearing loss, which is often the patient's chief complaint. The hearing loss is usually conductive, resulting from tympanic membrane rupture and/or changes in the ossicular chain secondary to fixation or erosion caused by the chronic inflammatory process. (1) Chronic otitis media is often accompanied by cholesteatoma. (2) Regardless of its origin, cholesteatoma is characterized by an accumulation of keratin debris, and it can cause local bone resorption. (2,3)

Neutrophils are phagocytes that play an important role in acute inflammation. (4) When a foreign body (e.g., a bacterium, fungus, virus, or tumor cell) invades tissue, many chemical substances are released from the cytoplasmic granules of neutrophils during phagocytosis. Myeloperoxidase (MPO) reacts with hydrogen peroxide ([H.sub.2],[0.sub.2]), which is itself a cytotoxic oxidant; its toxicity is accelerated by the reaction with MPO and chloride. (5,6) The complex formed by MPO and [H.sub.2][0.sub.2] oxidizes many substances, and it produces hypochlorous acid (HCIO) and chlorine monoxide (CIO). HCIO and CIO are strong oxidants that exert a defensive action against invading bacteria. It is known that patients with a genetic deficiency of MPO experience recurrent infections and that the neutrophils in their blood exert poor bactericidal activity. (7) On the other hand, reactive oxygen species (ROS), including free

radicals, have the potential to disturb cellular function and structure, (8) and ROS are involved in the development of inflammatory conditions. (9-11)

In this article, we describe our study (1) to examine the expression of MPO in chronic otitis media complicated by cholesteatoma using immunohistochemical staining techniques and (2) to investigate the relationship between MPO activity and the degree of conductive hearing loss in these patients.

Patients and methods

For this prospective investigation, we drew our study population from among all adults who had presented with chronic otitis media at the ENT Department at the Dr. Lutfi Kirdar Kartal Training and Research Hospital in Istanbul over a 2-year period. Following audiometric evaluation, we excluded all patients whose results identified a sensorineural or mixed hearing loss. We also excluded patients who had a history of ear surgery.

A total of 81 patients met our eligibility criteria. Of these, 51 patients--26 men and 25 women, aged 18 to 58 years (mean: 37.5)--had been diagnosed with cholesteatoma by physical examination and computed tomography, and they were assigned to the study group. The remaining 30 patients--13 men and 17 women, aged 18 to 52 years (mean: 32.7)--had chronic otitis media without cholesteatoma, and they were assigned to the control group.

All patients were scheduled for surgery. Surgery in the study group was performed according to the extent of the cholesteatoma. All the controls underwent a type I tympanoplasty.

In the study group, samples of cholesteatoma tissue obtained intraoperatively were sent to our pathology department, where they were analyzed immunohistochemically for MPO content, which serves as a marker for inflammation. The samples were fixed in 10% neutral buffered formalin and embedded in paraffin in sections 2 to 3 pm thick, and then they were incubated at 37[degrees]C for 24 hours. Sections were deparaffinized with xylene and rehydrated gradually with graded alcohol solutions. Heat-induced antigen retrieval was carried out in a microwave oven with a 10-mmol/L citrate buffer that had a pH of 6.0. The sections were immersed in 3% [H.sub.2][0.sub.2] for 15 minutes. Subsequently, they were incubated with the primary antibody to MPO at 1:4,000 dilution.

[FIGURE OMITTED]

In the immunohistochemical study, MPO positivity was monitored during a cytoplasmic dying process. The degree of inflammation in the cellular cytoplasm was graded as slight, moderate, or intense, depending on the intensity of the color brown of the MPO stain.

In the control group, samples of middle ear tissue were analyzed immunohistochemically for MPO content. Then we compared the preoperative audiometric findings and MPO activity (figure) in the two groups.

Statistical analysis. Statistical analysis was performed with the Statistical Package for the Social Sciences software (v. 10 for Windows; SPSS; Chicago). Descriptive statistical methods (frequency) were employed during the evaluation of the study data while the chi-square ([chi square]) test was used to compare qualitative data. Results were assessed with a 95% confidence level, and statistical significance was defined as p < 0.05.

Ethical considerations. Patients were told the scope and purpose of the study, and their informed consent was obtained.

Results

We found that MPO activity was present in all 51 study patients (100%) but in only 10 controls (33.3%); the difference was statistically significant (p < 0.01) (table 1). In the study group, the degree of MPO activity was slight in 6 patients (11.8%), moderate in 24 patients (47.1%), and intense in 21 patients (41.2%). In the control group, all 10 MPO-positive stains showed only a slight degree of activity.

In the study group, the degree of conductive hearing loss was evaluated as mild (20 to 40 dB), moderate (41 to 60 dB), or severe (61 to 80 dB). We found a statistically significant association between the degree of MPO activity and the degree of conductive hearing loss in the study group ([chi square] = 13.518; p < 0.001) (table 2).

Discussion

Only a limited number of studies have been performed on enzymatic antioxidants and cholesteatomas. Immunohistochemical and quantitative studies have demonstrated elevated levels of cytokines in cholesteatomas. (10,11) A depletion of enzymatic antioxidants has also been observed in cholesteatoma; in a study of 13 patients, Eskiizmir et al found various levels of superoxide dismutase, catalase, and glutathione peroxidase in cholesteatoma samples. (12) Also, the active role of lipopolysaccharides in the development and course of cholesteatomas was reported in previous studies. (13-16)

In a guinea pig experiment carried out by Watanabe et al, the presence of MPO was found in the lateral wall of the cochlea and in the cells of the organ of Corti 48 hours after bacterial lipopolysaccharide was injected into the middle ear. (17) The authors reported that MPO in these subjects had a negative effect on hearing.

Johnson et al perfused the renal arteries of rats with MPO and found glomerular damage due to the reaction between the MPO and [H.sub.2][0.sub.2]. (18) In another rat experiment, Lefkowitz et al found a relationship between MPO and the development of arthritis. (19) Kinoshita and Tomiyama showed that direct injection of the antigen into the endolymphatic sac region of guinea pigs caused expression of MPO at the endolymphatic sac, organ of Corti, and crista ampullaris. (20)

In our study, we found a statistically significant difference in the degree of MPO activity between the study and control groups, as well as an association between the intensity of MPO activity and the degree of conductive hearing loss in the study group. In light of these findings, we propose that migrating neutrophils cause conductive hearing loss by secreting MPO in patients with cholesteatoma. HCIO, the main microbicide, is produced by MPO. The prolonged inflammatory process seen in chronic otitis media may lead to excessive formation of reactive oxygen particles through MPO and eventually lead to the formation of a cholesteatoma and subsequent severe conductive hearing loss.

We believe that every step in the inflammatory process of cholesteatoma formation is worth studying so that we might eventually learn how to prevent cholesteatoma. Perhaps the utilization of systemically or locally administered antioxidant drugs in future studies will make it possible to write a new page about the development of cholesteatoma and severe conductive hearing loss.

References

(1.) da Costa SS, Rosito LP, Dornelles C. Sensorineural hearing loss in patients with chronic otitis media. Eur Arch Otorhinolaryngol 2009;266(2):221-4.

(2.) Peek FA, Huisman MA, Berckmans RJ, et al. Lipopolysaccharide concentration andbone resorption in cholesteatoma. Otol Neurotol 2003;24(5):709-13.

(3.) Thomsen J, Bretlau P, Balslev Jorgensen M. Bone resorption in chronic otitis media. The role of cholesteatoma, a must or adjunct? Clin Otolaryngol Allied Sci 1981;6(3):179-86.

(4.) Hazen SL, Hsu FF, Mueller DM, et al. Human neutrophils employ chlorine gas as an oxidant during phagocytosis. J Clin Invest 1996;98(6): 1283-9.

(5.) Kitahara M, Eyre H J, Simonian Y, et al. Hereditary myeloperoxidase deficiency. Blood 1981;57(5):888-93.

(6.) Hampton MB, Kettle AJ, Winterbourn CC. Inside the neutrophil phagosome: Oxidants, myeloperoxidase, and bacterial killing. Blood 1998;92(9):3007-17.

(7.) Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and ugly. Am J Physiol 1996;271(5 Pt l):C1424-37.

(8.) Takumida M, Zhang DM, Anniko M. Localization of nitric oxide synthase isoforms (I, II and III) in the endolymphatic sac of the guinea pig. ORL J Otorhinolaryngol Relat Spec 1997;59(6):311-16.

(9.) Watanabe K, Hess A, Bloch W, Michel O. Inhibition of inducible nitric oxide synthase lowers the cochlear damage by lipopolysaccharide in guinea pigs. Free Radio Res 2000;32(4):363-70.

(10.) Ottoviani F, Neglia CB, Berti E. Cytokines and adhesion molecules in middle ear cholesteatoma. A role in epithelial growth? Acta Otolaryngol 1999;119(4):462-7.

(11.) Tanaka Y, Kojima H, Miyazaki H, et al. Roles of cytokines and cell cycle regulating substances in proliferation of cholesteatoma epithelium. Laryngoscope 1999; 109(7 Pt 1): 1102-7.

(12.) Eskiizmir G, Yuceturk AV, Onur E, et al. The imbalance of enzymatic antioxidants in cholesteatoma. Acta Otolaryngol 2009; 129(11): 1187-91.

(13.) Nair SP, Meghji S, Wilson M, et al. Bacterially induced bone destruction: Mechanisms and misconceptions. Infect Immun 1996;64(7):2371-80.

(14.) Behling UH, Pham PH, Nowotny A. Biological activity of the slime and endotoxin of the periodontopathic organism Eikenella corrodens. Infect Immun 1979;26(2):580-4.

(15.) Raisz LG, Alander C, Eilon G, et al. Effects of two bacterial products, muramyl dipeptide and endotoxin, on bone resorption in organ culture. Calcif Tissue Int 1982;34(4):365-9.

(16.) lino Y, Hopps RM. The bone-resorbing activities in tissue culture of lipopolysaccharides from the bacteria Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Capnocytophaga ochracea isolated from human mouths. Arch Oral Biol 1984;29(1):59-63.

(17.) Watanabe K, Jinnouchi K, Yagi T. Immunoreactivity for myeloperoxidase (MPO) in the vestibule after the injection of bacterial lipopolysaccharide into the middle ear. Auris Nasus Larynx 2002;29(3):241-5.

(18.) Johnson RJ, Couser WG, Chi EY, et al. New mechanism for glomerular injury. Myeloperoxidase-hydrogen peroxide-halide system. J Clin Invest 1987;79(5):1379-87.

(19.) Lefkowitz DL, Gelderman MP, Fuhrmann SR, et al. Neutrophilic myeloperoxidase-macrophage interactions perpetuate chronic inflammation associated with experimental arthritis. Clin Immunol 1999;91 (2): 145-55.

(20.) Kinoshita T, Tomiyama S. Free radicals in inner ear immune responses; immunohistochemical study of myeloperoxidase [in Japanese]. Nihon Jibiinkoka Gakkai Kaiho 1994;97(9): 1608-12.

From the ENT Department, Recep Tayyip Erdogan University Faculty of Medicine, Rize, Turkey (Dr. Celebi Erdivanli); and the ENT Department, Dr. Lutfi Kirdar Kartal Training and Research Hospital, Istanbul (Dr. Sanli). The study described in this article was conducted at the Dr. Lutfi Kirdar Kartal Training and Research Hospital.

Corresponding author: Ozlem Celebi Erdivanli, MD, ENT Department, Recep Tayyip Erdogan UniversityTraining and Research Hospital, Islampasa Mahallesi, Rize, Turkey. Email: drozlemcelebi@mynet.com
Table 1. Incidence of MPO activity in the two groups
according to degree

                   n (%)

                Study group   Control group

MPO activity     (n = 51)       (n = 30)      p Value

None                 0          20 (66.7)      <0.01
Slight            6(11.8)       10(33.3)
Moderate         24 (47.1)          0
Intense          21 (41.2)          0

Table 2. Relationship * between the intensity of MPO
activity and the degree of conductive hearing loss in
the study group

                      Preoperative conductive
                         hearing loss, n (%)

MPO activity         Mild   Moderate     Severe

Slight (n = 6)        0     3 (50.0)    3 (50.0)
Moderate (n = 24)     0     15 (62.5)   9 (37.5)
Intense (n = 21)      0      2 (9.5)    19 (90.5)
Total (n = 51)        0     20 (39.2)   31 (60.8)

* There was a statistically significant association between
the degree of MPO activity and the degree of hearing loss
([chi square] = 13.518; p < 0.001).
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Article Details
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Title Annotation:ORIGINAL ARTICLE
Author:Erdivanli, Ozlem Celebi; Sanli, Arif
Publication:Ear, Nose and Throat Journal
Article Type:Clinical report
Geographic Code:7TURK
Date:Apr 1, 2015
Words:2258
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