Acta Oto-Laryngologica. 2014; 134: 791–795
ORIGINAL ARTICLE
Studies on distribution of a1-antitrypsin, lysozyme, lactoferrin, and mast cell enzymes in diseased middle ear mucosa
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JIN SUNG KIM, YONG JOO YOON & EUN JUNG LEE Department of Otolaryngology, Chonbuk National University Medical School and Research Institute of Clinical Medicine of Chonbuk National University – Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
Abstract Conclusion: Antimicrobial agents (AMAs) and mast cells in chronic otitis media (COM) may play a protective role in the pathogenesis of COM. Among them, our results indicated that lysozyme may be the most important parameter of the mucosa infectivity in COM. Objectives: AMAs include alpha 1-antitrypsin (a1-AT), lysozyme, and lactoferrin and exhibit innate immune activity that is known to play a critical role in mucosal defenses. In addition, proteases expressed in mast cells contribute to mucosal defenses. The aim of this study was to identify AMAs responsible for COM as well as their association with middle ear mucosal diseases. Methods: Middle ear mucosa (MEM) was intraoperatively collected by biopsy from patients, diagnosed as COM without or with cholesteatoma, and immediately processed for immunohistochemical study to evaluate expression of AMAs and mast cell proteases (chymase and tryptase). Results: The AMAs and mast cell enzymes were observed prominently in the chronic middle ear diseases whereas immunoreactivity of a1-AT and lysozyme was not significantly increased in the COM with cholesteatoma compared with that in COM without cholesteatoma. Lactoferrin was not detected in the diseased MEMs.
Keywords: Tryptase, chymase, cholesteatoma, antimicrobial agent, chronic otitis media
Introduction Infection is a very important factor in the pathogenesis of chronic otitis media (COM) and significantly influences the pathologic type of the middle ear mucosa (MEM), but the exact pathogenesis of COM is still unknown [1]. The middle ear is in part covered by a respiratory mucosa containing secretory cells [2]. It is known that the mucosal secretion produced by the epithelium and submucosal glands of MEM not only contributes to the mechanical mucociliary system of MEM in the function of the middle ear but also protects the mucosa from microbial infection [3]. Antimicrobial agents (AMAs) such as alpha 1-antitrypsin (a1-AT), lysozyme, lactoferrin, and defensins have been shown to play a role in the innate biochemical defense system [4–7]. The innate
immune defense strategies have the potential to prevent infections of the diseased MEM. Studies have suggested that expression of AMAs in the various tissues is induced by infection in mucosa and skin [8– 10]. However, little is known about the expression of AMAs in the middle ear under the conditions of otitis media and its role in persistent otitis media. In the pathogenesis of COM, much attention has been paid to the mechanisms of chronic reactions in which mast cells may be involved in inflammatory processes and the secretion of AMAs as secretagogues [11]. The increased expression of proteases in mast cells in skin and mucosa or diseases [12–15] may suggest that they contribute to the maintenance of the chronic inflammatory process of the MEM. On the basis of these considerations, we hypothesized that a mechanism by which chronic infections
Correspondence: Yong Joo Yoon MD PhD, 2-20 Keum-Am-Dong, Department of Otolaryngology, Chonbuk National University Medical School, Jeonju 561-180, South Korea. Tel: +82 63 250 1988. Fax: +82 63 250 1986. E-mail: [email protected]
(Received 4 February 2014; accepted 18 March 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.913198
792
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occur may be due to the differences in the expression levels of AMAs in the MEM. In the present study, we evaluated the density of AMAs and mast cell enzymes in diseased human MEMs using immunohistochemistry. Material and methods
Acta Otolaryngol Downloaded from informahealthcare.com by University of Utah on 12/02/14 For personal use only.
Tissue specimens We selected patients with COM, diagnosed as COM without cholesteatoma (five ear samples), COM with cholesteatoma (five ear samples), and postauricular skin (three samples). Chronically inflamed MEM of inactive type was obtained near the promontory during surgery from 10 patients with chronic middle ear diseases. After specimens of MEM were obtained by operation, the samples were treated in a similar manner. Immediately after surgical removal, the MEM specimens were fixed overnight in freshly prepared 4% paraformaldehyde in phosphate buffer (pH 7.4). Samples were then dehydrated in graded concentrations of ethanol and embedded in paraffin. This study was approved by the institutional review board of Chonbuk National University Hospital. Histochemistry and immunohistochemistry Paraffin-embedded specimens were sectioned to 4 mm thickness and mounted on albumin-coated glass slides. The tissue sections were deparaffinized twice in 100% xylene for 10 min, and then treated with 100%, 90%, 70%, and 30% ethanol for 10, 5, 5, and 5 min, respectively, before rehydration with distilled water. After deparaffinization and serial hydration in ethanol, sections were first stained with hematoxylin and eosin. Simultaneously, each second section was used for toluidine blue staining using a standard protocol. For the immunohistochemical studies, deparaffinized, dehydrated, and rehydrated tissue sections were treated with 3% hydrogen peroxide in ice-cold methanol for 30 min, followed by rinsing in phosphate-buffered saline (PBS). Non-specific binding was blocked by separately incubating sections with 10% normal goat serum in PBS for 10 min.
Immunoreactivity was detected using a standard avidin-biotin complex peroxidase method (Vectastain Universal Elite ABC kit; Vector Laboratories, Burlingame, CA, USA). Localization of immunoreactivity was carried out using goat anti-human polyclonal antibodies (against a1-AT, lysozyme and lactoferrin, chymase and tryptase). Sections were incubated with diluted primary antibodies overnight at 4 C. Control sections were incubated with preabsorbed antiserum in place of the primary antibody. The next day, slides were washed twice in PBS, and the secondary biotinylated antibody (Vectastain, Vector Laboratories) was then applied at room temperature for 45 min. Sections were again washed twice in PBS, before applying the tertiary antibody for 30 min at room temperature. The reaction product was visualized using 0.03% diaminobenzidine tetrahydrochloride, and sections were then counterstained with Meyer’s hematoxylin.
Results Differences were found in the expression of AMAs among samples obtained from diseased MEM. Immunohistochemical analyses demonstrated that AMAs and mast cells were expressed in the diseased MEM, associated with chronic inflammatory infections (Table I). Mast cells are more clearly visible with the immunostain than the toluidine blue stain (Figure 1). Immunoreactivity of a1-AT (Figures 2A and 3A) and lysozyme (Figures 2B and 3B) was observed in diseases of the MEM, and clear immunohistochemical staining was also seen in the submucosa of the MEM. Lysozyme (Figure 2B and 3B) was present mainly in the subepithelial layers of the diseased tissues of all samples. Unlike a1-AT and lysozyme, lactoferrin (Figures 2C and 3C) was not seen in the submucosa of the diseased MEM. The majority of the mast cells were found in the subepithelial space and adjacent to epithelium (Figure 2D, E and 3D). Higher magnification revealed the typical granular appearance of mast cells (Figure 3E). The immunostained AMAs were mainly observed with mast cells in the submucosa. Many chymase- and tryptase-positive mast cells were seen in the submucosa of diseased MEM. Interestingly,
Table I. Comparative study of antimicrobial agents and mast cell enzymes in diseased middle ear mucosa and skin. Sample tested
a1-AT
Lysozyme
Lactoferrin
Chymase
Tryptase
Skin
+++
Negative
Negative
++ or +++
+++
COM with cholesteatoma
++
+ or ++
–
+++
+ or ++
COM without cholesteatoma
+++
+++
?
+
++ or +++
a1-AT, alpha 1-antitrypsin; COM, chronic otitis media; +, mild; ++, moderate; +++, strong; ++++, very strong.
Antimicrobial agents in diseased middle ear mucosa Discussion
B
A
ep
The aim of this study was to assess the density of AMAs and mast cells in COM in relationship to different samples of COM. However, generally, in our study – unlike previous reports – AMAs were not found more frequently in cholesteatoma tissues than in COM without cholesteatoma. COM is defined as permanent inflammatory disease in the middle ear and mastoid cavity with the presence of tympanic membrane perforation, discharge from the ear, and often a proliferative process of the mucosal lining in the form of granulation tissue, polyp or cholesteatoma. Infection of MEM is resistant to medical treatment, including drying with cleaning and irrigation and administration of antibiotics. Therefore, early diagnosis and management of patients with acute or chronic middle ear disease have become key aims to treat middle ear diseases. Although infection and eustachian tube dysfunction are well known to be involved in the pathogenesis of otitis media, the protective factors responsible for the persistence of COM are not fully understood yet [1]. Study of the pathogenesis of chronic middle ear disease has been focused on alterations in mucosa, consisting of the epithelium, submucosa, various inflammatory cells, and connective tissue cells [1,14,16]. The present study revealed that expression of a1-AT, lysozyme, and mast cell proteases (tryptase and chymase) were significantly increased, but the expression of lactoferrin was not detectable. Lysozyme was mainly expressed in the subepithelial space. The majority of the mast cells were found in the subepithelial space and adjacent to epithelium. The immunostained AMAs were mainly observed
ep
C
D
ep
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Figure 1. Comparative histological and immunohistological sections of middle ear mucosa; epithelium (ep), submucosa (sm), in a patient with chronic otitis media for mast cells using toluidine blue stain (A) and immunohistochemical stain for chymase (B). Mast cells are more clearly visible with the immunostain (D) than the toluidine blue stain (C). In the submucosa, most mast cells were located in the subepithelial space (solid arrows) next to epithelium, although some cells were scattered in the basal and suprabasal cell layers of the epithelium (open arrows).
cholesteatoma tissue showed high levels of chymasepositive mast cells, but normal postauricular skin (Figure 3F) showed low levels of chymase-positive mast cells. The density of chymase-positive mast cells was higher in tissue samples from the group with cholesteatoma. These results indicate that increased expression of chymase in mast cells may be related to the pathogenesis of the cholesteatoma. The expression of AMAs and mast cell proteases was not lower in tissue samples from the group with a COM than in those from the group with cholesteatoma. No immunostaining was observed in the control samples.
A
B
D
C
E
Figure 2. Immunohistochemical demonstration of immunoreactive density using a1-antitrypsin (A), lysozyme (B), lactoferrin (C), chymase (D), and tryptase (E) antibody in the middle ear mucosa of a patient with chronic otitis media without cholesteatoma. Immunoreactivity for a1-antitrypsin (A), lysozyme (B), chymase (D), and tryptase (E) was strongly positive, but the expression of lactoferrin (C) was not detectable. Lysozyme was seen mainly in the subepithelial space (arrow), and are involved in the inflammatory response of the middle ear mucosa.
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B
Acta Otolaryngol Downloaded from informahealthcare.com by University of Utah on 12/02/14 For personal use only.
D
F
C
E
G
Figure 3. Immunohistochemical demonstration of immunoreactive density using a1-antitrypsin (A), lysozyme (B), lactoferrin (C), chymase (D), and tryptase (E) antibodies in the cholesteatoma samples and chymase (F) and tryptase (G) antibodies in the postauricular skin samples. Immunoreactivity for the a1-antitrypsin (A) and lysozyme (B) was elevated in the mucosa of chronic otitis media (COM with cholesteatoma compared with COM without cholesteatoma, but the expression of lactoferrin (C) was not detectable. In cholesteatoma, chymase-positive mast cells (D) were also detected in either the epithelium or the stroma. Many chymase-positive mast cells are seen in the subepithelial stroma (D, arrow). Cholesteatoma tissue showed high levels of chymase-positive mast cells, but postauricular skin showed low levels of chymase-positive mast cells (F). Higher magnification revealed the typical granular appearance of mast cells (E, arrowheads).
with mast cells in the submucosa. Interestingly, cholesteatoma tissue showed high levels of chymasepositive mast cells, but postauricular skin showed low levels of chymase-positive mast cells. However, generally, in our study, unlike previous reports, AMAs were not more frequently found in cholesteatoma tissues than in COM without cholesteatoma. The density of chymase-positive mast cells was higher in tissue samples from the group with cholesteatoma. These results indicate that increased chymase expression in mast cells may be related to the pathogenesis of cholesteatoma. AMAs, such as a1-AT, lysozyme, and lactoferrin, act as antimicrobial agents and/or physiological regulators with respect to both inflammatory and immune responses for host defense. Most AMAs are usually up-regulated in response to acute or chronic inflammation [8]. Patients with inflammatory diseases have a reduced amount of the AMAs in their mucosa, which correlates with an impaired innate defense of human mucosa [13]. Studies [8] have indicated that AMAs play potential roles in the chronicity associated with inflammatory diseases. Therefore, the observed distribution of AMAs in the diseased MEM can be a useful basis for understanding of mucosal immune defenses. Harada et al. [9] have reported that lysozyme concentration is
significantly elevated in middle ear fluid of patients with otitis media with effusion (OME), compared with healthy controls. A study has also demonstrated that AMAs are increased in MEM with COM [3]. In the present study, lysozyme immunoreactivity in middle ear cholesteatoma was not higher than in COM without cholesteatoma. Over-distribution of lysozyme locally in the middle ear may be related to the pathological condition of COM, which is a COM characterized by the presence of a high level of inflammatory cells containing mast cells. The distribution of lysozyme in MEM significantly and positively correlated with infectivity of COM patients. Studies with the diseased tissues have demonstrated the altered distribution of the antimicrobial peptides, lysozyme and lactoferrin [8,9]. In past decades, AMA activity in chronic diseases has been studied, while new parameters are still being developed. Lysozyme is likely to be specific in the assessment of middle ear inflammation. It can be assessed by pathologic study of diseases; assessment of the lysozyme in the diseased MEM is of great clinical importance in the treatment of patients with COM. Evalution of the expression of AMAs in chronic middle ear disease appears to be an attractive approach for discriminating between COMs with and without cholesteatoma.
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Antimicrobial agents in diseased middle ear mucosa Mast cells are considered to be most highly involved in the pathological condition of chronic inflammatory diseases [12–15]. The pathological conditions of middle ear disease have been demonstrated as active inflammation of the middle ear with production of various AMAs that induce migration of mast cells in the MEM [7]. Increased mast cells in the MEM after infection reflect the fact that mast cells are highly activated and participate in various immune and inflammatory responses [17–19]. In addition, many mast cells are found in the MEM, indicating that it is potentially related to the pathological condition of COM. In the present study, tryptase-positive mast cells were barely detected but chymase-positive cells were highly expressed in the cholesteatoma, which is apparently related to its pathogenesis. The present study is the first to demonstrate AMAs and proteases in mast cells in the human MEM. Different expression patterns of AMAs in various chronic infectious middle ear diseases were clearly observed in this experiment. Expression of AMAs was identified nearly at constant levels in all COM, including COM with cholesteatoma. AMAs may correlate well with inflammatory activity in middle ear diseases. Lysozyme and lactoferrin were not detected in the epithelium and submucosal glands of the normal MEM. Immunostaining of a1-AT and lysozyme in diseased MEM revealed strong expression, but positive staining for lactoferrin was not identified for COM. The density of AMAs is known to vary considerably among different middle ear diseases. Further investigation on the interactions of AMAs with mast cells in COM is warranted to provide a more complete picture of this inflammatory process.
Conclusion In conclusion, the expression of AMAs and mast cell proteases in COM tissue was found to differ depending on the disease. These findings suggest that AMAs and mast cell enzymes contribute to the maintenance of the inflammatory process and that assessment of the lysozyme with mast cells in the diseased MEM is of great clinical importance in patients with COM. Further research into the development of agents is needed for increased understanding of the chronicity of COM.
Acknowledgment This paper was supported by the research fund of Chonbuk National University and a fund of the
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Biomedical Research Institute, Chonbuk National University Hospital. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References [1] Bluestone MD. Pathogenesis of otitis media: role of eustachian tube. Pediatr Infect Dis J 1996;15:281–91. [2] Lim DJ. Functional morphology of the lining membrane of the middle ear and eustachian tube: an overview. Ann Otol Rhinol Laryngol 1974;83:5–26. [3] Lim DJ, DeMaria TF. Immunobarriers of the tubotympanum. Acta Otolaryngol 1987;103:355–62. [4] Yoon YJ, Jeong WC, Kim YK. Immunohistochemical localization of alpha 1-antitrypsin in the rat eustachian tube and tympanic membrane. Korean J Otolaryngol 1999;42:279–83. [5] Hanamure Y, Lim DJ. Normal distribution of lysozyme and lactoferrin-secreting cells in the chinchilla tubotympanum. Am J Otolaryngol 1986;7:410–25. [6] Yoon YJ. Presence of hBD-1 and hBD-2 in human cerumen and external auditory canal skin. Acta Otolaryngol 2008;128:871–5. [7] Yoon YJ, Lee EJ. Spatial distribution of antimicrobial peptides and mast cells in the skin of the external auditory canal. J Laryngol Otol 2011;125:e6. [8] Fischbach W, Becker W, Mossner J, Koch W, Reiners C. Faecal alpha-1-antitrypsin and excretion of 111-Indium granulocytes in assessment of disease activity in chronic inflammatory bowel diseases. Gut 1987;28:386–93. [9] Harada T, Juhn SK, Adams GL. Lysozyme levels in middle ear effusion and serum in otitis media. Arch Otolaryngol Head Neck Surg 1990;116:54–6. [10] Nizet V, Ohtake T, Lauth X, Trowbridge J, Rudisill J, Dorschner RA, et al. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 2001;414:454–7. [11] Church MK, Lowman MA, Robinson C, Holgates ST, Benyon RC. Interaction of neuropeptides with human mast cells. Int Arch Allergy Appl Immunol 1989;88:70–8. [12] Yoon YJ, Jeong WC, Jeong KS. Distribution of mast cell in the rat tympanic membrane and eustachian tube. Korean J Otolaryngol 2000;43:1270–82. [13] Watanabe T, Kawauchi H, Fujiyoshi T, Mogi G. Distribution of mast cells in the tubotympanum of guinea pigs. Ann Otol Rhinol Laryngol 1991;100:407–12. [14] Sankovic S, Degenc R, Bojic P. Mast cells in chronic inflammation of the middle ear mucosa. Rev Laryngol Otol Rhinol 2005;126:15–18. [15] Albino AP, Reed JA, Bogdany JK, Sassoon J, Parisier SC. Increased numbers of mast cells in human middle ear cholesteatomas: implications for treatment. Am J Otol 1998;19:266–72. [16] Telian SA, Schmalbach CE. 2003. Chronic otitis media. In Snow JB, Ballenger JJ, editors. Ballenger’s otorhinolaryngology head neck surgery. 16th edn. Hamilton, ON: BC Decker. p 261–93. [17] Caughey GH. Mast cell tryptases and chymases in inflammation and host defense. Immunol Rev 2007;217:141–54. [18] Metz M, Grimbaldeston MA, Nakae S, Piliponsky AM, Tsai M, Galli A. Mast cell in promotion and limitation of chronic inflammation. Immunol Rev 2007;217:304–28. [19] Dawicki W, Marshall JS. New and emerging roles for mast cells in host defence. Curr Opin Immunol 2007;19:31–8.
ORIGINAL ARTICLE
Studies on distribution of a1-antitrypsin, lysozyme, lactoferrin, and mast cell enzymes in diseased middle ear mucosa
Acta Otolaryngol Downloaded from informahealthcare.com by University of Utah on 12/02/14 For personal use only.
JIN SUNG KIM, YONG JOO YOON & EUN JUNG LEE Department of Otolaryngology, Chonbuk National University Medical School and Research Institute of Clinical Medicine of Chonbuk National University – Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
Abstract Conclusion: Antimicrobial agents (AMAs) and mast cells in chronic otitis media (COM) may play a protective role in the pathogenesis of COM. Among them, our results indicated that lysozyme may be the most important parameter of the mucosa infectivity in COM. Objectives: AMAs include alpha 1-antitrypsin (a1-AT), lysozyme, and lactoferrin and exhibit innate immune activity that is known to play a critical role in mucosal defenses. In addition, proteases expressed in mast cells contribute to mucosal defenses. The aim of this study was to identify AMAs responsible for COM as well as their association with middle ear mucosal diseases. Methods: Middle ear mucosa (MEM) was intraoperatively collected by biopsy from patients, diagnosed as COM without or with cholesteatoma, and immediately processed for immunohistochemical study to evaluate expression of AMAs and mast cell proteases (chymase and tryptase). Results: The AMAs and mast cell enzymes were observed prominently in the chronic middle ear diseases whereas immunoreactivity of a1-AT and lysozyme was not significantly increased in the COM with cholesteatoma compared with that in COM without cholesteatoma. Lactoferrin was not detected in the diseased MEMs.
Keywords: Tryptase, chymase, cholesteatoma, antimicrobial agent, chronic otitis media
Introduction Infection is a very important factor in the pathogenesis of chronic otitis media (COM) and significantly influences the pathologic type of the middle ear mucosa (MEM), but the exact pathogenesis of COM is still unknown [1]. The middle ear is in part covered by a respiratory mucosa containing secretory cells [2]. It is known that the mucosal secretion produced by the epithelium and submucosal glands of MEM not only contributes to the mechanical mucociliary system of MEM in the function of the middle ear but also protects the mucosa from microbial infection [3]. Antimicrobial agents (AMAs) such as alpha 1-antitrypsin (a1-AT), lysozyme, lactoferrin, and defensins have been shown to play a role in the innate biochemical defense system [4–7]. The innate
immune defense strategies have the potential to prevent infections of the diseased MEM. Studies have suggested that expression of AMAs in the various tissues is induced by infection in mucosa and skin [8– 10]. However, little is known about the expression of AMAs in the middle ear under the conditions of otitis media and its role in persistent otitis media. In the pathogenesis of COM, much attention has been paid to the mechanisms of chronic reactions in which mast cells may be involved in inflammatory processes and the secretion of AMAs as secretagogues [11]. The increased expression of proteases in mast cells in skin and mucosa or diseases [12–15] may suggest that they contribute to the maintenance of the chronic inflammatory process of the MEM. On the basis of these considerations, we hypothesized that a mechanism by which chronic infections
Correspondence: Yong Joo Yoon MD PhD, 2-20 Keum-Am-Dong, Department of Otolaryngology, Chonbuk National University Medical School, Jeonju 561-180, South Korea. Tel: +82 63 250 1988. Fax: +82 63 250 1986. E-mail: [email protected]
(Received 4 February 2014; accepted 18 March 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.913198
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occur may be due to the differences in the expression levels of AMAs in the MEM. In the present study, we evaluated the density of AMAs and mast cell enzymes in diseased human MEMs using immunohistochemistry. Material and methods
Acta Otolaryngol Downloaded from informahealthcare.com by University of Utah on 12/02/14 For personal use only.
Tissue specimens We selected patients with COM, diagnosed as COM without cholesteatoma (five ear samples), COM with cholesteatoma (five ear samples), and postauricular skin (three samples). Chronically inflamed MEM of inactive type was obtained near the promontory during surgery from 10 patients with chronic middle ear diseases. After specimens of MEM were obtained by operation, the samples were treated in a similar manner. Immediately after surgical removal, the MEM specimens were fixed overnight in freshly prepared 4% paraformaldehyde in phosphate buffer (pH 7.4). Samples were then dehydrated in graded concentrations of ethanol and embedded in paraffin. This study was approved by the institutional review board of Chonbuk National University Hospital. Histochemistry and immunohistochemistry Paraffin-embedded specimens were sectioned to 4 mm thickness and mounted on albumin-coated glass slides. The tissue sections were deparaffinized twice in 100% xylene for 10 min, and then treated with 100%, 90%, 70%, and 30% ethanol for 10, 5, 5, and 5 min, respectively, before rehydration with distilled water. After deparaffinization and serial hydration in ethanol, sections were first stained with hematoxylin and eosin. Simultaneously, each second section was used for toluidine blue staining using a standard protocol. For the immunohistochemical studies, deparaffinized, dehydrated, and rehydrated tissue sections were treated with 3% hydrogen peroxide in ice-cold methanol for 30 min, followed by rinsing in phosphate-buffered saline (PBS). Non-specific binding was blocked by separately incubating sections with 10% normal goat serum in PBS for 10 min.
Immunoreactivity was detected using a standard avidin-biotin complex peroxidase method (Vectastain Universal Elite ABC kit; Vector Laboratories, Burlingame, CA, USA). Localization of immunoreactivity was carried out using goat anti-human polyclonal antibodies (against a1-AT, lysozyme and lactoferrin, chymase and tryptase). Sections were incubated with diluted primary antibodies overnight at 4 C. Control sections were incubated with preabsorbed antiserum in place of the primary antibody. The next day, slides were washed twice in PBS, and the secondary biotinylated antibody (Vectastain, Vector Laboratories) was then applied at room temperature for 45 min. Sections were again washed twice in PBS, before applying the tertiary antibody for 30 min at room temperature. The reaction product was visualized using 0.03% diaminobenzidine tetrahydrochloride, and sections were then counterstained with Meyer’s hematoxylin.
Results Differences were found in the expression of AMAs among samples obtained from diseased MEM. Immunohistochemical analyses demonstrated that AMAs and mast cells were expressed in the diseased MEM, associated with chronic inflammatory infections (Table I). Mast cells are more clearly visible with the immunostain than the toluidine blue stain (Figure 1). Immunoreactivity of a1-AT (Figures 2A and 3A) and lysozyme (Figures 2B and 3B) was observed in diseases of the MEM, and clear immunohistochemical staining was also seen in the submucosa of the MEM. Lysozyme (Figure 2B and 3B) was present mainly in the subepithelial layers of the diseased tissues of all samples. Unlike a1-AT and lysozyme, lactoferrin (Figures 2C and 3C) was not seen in the submucosa of the diseased MEM. The majority of the mast cells were found in the subepithelial space and adjacent to epithelium (Figure 2D, E and 3D). Higher magnification revealed the typical granular appearance of mast cells (Figure 3E). The immunostained AMAs were mainly observed with mast cells in the submucosa. Many chymase- and tryptase-positive mast cells were seen in the submucosa of diseased MEM. Interestingly,
Table I. Comparative study of antimicrobial agents and mast cell enzymes in diseased middle ear mucosa and skin. Sample tested
a1-AT
Lysozyme
Lactoferrin
Chymase
Tryptase
Skin
+++
Negative
Negative
++ or +++
+++
COM with cholesteatoma
++
+ or ++
–
+++
+ or ++
COM without cholesteatoma
+++
+++
?
+
++ or +++
a1-AT, alpha 1-antitrypsin; COM, chronic otitis media; +, mild; ++, moderate; +++, strong; ++++, very strong.
Antimicrobial agents in diseased middle ear mucosa Discussion
B
A
ep
The aim of this study was to assess the density of AMAs and mast cells in COM in relationship to different samples of COM. However, generally, in our study – unlike previous reports – AMAs were not found more frequently in cholesteatoma tissues than in COM without cholesteatoma. COM is defined as permanent inflammatory disease in the middle ear and mastoid cavity with the presence of tympanic membrane perforation, discharge from the ear, and often a proliferative process of the mucosal lining in the form of granulation tissue, polyp or cholesteatoma. Infection of MEM is resistant to medical treatment, including drying with cleaning and irrigation and administration of antibiotics. Therefore, early diagnosis and management of patients with acute or chronic middle ear disease have become key aims to treat middle ear diseases. Although infection and eustachian tube dysfunction are well known to be involved in the pathogenesis of otitis media, the protective factors responsible for the persistence of COM are not fully understood yet [1]. Study of the pathogenesis of chronic middle ear disease has been focused on alterations in mucosa, consisting of the epithelium, submucosa, various inflammatory cells, and connective tissue cells [1,14,16]. The present study revealed that expression of a1-AT, lysozyme, and mast cell proteases (tryptase and chymase) were significantly increased, but the expression of lactoferrin was not detectable. Lysozyme was mainly expressed in the subepithelial space. The majority of the mast cells were found in the subepithelial space and adjacent to epithelium. The immunostained AMAs were mainly observed
ep
C
D
ep
sm sm Acta Otolaryngol Downloaded from informahealthcare.com by University of Utah on 12/02/14 For personal use only.
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Figure 1. Comparative histological and immunohistological sections of middle ear mucosa; epithelium (ep), submucosa (sm), in a patient with chronic otitis media for mast cells using toluidine blue stain (A) and immunohistochemical stain for chymase (B). Mast cells are more clearly visible with the immunostain (D) than the toluidine blue stain (C). In the submucosa, most mast cells were located in the subepithelial space (solid arrows) next to epithelium, although some cells were scattered in the basal and suprabasal cell layers of the epithelium (open arrows).
cholesteatoma tissue showed high levels of chymasepositive mast cells, but normal postauricular skin (Figure 3F) showed low levels of chymase-positive mast cells. The density of chymase-positive mast cells was higher in tissue samples from the group with cholesteatoma. These results indicate that increased expression of chymase in mast cells may be related to the pathogenesis of the cholesteatoma. The expression of AMAs and mast cell proteases was not lower in tissue samples from the group with a COM than in those from the group with cholesteatoma. No immunostaining was observed in the control samples.
A
B
D
C
E
Figure 2. Immunohistochemical demonstration of immunoreactive density using a1-antitrypsin (A), lysozyme (B), lactoferrin (C), chymase (D), and tryptase (E) antibody in the middle ear mucosa of a patient with chronic otitis media without cholesteatoma. Immunoreactivity for a1-antitrypsin (A), lysozyme (B), chymase (D), and tryptase (E) was strongly positive, but the expression of lactoferrin (C) was not detectable. Lysozyme was seen mainly in the subepithelial space (arrow), and are involved in the inflammatory response of the middle ear mucosa.
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B
Acta Otolaryngol Downloaded from informahealthcare.com by University of Utah on 12/02/14 For personal use only.
D
F
C
E
G
Figure 3. Immunohistochemical demonstration of immunoreactive density using a1-antitrypsin (A), lysozyme (B), lactoferrin (C), chymase (D), and tryptase (E) antibodies in the cholesteatoma samples and chymase (F) and tryptase (G) antibodies in the postauricular skin samples. Immunoreactivity for the a1-antitrypsin (A) and lysozyme (B) was elevated in the mucosa of chronic otitis media (COM with cholesteatoma compared with COM without cholesteatoma, but the expression of lactoferrin (C) was not detectable. In cholesteatoma, chymase-positive mast cells (D) were also detected in either the epithelium or the stroma. Many chymase-positive mast cells are seen in the subepithelial stroma (D, arrow). Cholesteatoma tissue showed high levels of chymase-positive mast cells, but postauricular skin showed low levels of chymase-positive mast cells (F). Higher magnification revealed the typical granular appearance of mast cells (E, arrowheads).
with mast cells in the submucosa. Interestingly, cholesteatoma tissue showed high levels of chymasepositive mast cells, but postauricular skin showed low levels of chymase-positive mast cells. However, generally, in our study, unlike previous reports, AMAs were not more frequently found in cholesteatoma tissues than in COM without cholesteatoma. The density of chymase-positive mast cells was higher in tissue samples from the group with cholesteatoma. These results indicate that increased chymase expression in mast cells may be related to the pathogenesis of cholesteatoma. AMAs, such as a1-AT, lysozyme, and lactoferrin, act as antimicrobial agents and/or physiological regulators with respect to both inflammatory and immune responses for host defense. Most AMAs are usually up-regulated in response to acute or chronic inflammation [8]. Patients with inflammatory diseases have a reduced amount of the AMAs in their mucosa, which correlates with an impaired innate defense of human mucosa [13]. Studies [8] have indicated that AMAs play potential roles in the chronicity associated with inflammatory diseases. Therefore, the observed distribution of AMAs in the diseased MEM can be a useful basis for understanding of mucosal immune defenses. Harada et al. [9] have reported that lysozyme concentration is
significantly elevated in middle ear fluid of patients with otitis media with effusion (OME), compared with healthy controls. A study has also demonstrated that AMAs are increased in MEM with COM [3]. In the present study, lysozyme immunoreactivity in middle ear cholesteatoma was not higher than in COM without cholesteatoma. Over-distribution of lysozyme locally in the middle ear may be related to the pathological condition of COM, which is a COM characterized by the presence of a high level of inflammatory cells containing mast cells. The distribution of lysozyme in MEM significantly and positively correlated with infectivity of COM patients. Studies with the diseased tissues have demonstrated the altered distribution of the antimicrobial peptides, lysozyme and lactoferrin [8,9]. In past decades, AMA activity in chronic diseases has been studied, while new parameters are still being developed. Lysozyme is likely to be specific in the assessment of middle ear inflammation. It can be assessed by pathologic study of diseases; assessment of the lysozyme in the diseased MEM is of great clinical importance in the treatment of patients with COM. Evalution of the expression of AMAs in chronic middle ear disease appears to be an attractive approach for discriminating between COMs with and without cholesteatoma.
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Antimicrobial agents in diseased middle ear mucosa Mast cells are considered to be most highly involved in the pathological condition of chronic inflammatory diseases [12–15]. The pathological conditions of middle ear disease have been demonstrated as active inflammation of the middle ear with production of various AMAs that induce migration of mast cells in the MEM [7]. Increased mast cells in the MEM after infection reflect the fact that mast cells are highly activated and participate in various immune and inflammatory responses [17–19]. In addition, many mast cells are found in the MEM, indicating that it is potentially related to the pathological condition of COM. In the present study, tryptase-positive mast cells were barely detected but chymase-positive cells were highly expressed in the cholesteatoma, which is apparently related to its pathogenesis. The present study is the first to demonstrate AMAs and proteases in mast cells in the human MEM. Different expression patterns of AMAs in various chronic infectious middle ear diseases were clearly observed in this experiment. Expression of AMAs was identified nearly at constant levels in all COM, including COM with cholesteatoma. AMAs may correlate well with inflammatory activity in middle ear diseases. Lysozyme and lactoferrin were not detected in the epithelium and submucosal glands of the normal MEM. Immunostaining of a1-AT and lysozyme in diseased MEM revealed strong expression, but positive staining for lactoferrin was not identified for COM. The density of AMAs is known to vary considerably among different middle ear diseases. Further investigation on the interactions of AMAs with mast cells in COM is warranted to provide a more complete picture of this inflammatory process.
Conclusion In conclusion, the expression of AMAs and mast cell proteases in COM tissue was found to differ depending on the disease. These findings suggest that AMAs and mast cell enzymes contribute to the maintenance of the inflammatory process and that assessment of the lysozyme with mast cells in the diseased MEM is of great clinical importance in patients with COM. Further research into the development of agents is needed for increased understanding of the chronicity of COM.
Acknowledgment This paper was supported by the research fund of Chonbuk National University and a fund of the
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Biomedical Research Institute, Chonbuk National University Hospital. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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