LOCALIZATION AND DETECTION OF NITRIC OXIDE SYNTHASE ISOFORMS IN HUMAN MIDDLE-EAR CHOLESTEATOMAS: POSSIBLE RELATION TO NITRIC OXIDE PRODUCTION AN D INFLAMMATORY BONE ABSORPTION ACTIVITIES

Marin Miyasato, Sachio Takeno, Takayuki Taruya, Katsuhiro Hirakawa, Takashi Ishino, Keishin Goh

Department of Otolaryngology, Head and Neck Surgery, Division of Clinical Medical Science, Programs for Applied Biomedicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

Introduction

Middle-ear cholesteatoma histologically consists of an epithelial matrix that produces the accumulation of keratin debris and underlying connective tissue layers.1 The aggressive absorption process of the bone structure adjacent to the matrix is one of the most distinct clinical findings of middle-ear cholesteatoma.2 We have recently shown that a part of the ligand-receptor system and the cytokine network including the receptor activator of NF-ΚappaB ligand (RANKL), IL-6, and TGF-beta are deeply involved in the mechanisms through the inflammatory manifestation and bone absorption activities.

In the present study, we focused on possible roles of nitric oxide (NO) in the middle-ear inflammation triggered by cholesteatoma proliferation. Nitric oxide plays a variety of roles in the human upper respiratory airways in relation to airway defense mechanisms, as well as being an inflammatory mediator.3 We examined the immunohistochemical localization and mRNA expression of RANKL and nitric oxide synthase (NOS) isoforms to understand possible relationship between inflammatory manifestation and bone absorption activities using cell-culture methods.

Methods

Patients ’ enrollment and tissue preparation

Cholesteatoma tissues were collected from adult patients who had undergone initial surgery for acquired cholesteatoma at the Hiroshima University Hospital.

The degrees of immunohistological expression of inducible NOS (iNOS), endothelial NOS (eNOS), RANKL and Ki-67, a proliferation marker were semi-quantitatively assessed using in vivo pathological specimens. The LSAB™2 system (Dako) was employed as the secondary antibody. The stained cells were observed with a Nikon light microscope and digital photomicrographs were taken for analysis.

The mRNA expression of three NOS isoforms, RANKL, and TGF-beta was quantitatively analyzed by real-time RT-PCR. Cellular RNA was isolated using RNeasy mini kits (Qiagen). For cDNA synthesis, total RNA was reverse-transcribed to cDNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems). Gene expression was measured on a 7300 real-time PCR system using TaqMan Gene Expression Assays. The mRNA levels for targeted genes were normalized to the value of GAPDH by calculating the change in Ct (ΔCt) for each sample.

Cell culture of cholesteatoma cells

We have successfully established a cell culture of cholesteatoma epithelial cells by elimination of fibroblasts contamination using the trypsinization technique and employment of a defined serum-free medium. Briefly, the cholesteatoma epithelium was dissected from the underlying submucosal tissue, and was then incubated in 0.1% collagenase at 37°C for about one hour. Following cell dissociation and centrifugation, the resulting cell pellet was resuspended in a culture medium and seeded onto 75 mm2 cell-culture flasks precoated with type-I collagen (Asahi Techno Glass, Tokyo, Japan). The cell culture was maintained in Defined Keratinocyte-Serum-Free Medium (Invitrogen, Tokyo, Japan) with growth supplement and antibiotics. Upon reaching cell confluence, a secondary culture of cholesteatoma cells was performed. The epithelial nature of the cells was confirmed by phase contrast microscopy. In order to standardize for both the period and nature of the cultures, the third generation of cholesteatoma cell cultures from each subject was used.

Results

Immunohistological findings

A positive expression of RANKL was predominantly detected mainly in the basal and para-basal layers of the matrix accompanying severe inflammatory cell infiltration. The Ki-67 LI significantly increased in the group with higher RANKL expression. We also examined the immunohistological localization of iNOS and eNOS in the cholesteatoma matrix. In a patient with minimal inflammation, a positive iNOS immunoreactivity was clearly observed in the cholesteatoma epithelial cells, mainly in their cytoplasm. A weak expression of eNOS isoform was also detected in the matrix. On the other hand, a higher iNOS immunoreactivity tended to be observed in the matrix accompanying severe inflammatory cell infiltration in the submucosal layer.

Real-time RT-PCR analysis of cultured cells

The use of defined Keratinocyte-SFM and collagen-coated dishes has demonstrated superior cell growth of primary cholesteatoma with maintaining epithelial morphology and biological markers. A positive expression of RANKL was detected mainly in the perinuclear cytoplasm of most cultured cells. Ki-67 antigen was also found to be positive in the nuclei of the cultured cells in various degrees. A real-time PCR analysis indicated that the cultured cholesteatoma cells showed distinct expression of TGF-beta and RANKL mRNA. Relatively low, but constitutive levels of NOS isoform mRNA were also noted. Among the three NOS isoforms, iNOS showed higher levels compared to the other two isoforms.

Discussion

Several theories have been proposed to explain the destructive properties of middle-ear cholesteatoma. A higher population of the osteoclast progenitor cell lineage and macrophages was immunohistochemically demonstrated in the cholesteatoma tissue compared with normal auditory canal skin.4 The balance between osteoblasts and osteoclasts plays a major role in bone formation and resorption. As shown in the present study, the combination of RANK, RANKL, and osteoprotegrin (OPG) is considered to be deeply involved in the disease mechanisms. These results suggest that epithelial proliferative activities and inflammatory manifestation could be possible factors responsible for the markedly increased bone resorption observed in cholesteatoma patients. It has been considered that the activity of cholesteatoma proliferation is greatly affected by difference in micro-environment. Because we found a tendency for the expression of Ki-67 antigen to be augmented in severe inflammatory conditions, we speculate that these factors which often coincide as a result of bacterial inflammation may act to regulate cell proliferation in the cholesteatoma epithelium through paracrine fashion.

In the present study, we hypothesize that NO production in the middle ear has a possible role in some of the clinical features of cholesteatoma such as epithelial hyperproliferation and bone resorption, as is the case for IL-6 and TGF-beta.5 NO is produced in human respiratory tract broadly by activation of NOS. We found that human cholesteatoma epithelial cells also express two different NOS isoforms. In addition, iNOS immunoreactivity tends to be augmented in the area accompanying increased RANKL expression and severe inflammatory cell infiltration. RT-PCR analysis of cultured cholesteatoma cells has also shown constitutive iNOS and eNOS expression. Further elucidation of NO synthesis and regulatory mechanisms in the middle ear may imply valuable approaches to assess underlying inflammatory status as well as new treatment modalities to healing processes with restoration of mucociliary clearance abilities.

Acknowledgement

This study was supported in part by Grants-in-Aid for Scientific Research, administered by the Ministry of Education, Science, Sports and Culture, Japan.

The study protocol was approved by the Institutional Review Board at the Hiroshima University School of Medicine.

References

1.Bujía J, Schilling V, Holly A, Stammberger M, Kastenbauer E. Hyperproliferation-associated keratin expression in human middle ear cholesteatoma. Acta Otolaryngol 113:364–368, 1993

2.Jeong JH, Park CW, Tae K, Lee SH, Shin DH, Kim KR, Park YW. Expression of RANKL and OPG in middle ear cholesteatoma tissue. Laryngoscope 116:1180–1184, 2006

3.Jung JY, Lin AC, Ramos LM, Faddis BT, Chole RA. Nitric oxide synthase I mediates osteoclast activity in vitro and in vivo. J Cell Biochem 89:613–621, 2003

4.Chole RA. The molecular biology of bone resorption due to chronic otitis media. Ann N Y Acad Sci 830:95–109, 1997

5.Jung JY, Pashia ME, Nishimoto SY, Faddis BT, Chole RA. A possible role for nitric oxide in osteoclastogenesis associated with cholesteatoma. Otol Neurotol 25:661–668, 2004


Address for correspondence: Sachio Takeno, MD, PhD, Department of Otolaryngology, Hiroshima University School of Medicine, Kasumi 1–2-3, Minami-ku, Hiroshima 734–8551, Japan. takeno@hiroshima-u.ac.jp

Cholesteatoma and Ear Surgery – An Update, pp. 281–283

Edited by Haruo Takahashi

2013 © Kugler Publications, Amsterdam, The Netherlands