MECHANISM OF BONE DESTRUCTION IN CHOLESTEATOMA
Introduction
Cholesteatoma destructs bony structures of temporal bone to cause complications such as inner ear dysfunction, facial nerve palsy and so on. However, the mechanism of the bone destructive feature of cholesteatoma remains to be elucidated. It was recently reported that the RANKL (receptor activator of NF-κB ligand)-Th17 system contributed to bone destruction in rheumatoid arthritis.1 The RANKL-Th17 system was focused on as a possible mechanism of bone destruction in cholesteatoma.2,3
Material and method
Subjects were 33 patients with cholesteatoma and three patients with suppurative chronic otitis media (OMC) who were operated in Fukuoka University Hospital. Cholesteatoma was taken with surrounded granulation during surgery. For PCR, materials were snap-frozen using liquid nitrogen and preserved with the freezer of -80oC. Total RNA was obtained using TRIzol reagent (Invitrogen®) and quantitated. First-strand cDNA synthesis was done using SuperScript II (Invitrogen®). Real-time PCR of cDNA products was performed. Values were normalized by relative quantification. Valuesobtained were expressed as fold changes of gene expression. For flowcytometry, fresh materials were prepared as single cell and stained by antibodies. FACSCanto™II flowcytometer and FACSDiva™ software were employed.
Results
Immunity cytokine expression in cholesteatoma
Active inflammation was assessed by a clinical scoring system focused on three points: state of granulation (0: scar, 1; mild; 2: moderate; 3: marked); pre-operative ear discharge (0: none; 1: wet; 2: seromucous; 3: purulent); and cellular infiltration by pathohistological findings (0: none/few; 1: mild; 2: moderate; 3: severe). Active signs of granulation were evaluated by extent of redness, swelling and easy bleeding.
The value of tumor necrosis factor alpha (TNFα) was measured by PCR in order to compare the clinical score. The relative ratio of TNFα/GADPH was a hundred times larger than when the clinical score was equal or higher than three. Thereafter, we classified the inflammation phase by TNFα value of 102. Since macrophage is reported to secrete TNFα, macrophage might be activated in a severe inflammation phase of cholesteatoma.
Acquired immunity in cholesteatoma was investigated by flowcytometry. Cells were gated by T-cell receptor beta (TCR-β). Two populations with cell surface markers CD4 and CD8 were noted (data not shown). CD69 is a T cell early activate marker. It was expressed only in CD4-positive cells. It indicated that helper T cells (Th cell) were activated as acquired immunity in cholesteatoma.
Cytokine expression in cholesteatoma was investigated by real-time PCR. Interferon gamma (IFNγ), IL-23 and IL-17 were expressed more in the TNFα high group than in those in the low group. IFNγ enhances activation of macrophage which secretes IL-23. IL-23 induces differentiation of Th17 which releases IL-17.
Bone destruction-related substances in cholesteatoma
Bone destruction related substances were investigated in severe inflammatory cholesteatoma by real-time PCR. RANKL and one of the proteins of type I matrix metalloproteinase (MMP-1)2 were found significantly more in the TNFα-high group. This indicated that bone was being destructed in the severe inflammatory phase of cholesteatoma. By flowcytometry, RANKL was found only in CD4-positive cells. This may suggest that Th17 cells contain RANKL.
Specificity of the RANKL-Th17 system in cholesteatoma
Relative mRNA expression of various substances was compared between cholesteatoma of the TNFα-high group and granulation of suppurative chronic otitis media. The value of RANKL was higher in cholesteatoma than that of OMC. It is indicated that RANKL-Th17 system is induced more specifically in cholesteatoma than in other otitis media.
Discussion
Figure 1 shows a hypothesis of bone destruction in cholesteatoma from the present results. Once inflammation occurred, macrophage was activated as an innate immune system in the severe inflammatory phase of cho-lesteatoma and secretion of IL-23 and IFNγ. IL-23 from activated-macrophage induces differentiation of Th0 to Th17. Activated Th17 cell expresses bone destructive factor, RANKL, on itself, and releases IL-17. Both RANKL and IL 17 involve bone destruction in a certain way.4 Resulting from bone destruction, MMP-1 can be detected.5 Since the value of RANKL was higher in cholesteatoma than that of OMC, the RANKL-Th17 system is more activated in cholesteatoma. Further studies are necessary to investigate why cholesteatoma predominantly induces the RANKL-Th17 system compared to other otitis media. There is a possibility that an inhibitory agent against the RANKL-Th17 system suppresses the bone destruction feature of cholesteatoma.
Fig. 1. Hypothesis of bone destruction in cholesteatoma. Mφ: macrophage.
1.Sato K et al. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction. J Exp Med 203(12):2673–282, 2006
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(7):1180–1184, 2006
3.Kuczkowski J, Sakowicz-Burkiewicz M, Iżycka-Świeszewska E. Expression of the receptor activator for nuclear factor-κB ligand and osteoprotegerin in chronic otitis media. Am J Otolaryngol 31(6):404–409, 2010
4.Haruyama T, Furukawa M, Kusunoki T, Onoda J, Ikeda K. Expression of IL-17 and its role in bone destruction in human middle ear cholesteatoma. ORL J Otorhinolaryngol Relat Spec 72(6):325–331, 2010
5.Choi HM, et al. Adiponectin may contribute to synovitis and joint destruction talloproteinase-1, and matrix metalloproteinase-13 expression in fibroblast-like synoviocytes more than proinflammatory mediators. Arthritis Research & Therapy 11(6):R161, 2009
Address for correspondence: Yoshiki Ohnishi, y.ohnishi@minf.med.fukuoka-u.ac.jp
Cholesteatoma and Ear Surgery – An Update, pp. 203–205
Edited by Haruo Takahashi
2013 © Kugler Publications, Amsterdam, The Netherlands