Activities of matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in idiopathic hemotympanum and otitis media with effusion

Abstract

Conclusion: The expression profile of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-2 (TIMP-2) was specific to the type of middle ear effusion. Further studies are necessary for elucidating its correlation with the sequelae of otitis media with effusion (OME) and idiopathic hemotympanum.

Objectives: We aimed to investigate the relative activities of gelatinases (MMP-2 and -9), stromelysin-1 (MMP-3), and matrilysin-1 (MMP-7), as well as measuring TIMP-2 levels in the serous and mucous effusions of OME and hemorrhagic effusion of the idiopathic hemotympanum.

Materials and methods: Middle ear effusions were collected from patients with OME and idiopathic hemotympanum, and were classified as mucoid, serous, or hemorrhagic. MMP activity in the effusion samples was examined by gelatin and casein zymography. Levels of TIMP-2 were measured by ELISA. Human temporal bone sections, with and without otitis media (OM), were examined histologically.

Results: One case showed tympanic membrane thinning in the OM group, but none in the control group. While MMP-2 was present in all effusions, the active form of MMP-2 was found only in mucous effusions. MMP-3 and MMP-7 activity was detected only in the mucous effusions. MMP-9 exhibited activity in all effusions, with the highest levels in mucous effusions. TIMP-2 levels were markedly elevated in serous effusions.

Figure 1

Archival human temporal bone sections with or without otitis media (OM). Horizontal sections crossing middle of manubrium were stained with hematoxylin and eosin. The tympanic membrane shows a typical conical shape (*) in the normal subject (A) while it is retracted and thinner in the case with OM (C). Blood vessels and connective tissue lateral to manubrium are not seen (arrow) in the case with OM comparing with the normal subject. The fibrous layer (FL) of tympanic membrane is thin and irregular in the case with OM (D), which is intact in the normal subject (B). Arrow heads: middle ear effusion. H & E stain. Original magnification: A and C, x10; B and D, x100. Scale bar: 0.5 mm. EAC: external auditory canal. ME: middle ear. L: lateral side. P: posterior side.

Figure 2

Gelatin zymography (A) demonstrates MMP-2 (72 kD) activity in all samples. Increased activity of MMP-2 (72 kD) and MMP-9 (92 kD) is present in mucous effusions in comparison with serous and hemorrhagic effusions. The active form of MMP-2 (66 kD) is noted only in mucous effusions, while the active form of MMP-9 (84 kD) is noted in both the mucous and the hemorrhagic effusions. In contrast, casein zymography (B) shows enzyme activities of MMP-3 (29 kD) and MMP-7 (57 kD) only in mucous effusions. EDTA treatment (lane E) completely abloshed these enzyme activities.

Figure 3

(A) Denstinometry of zymograms demonstrating different patterns of enzyme activities according to the type of effusion. It is noted that the activity of MMP-9 is highly elevated in all effusions, and is highest in mucous effusions. In contrast, levels of MMP-2 activity were not significantly different. The activities of MMP-3 and 7 are significantly up-regulated in the mucous effusion than in other effusions. (B) TIMP-2 levels according to the type of effusion. It was measured by ELISA, based on a two site sandwich format. The level of TIMP-2 is markedly elevated in serous effusions. Values are given as mean ± standard deviation. ND: not detected. n=5. *: p<0.05.