Matrix metalloproteinase-8/collagenase-2 in childhood otitis media with effusion

Abstract

Background. Matrix metalloproteinases (MMPs), gelatinases, have been associated with otitis media with effusion (OME), but the role of collagenase-2/matrix metalloproteinase-8 (MMP-8) in OME has not been studied previously. We studied the levels, isoenzyme distribution, and activation of MMP-8 in childhood OME, and also the levels of pro- and active forms of MMP-2 and -9 as well as 120 kDa gelatinase complexes were assessed.

Methods. Seventy middle ear fluid (MEF) samples were collected from 54 children with OME and classified to mucoid (n = 39) or serous (n = 31). MMPs were studied from MEF samples by time-resolved immunofluorometric assay, Western immunoblotting, and gelatin-zymography.

Results. MMP-8 was found in its active form in MEF of children with OME. MMP-8 levels were significantly higher in mucous relative to serous OME. The pro- or active MMP-2 and -9 were found in MEF, but no MEF type-specific differences were found.

Conclusion. Our results suggest that MMP-8 may play a role in the pathogenesis of childhood OME. New therapeutic strategies with MMP inhibitors targeting MMP-8, but allowing MMP-8 to carry out the protective action, may play a role in the future treatment of otitis media and OME. However, further studies of this topic are needed.

Key words::

• Matrix metalloproteinase
• matrix metalloproteinase-8
• MMP
• MMP-8
• MMP inhibitor
• otitis media
• otitis media with effusion

Abbreviations
IFMA	=	immunofluorometric assay
MEE	=	middle ear effusion
MEF	=	middle ear fluid
MMP	=	matrix metalloproteinase
MMP-2	=	matrix metalloproteinase-2
MMP-8	=	matrix metalloproteinase-8
MMP-9	=	matrix metalloproteinase-9
NGAL	=	neutrophil gelatinase associated lipocalin
OME	=	otitis media with effusion
proMMP-2	=	promatrix metalloproteinase-2
proMMP-8	=	promatrix metalloproteinase-8
proMMP-9	=	promatrix metalloproteinase-9

Key messages

• Matrix metalloproteinase-8 (MMP-8)/collagenase-2 was found in its active form in middle ear fluid of children with otitis media with effusion.

• MMP-8 levels were significantly higher in mucoid than in serous middle ear fluid of children with otitis media with effusion.

• New therapeutic strategies with MMP inhibitors targeting MMP-8 at an early phase of the disease, allowing MMP-8 to carry out the protective action, may play a role in modulating the course of otitis media and otitis media with effusion in the future.

Introduction

Otitis media with effusion (OME) is a chronic inflammation and fluid accumulation in the middle ear lasting more than 3 months (1). Although OME is extremely common in children, the precise etiopathogenesis or healing mechanisms have remained unclear. Spontaneous healing of OME in children happens, but the process may take several months. Except insertion of tympanostomy tubes no other treatment options exist for OME. OME can lead to structural changes in tympanic membrane and impact hearing function (1). Tissue-destructive proteases of both the serine and metalloproteinase (MMP) classes have been suggested to play a role in the pathogenesis of otitis media (OM) (2). Changes in the tympanic membrane impact hearing function (1). There are controversies regarding whether simple OME can lead to structural changes in the tympanic membrane without recurrent inflammation (1). It has been suggested that gelatinases, MMP-2 and-9, may be involved in tympanic membrane damage and prognosis of OME (1, 3). However, the role of collagenase-2, matrix metalloproteinase-8 (MMP-8) has not been studied previously in OME.

The genetically distinct but structurally related MMPs are zinc-dependent metalloendopeptidases, which can be classified based on their primary structures and substrate specificities into several groups, such as collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -10, -11, -19), matrilysins (MMP-7, -26), membrane-type MMPs (MT-MMPs) (MMP-14, -15, -16, -17, -24, -25), and other MMPs (4,5). MMPs can degrade almost all components of the extracellular matrix and basement membrane as well as process serpins, growth factors, pro- and anti-inflammatory cytokines, chemokines, and apoptotic signals to modulate the immune response (4–6). The decisive substrate cleavages of MMPs can direct their function to be either surrogately tissue-destructive or defensive and anti-inflammatory (5,7–9). Most MMPs are secreted as latent, inactive forms, proMMPs (4,5). Their extracellular or membrane-associated activation involves a complex conformational change, induced by oxidants or proteases (5). We set up this work to study the role of MMP-8, its levels, isoenzyme distribution, and degree of activation, in mucoid and serous effusions of OME. Also levels of pro- and active forms of MMP-9 and -2 as well as high molecular size 120 kDa gelatinase complexes were measured.

Patients and methods

Children

The study population comprised 54 children (32 boys), median age 5.4 years (range 2.8–9.8 years) with OME, who had been admitted for elective surgery (adenoidectomy or adenotonsillectomy) either to the Institute of Otorhinolaryngology of the University of Debrecen or to the County Hospital of Miskolc from November 2006 to April 2007 (10). All children were free of acute respiratory infections at least during the preceding 3 weeks and at the time of surgery. Children were admitted for grommet insertion for OME with or without adenoidectomy or adenotonsillectomy.

All children had conductive hearing loss documented by pure tone audiometry and type B tympanograms for a minimum of 4 weeks prior to operation. A total of 21 (39%) children had a bilateral disease, but 33 (61%) children had middle ear effusion (MEE) only in one of the ears; altogether 70 middle ear fluid (MEF) samples were available. The study protocol was accepted by the local ethical committee of the University of Debrecen. A written informed consent was obtained from each child's guardians.

Sample collection

MEF were obtained under general anesthesia. After mechanical cleaning of the external auditory canal, a myringotomy was performed on the anterior part of the tympanic membrane, and specimens of MEF were aspirated with suction to a sterile glass tip. Samples were characterized as mucoid or serous effusions based on the viscosity of the fluid. Samples were rinsed out from the collector with 1 mL of PBS into sterile tubes, frozen immediately, and stored at −70°C for 2 weeks to 7 months before processing. Samples were transported on dry ice to the University of Helsinki in Finland, where the MMP analyses were carried out.

Time-resolved immunofluorometric assay (IFMA)

MMP-8 concentration was determined by a time-resolved immunofluorometric assay (Medix Biochemica, Kauniainen, Finland) (11,12). The monoclonal MMP-8-specific antibodies 8708 and 8706 (Medix Biochemica, Kauniainen, Finland) were used as a catching antibody and a tracer antibody, respectively. The tracer antibody was labeled using europium-chelate. The assay buffer contained 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 5 mM CaCl₂, 50 µM ZnCl₂, 50 µM ZnCl₂, 0.5% BSA, 0.05% sodium azide, and 20 mg/L diethylenetriaminepentaacetic acid (DTPA). Samples were diluted in assay buffer and incubated for 1 hour, followed by incubation for 1 hour with tracer antibody. Enhancement solution was added, and after 5 min fluorescence was measured using a 1234 Delfia Research Fluorometer (Wallac, Turku, Finland). The specificity of the monoclonal antibodies against MMP-8 corresponded to that of polyclonal MMP-8 (13,14). The interassay coefficient of variation (CV) % was 7.3% (n = 28) and detection limit for the assay 0.08 µg/mL (12). Data are expressed as ng/mL.

Western immunoblotting

The molecular forms and degree of activation of MMPs in middle ear effusion were analyzed by Western immunoblotting analysis using specific rabbit polyclonal antisera to human MMP-2, -8, and -9 as previously described (13,15). After SDS-PAGE under non-reducing conditions, the proteins in the gel were electrotransferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Richmond, CA, USA). After blocking with 3% gelatin, the membrane was first reacted with the primary antibody diluted 1:500 and then with an alkaline phosphatase-conjugated secondary antibody. Immunoreactive proteins were visualized with nitro blue tetrazolium (Sigma, St Louis, MO, USA) and 5-bromo-4-chloro-3-indolyl-phosphate (Sigma, St Louis, MO). MMP immunoreactivities were in the linear range of the reaction assessed as previously described (16). Quantitation was performed by Bio-Rad Model GS-700 Imaging Densitometer using the Analysis program (16). Data are expressed as densitometric units (DU). Human polymorphonuclear neutrophil (PMN) extract (14) and rheumatoid synovial culture medium were used as positive controls for PMN-type and mesenchymal-type MMP-8 isoforms (17). Recombinant human MMP-2 and -9 were used as positive controls for MMP-2 and -9 (17,18).

Gelatin-zymography

Molecular forms and degree of activation of gelatinase A (72 kDa MMP-2) and gelatinase B (92 kDa MMP-9) and 120 kDa high molecular size gelatinase complexes in OME samples were analyzed by zymography using 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) (containing 1 mg/mL gelatin (Sigma, St Louis, MO) as a substrate, as previously described (15,18)). After electrophoresis, the gels were washed for 30 min with Tris-HCl buffer, pH 7.5, containing 2.5% Tween 80, 0.02% NaN₃, and then for 30 min with the same buffer supplemented with 0.5 mM CaCl₂ and 1 µM ZnCl₂. Finally, the gels were incubated in 50 mM Tris-HCl buffer, pH 7.5, containing 0.02% NaN₃, 0.5 mM CaCl₂, and 1 µM ZnCl₂, overnight at 37°C. The reaction was interrupted by staining the gels with 0.1% Coomassie brilliant blue R250 and destaining (18). The gelatinolytic activity was visualized as a clear band against a blue background. The band intensities were quantified densitometrically and were in the linear range of the reaction assessed as previously (18,19). The gelatinolytic activities are expressed as densitometric units, DU. Human recombinant 92 kDa neutrophil MMP-9 and recombinant 72 kDa MMP-2 were used as positive controls (18). The gelatinases A (MMP-2), B (MMP-9), and 120 kDa high molecular size gelatinase complexes representing MMP-9 neutrophil gelatinase-associated lipocalin (NGAL) detected by zymography were identified by Western immunoblotting and by specific polyclonal and monoclonal antibodies as described previously (18,20).

Statistics

Data were analyzed by using GraphPad Prism version 4.0 (GraphPad Inc., San Diego, CA, USA). Data of two groups were compared by Mann-Whitney test. The results are presented as median (range). A P value less than 0.05 was considered statistically significant.

Results

MMP-8 in OME

Median (range) MMP-8 levels were significantly higher in mucoid MEF (n = 39) than in serous MEF (n = 31) of children with OME [5,621 ng/mL (60.00–21,056) versus 3,055 ng/mL (77.42–14,012), P = 0.028] (Figure 1). Western blot analysis showed that MMP-8 was found in its active form in MEF of children with OME (Figure 2). Western immunoblot analysis of MMP-8 isoenzyme distribution revealed that the major MMP-8 species detected in mucoid and serous middle ear effusions was 65–75 kDa neutrophil (PMN)-type MMP-8 (Figure 2). Very low or hardly any immunoreactivities corresponding to fibroblast-type or mesenchymal cell-type 45–55 kDa MMP-8 could be detected.

Figure 1. MMP-8 levels analyzed by IFMA in effusions of children with otitis media (n = 70), of which 39 were classified to mucoid and 31 to serous effusions. Median MMP-8 levels were significantly higher in mucoid middle ear fluid (MEF) than in serous MEF, analyzed by Mann-Whitney test.

Figure 2. Representative Western immunoblot analysis of MMP-8 (collagenase-2) in middle ear effusions. Lanes 2–4 represent mucous and lanes 5–7 serous middle ear effusions. Lane 8 represents human neutrophil (PMN) and lane 9 human rheumatoid synovial fibroblast (F) culture media fluid used as positive controls for pro- and active PMN and mesenchymal (Mes)-type MMP-8 isoforms, respectively, of which mobilities are indicated on the right. Mobilities of the molecular weight markers (MW) are indicated on the left (lane 1).

MMP-2 and MMP-9 in OME

Pro- and active MMP-2 and -9 were found both in mucoid and serous MEF (Figure 3, Table I). However, the levels of pro- and active MMP-2 as well as pro- and active MMP-9 together with 120 kDa high molecular size gelatinase species identified as MMP-9-NGAL-complexes showed no statistical significant difference when compared to each other (Table I).

Figure 3. Representative gelatin-zymography analysis of molecular forms and degree of activation of gelatinases (MMP-2 and -9) in middle ear effusion. Lanes 2–4 represent mucous and lanes 5–7 serous middle ear effusion. Lane 8 represents human 92 kDa MMP-9 and lane 9 represents human 72 kDa MMP-2. Mobilities of 92 kDa proMMP-9, 82 kDa active MMP-9, 72 kDa proMMP-2, and active 62 kDa MMP-2, as well as 120 kDa gelatinase (MMP-9-NGAL) complex, are indicated on the right. Mobilities of the molecular weight markers (MW) are indicated on the left (lane 1).

Table I. Gelatinolytic activity in middle ear fluid (n = 70) of children with early phase of otitis media with effusion (OME). Gelatinolytic activity was assayed by gelatin-zymography.

	Mucoid middle ear fluid (n = 39)	Serous middle ear fluid (n = 31)	P
ProMMP-9	5.7 (0–9.3)	6.2 (0–10.1)	0.717
Active MMP-9	2.6 (0–8.7)	1.7 (0–9.7)	0.957
ProMMP-2	0.0 (0–7.9)	0.0 (0–7.3)	0.497
Active MMP-2	0.0 (0–8.5)	0.0 (0–5.7)	0.849
120 kDa gelatinase	5.1 (0–8.9)	6.1 (0–8.8)	0.829

Data are expressed as median (range) of densitometric units and analyzed by Mann-Whitney test.

Discussion

We demonstrated that mainly PMN-derived MMP-8 in its active form is present in MEF of children with OME. Additionally the MMP-8 levels were significantly increased in mucous middle ear effusion (MEE) relative to serous MEE, reflecting more severe inflammatory response and neutrophil activity in mucoid than in serous OME as previously found with neutrophil elastase-alpha 1-antitrypsin in children with mucoid or serous OME (21).

Moon and co-workers suggested that MMP activity of middle ear effusions, in combination with other pro-inflammatory mediators, can cause structural damage to the tympanic membrane thus impairing the hearing function (1). The extracellular matrix components, including collagen fibers, support the tympanic membrane, and its processing results in structural changes (22). It has been recently found that fibrillar collagen types I, II, and III, the primary substrates of MMP-8, are present in tympanic membrane (23,24), and the collagen content of the tympanic membrane is modified during the inflammatory and healing process (23). However, the enzymes involved in degrading the extracellular matrix of the tympanic membrane are not well known. Increased activity of MMPs other than MMP-8 has earlier been found in MEEs and in cholesteatoma tissue (3,25,26). Our results indicate that also collagenase-2/MMP-8, capable of efficiently breaking down native fibrillar collagens and other non-matrix bioactive substrates including cytokines, chemokines, apoptotic signals, and complement components (7), may play a significant role in OME, also possibly contributing to structural and functional damage of the tympanic membrane. On the other hand, MMPs may also be a part of the healing process of OME and collagen turnover of tympanic membrane (23).

In the present study pro- and active MMP-2 and -9 were found in effusion of mucoid and serous MEF. No differences between MMP-2 and -9 were found between mucoid and serous MEF. These findings differ from earlier studies. An earlier study has demonstrated that the activity of both MMP-2 and -9 was higher in thick than in thin effusion (3). Also high levels of active forms of MMP-2 and -9 have been found in mucoid MEF (1). However, in that study the mean duration of OME was longer than in our study, and also the patients were older: mean age 18 years compared to our study children's median age 5 years. The OME duration and age differences may at least partly explain the differences, while OME is known to be a fluctuating condition that varies with time and age. Therefore having only one sample/ear and no follow-up, the differences between measurements cannot be avoided. Notably, the follow-up in OME with repeated MEF samples is difficult because spontaneous recovery from the disease also happens.

Antonelli and co-workers demonstrated significant inhibition of MMPs by a protease inhibitor in MEEs (2). The use of a broad-spectrum MMP inhibitor has been shown to alter the outcome of acute otitis media and OME (27). Therapeutic strategies with anti-MMP molecules are currently being developed and may play a role in modulating the course of non-neoplastic otorhinolaryngological diseases (27). Based on our results also MMP-8 inhibitors could possibly play a role as a target of new therapeutic strategy of OME. Tetracyclines and non-antimicrobial chemically modified tetracyclines can inhibit MMPs (28–30). Doxycycline and lymecycline have been found to inhibit MMP-8 and proMMP-8 activation by different mechanisms (13,17,28,31,32) and also MMP-9 (30). Recently a synthesized peptidomimetic gelatinase B inhibitor Regasepin1 was found also to inhibit, almost to the same degree, the neutrophilic enzymes MMP-8 and MMP-9 (33). Because of the pro- and anti-inflammatory characteristics of MMP-8 and MMP-9 (6,8) the less efficient synthetic broad-spectrum MMP inhibitors, like doxycycline or lymecycline, may be favorable to reduce mainly the pathological excessive action of MMP-8 and MMP-9 allowing them to carry out their anti-inflammatory, protective actions (5,6,8,30,32,34), because MMPs may also be a part of the healing process (5–7).

OME is in children a common disease, which is frequently treated with surgical insertion of tympanostomy tubes. Also OME can last long time, over 2 years (1). In addition OME can impair the hearing function, which eventually may disturb the development of the children (35,36). Also while OME is common, it leads to an economic burden not only on children's parents but also on the surrounding society. For all these reasons new treatment strategies for otitis media and OME will be welcome. We suggest that the new therapeutic strategy of otitis media and OME in the future consisting of broad-spectrum MMP inhibitor targeting MMP-8 and selectively reducing the pathological excess of MMPs, but also allowing MMP-8 to carry out its defensive actions (5–8) at an early phase of the disease, could better the prognosis of otitis media and OME. More studies of this topic are needed.

As a conclusion, we have demonstrated that MMP-8 in its active form is present in high, pathological levels in MEF of children with OME. Furthermore, the MMP-8 levels are significantly higher in mucous MEF compared to serous MEF. New therapeutic strategies with MMP inhibitors, safe for children and targeting MMP-8 at early phase of the disease allowing MMP-8 to carry out the protective action, may play a role in modulating the course of otitis media and OME in the future.

Acknowledgements

The authors thank Maiju Kivistö for secretarial work.

Declaration of interest: The study was supported by grants from the Academy of Finland and the Helsinki University Central Hospital Research Foundation. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.