Mucin gene expression in the effusions of otitis media with effusion

1. Introduction 

Otitis media with effusion (OME) is the most common cause of deafness in children in the developed world. The primary event is inflammation of the middle ear mucosa, usually due to the presence of bacteria. This leads to the release of inflammatory mediators, which cause secretion of a mucin-rich effusion by up-regulating mucin genes. Mucins constitute a heterogeneous group of high molecular weight, polydisperse and highly glycosylated molecules. Recent developments in cloning human mucin genes now enable us to approach the peptide moiety of these macromolecules. Twelve different mucin genes (MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC11 and MUC12) have been described in the literature to date [1], [2]. MUC10 is described only in mouse and its human equivalent is not known. Expression of human mucin genes is studied in various organs and it is known that MUC2, MUC4, MUC5AC and MUC5B are expressed in the bronchus [3]. As for mucin expression in the nasal tissues, it has been reported that MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 were expressed both in the normal and vasomotor rhinitis inferior turbinates [4]. Regarding the middle ear, MUC5B mucin gene expression was identified in human middle ear with chronic otitis media [5] and expression of MUC1, MUC2, MUC5AC and MUC5B genes are detected in cultured human middle ear epithelial cells [6]. However, the information of mucin gene expression in otitis media with effusion is lacking, partly because it is difficult to obtain epithelial cells from the patients with otitis media with effusion.

Cytologic studies of middle ear effusion have shown that considerable numbers of epithelial cells can be observed [7]. Although middle ear mucosa itself is the most reliable source of information to study middle ear pathology, it is not easy to obtain middle ear mucosa in the clinical practice. We thought that it may be possible to obtain information on mucin gene expression from analyzing middle ear effusion because effusions contain exfolitative epithelial cells [7].

In this study, we tried to clarify (1) if mucin gene expression can be detected in the middle ear effusion; and if so (2) which mucin genes are expressed in the effusions. For these purposes, we performed RT-PCR analysis for mucin gene expression. Moreover, we examined mucin gene expression in the bronchial epithelial cell line BEAS-2B [8] and compared it with that in the middle ear.

2. Materials and methods 

For analysis by RT-PCR, middle ear effusions were obtained from five patients with otitis media with effusion. The profile of these patients is shown in Table 1. All the patients underwent clinically indicated operations. The diagnosis of otitis media with effusion was based on a compatible history, pneumatic otoscopic examination and the following two criteria: conductive hearing loss on pure tone audiogram and type B tympanogram.

Table 1. List of effusions examined by RT-PCR

	Case	Age	Sex	Characteristics of effusions	Duration of effusions (months)
	1	10	Female	Mucopurulent	5
	2	7	Male	Serous	2
	3	10	Female	Mucopurulent	1
	4	20	Male	Serous	4
	5	74	Female	Serous	2

RNAs were extracted from middle ear effusion and BEAS-2B cells as a control. The expression of mucin genes was analyzed by RT-PCR. Immediately after specimens were obtained, total RNA was obtained using Katrimox 14 (Takara Biochemicals, Tokyo, Japan).

The primers of RT-PCR analysis used in this study are summarized in Table 2. The first-strand cDNA was synthesized using an oligo dT primer and GeneAmp RNA PCR kit (Takara Biochemicals). After incubation at 42°C for 60 min, the samples were heated for 5 min at 99°C to terminate the reactions and were stored in 40 μl of H2O at −20°C until used. Positive control reactions consisted of amplification of β-actin to confirm successful first-strand cDNA synthesis and to ensure that first-strand cDNA synthesis was quantitatively similar among different RNA samples. For RT-PCR of mucin mRNA expression, 1 μl of diluted cDNA were amplified in 50 μl of reaction mixtures containing 0.5 μM of each oligonucleotide primer, 200 μmol/l dNTPs, 1×Taq DNA polymerase buffer and 2 U of Tap DNA polymerase (Takara Biochemicals), overlaid with mineral oil. Reaction conditions used with a thermal cycler were as follows: 95°C for 5 min; 35 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 1 min; 72°C for 10 min. PCR products were electrophoresed on a 2% agarose gel, denatured and neutralized and then transferred onto a nylon membrane (NEN Research Products, MA). The membranes were fixed with UV light.

Table 2. RT-PCR primers and probes for Southern hybridization for mucin genes

	mRNA size	Primer sequences
	MUC1 533 bp	U: 5′-GCCAGGGCTACCACAACCCCAGCC-3′
		L: 5′-CACCCCAGCCCCAGATGGGCAGA-3′
		P: 5′-GCCTTCCGAGAAGGTACCATCAATGTCCAC-3′
	MUC2 375 bp	U: 5′-CATGGAGGCCCCCGGGGAGTGCTG-3′
		L: 5′-TGTCCCGCAGGACCCGGAGCAATG-3′
		P: 5′-ATGCCCAATGGATGCTGCAAGACCTGCACC-3v
	MUC3 260 bp	U: 5′-ACCACCTCTGAGACCCCCTCACACAG-3′
		L: 5′-GGCCACGCCACCTCTCTTCTTGTC-3′
		P: 5′-ACCACCTCACACAGTACTCCCAGCTACACTACC-3′
	MUC4 478 bp	U: 5′-GGCCACGCCACCTCTCTTCTTGTC-3′
		L: 5′-GGATGCTGAGGAAGCGTCGGTGAC-3′
		P: 5′-TCACGCCACCCCTCTTCATGTCACCAGCCT-3′
	MUC5AC 438 bp	U: 5′-CACCGGCCTCACCCGACGCCCACC-3′
		L: 5′-GATGGGGCCGGCCTCCCGGAGAGC-3′
		P: 5′-ACTGTGATCCCCCCACTGCTGTTCTATGAG-3′
	MUC5B 698 bp	U: 5′-CCAGGGTCAGCCCCCGCCGTGCAA-3′
		L: 5′-CGGGCAGGAGCCCTCGCAGAAGGT-3′
		P: 5′-CAGCCAGTCCAGCTGAATGAAACCTGGGTC-3′
	MUC6 498 bp	U: 5′-GCCACCAGCAGCAGGCCACCACCA-3′
		L: 5′-GTGGGTCACAGGGGTGCTGGTGTC-3′
		P: 5′-AGGATCAGCCAAGCCCACAGCTCAATCAGC-3′
	MUC7 362 bp	U: 5′-CTGGACTGCTAGCTCACCAGAAGCCG-3′
		L: 5′-GGGTGGGGCAGCTGTGGTGTCTTG-3′
		P: 5′-AGCAGTGTGGTCAACCCTACCTTAGTGGCT-3′
	MUC8 240 bp	U: 5′-GACAGGGTTTCTCCTCATTG-3′
		L: 5′-CGTTTATTCCAGCACTGTTC-3′
		P: 5′-ACTAATAGTCTGCTGTGTGAATCCACCGCT-3′
	MUC9 510 bp	U: 5′-CACTGGGGCAATGACCATGA-3′
		L: 5′-ACAAAGCGGTTGTCAAAAGC-3′
		P: 5′-CTTAGACAGAATACAGTGGCCCCTA-3′
	MUC11 272 bp	U: 5′-ACCTCACCCATCAGTTCAGG-3′
		L: 5′-CCTGGGCTGCTGTGGAAGGT-3′
		P: 5′-CGACCAGGCTCAACGCACACAACAG3′
	MUC12 530 bp	U: 5′-CGGTGACTCCGGGCTTTGAC-3′
		L: 5′-CTGGATGTGGAGCTCTGTGC-3′
		P: 5′-CATTGATCATCCTAATCATCTTATT-3′
	β-Actin 246 bp	U: 5′-ATGGATGATGATATCGCCGCG-3′
		L: 5′-ATGTCGTCCCAGTTGGTGACGAT-3′
		P: 5′-GTCGCCCACATAGGAATCCTTCTGACCCAT-3′

In experiments designed to validate the PCR products, oligonucleotide probes internal to the amplified fragments were synthesized. Some 5 pmole of each probe were end-labeled with 500 μCi of [γ-32P]ATP using 10 U of T4 polynucleotide kinase. The nylon membranes were prehybridized in 20 ml of buffer composed of 0.25% SDS, 1.2×SSC, 2×Denhardt's and 100 μg/ml salmon sperm DNA at 50°C for 2 h. Hybridization was conducted in the presence of each labeled oligonucleotide probe in the same solution as in prehybridization by incubation at 50°C for 3 h. The blots were washed in 2×SSC, 0.05% SDS.

3. Results 

In middle ear effusions, MUC1, MUC4, MUC5AC, MUC6, MUC7, MUC8, MUC9 and MUC11 were expressed to various degrees (Fig. 1). MUC5AC expression was detected clearly in lane No. 1 and very weakly in lane No. 3 (hardly seen in the figure but recognizable on the original film). MUC2, MUC3, MUC5B or MUC12 were not expressed in any samples examined. Among the eight mucin genes of which expression was observed, the bands intensity was highest in MUC4 and MUC11, but this does not necessarily mean that these two are the dominant mucin in the middle ear effusion. Since PCR primers for MUC4 and MUC11 were designed from tandem repeat regions, the PCR products were not single bands but dispersed smear.

View Large Image Download to PowerPoint

Fig. 1. RT-PCR of 12 mucin genes mRNA and β-actin mRNA. cDNA was synthesized using an oligo dT primer from total RNA from each specimen. The expression of 12 mucin mRNA and β-actin was examined using 13 different pairs of PCR primers and different internal control probes to verify the PCR products by Southern blot analysis.

The mucin gene expression in BEAS-2B cells were similar to that in middle ear effusions. The major difference was the absence of MUC6 gene expression in the BEAS-2B cells.

4. Discussion 

The first objective of our study was to know if mucin gene expression can be detected in the middle ear effusions. In the middle ear effusions in the patients with otitis media with effusion, epithelial cells can be found in addition to lymphocytes, neutrophils, eosinophils and macrophages [7]. In this present study, we succeeded in amplifying mucin gene mRNA in the middle ear effusion, which means that the number of epithelial cells in the effusions are sufficient for the detection by PCR. Previously, mucin gene expression has been studied using middle ear mucosa of the chronic otitis media patients [5], cultured human middle ear epithelia cells [6] or rat middle ear [9], [10]. This report is the first to utilize middle ear effusions as the source of mRNA detection. The advantage of our method is that it is easy to obtain the materials and that the result obtained through this method better reflect the pathology of middle ear mucosa compared with culture method. However, our method has some limitations. Since epithelial cells in the effusions are exfolitative cells, it is likely surface epithelial cells may not contain submucosal gland cells. Moreover, mRNAs contained in the epithelial cells in the effusions may likely be degraded to some extent.

Our second objective was to clarify which mucin genes are expressed in the middle ear effusions in the patients with otitis media with effusion. In middle ear effusions, MUC1, MUC4, MUC5AC, MUC6, MUC7, MUC8, MUC9 and MUC11 were expressed to various degrees. MUC5AC expression was detected in only one sample. MUC2, MUC3, MUC5B or MUC12 were not expressed in any samples examined. Moon et al. [6] reported the expression of MUC1, MUC2, MUC5AC and MUC5B genes in cultured human middle ear epithelial cells, though the expression of the latter three was weak. Audie et al. [3] showed that MUC5AC expression is higher than MUC2 expression in bronchus by in situ hybridization. Thus, MUC2 is not a major mucin in the upper airway. Among the mucin genes, MUC2 and MUC5AC are known to be upregulated by inflammatory mediators, such as cytokines, lipopolysaccharide and neutrophil elastase. Lin et al. [10] reported that rat Muc2 mucin gene expression was induced in rat middle ear by tumor necrosis factor-α. At least in vitro, MUC5AC mucin secretion is upregulated by TNF-α and IL-6 [11], [12]. In the present study, MUC2 gene was not detected in any sample and MUC5AC was expressed clearly in lane No. 1 and weakly in lane No. 3. These two effusions were both mucopurulent in appearance and the other three that did not express MUC5AC were serous. Tsuboi et al. [13] recently examined pattern changes of mucin gene expression with pneumococcal otitis media in rats and found that rat Muc5 mRNA, which is equivalent to human MUC5AC, is expressed upon infection for prolonged period of time (>14 days). Inflammatory mediators or bacteria in the two effusions (lane Nos. 1 and 3) may have upregulated MUC5AC gene expression.

Although the sample size is small in the present study, duration of the effusion does not seem to correlate to mucin gene expressions. Age may be a factor influencing mucin gene expression in the effusion. Since most of the effusion in adults are serous and are seldom accompanied by infection, it may be unlikely that MUC2 or MUC5AC mucin genes are expressed in the effusions of the adult patients.

Hutton et al. [14] reported that human middle ear effusions contained heterogeneous mucin protein cores and their result suggested the presence of MUC5B epitopes in the effusions. Kawano et al. [5] identified MUC5B mucin gene in human middle ear with chronic otitis media. Like these, MUC5B gene product may be a major component in the mucin of middle ear effusions. The absence of MUC5B mRNA from the present study could be interpreted as follows. According to the in situ hybridization study by Aust et al. [4], MUC5B mRNA was localized almost exclusively in submucosal glands. Since we examined possible exfolitative cells, submucosal gland cells that may contain MUC5B mRNA were not contained in the effusions examined in the present study.

In the present study, we used BEAS-2B cells as a control and compared the result of middle ear effusion with that of the BEAS-2B cells. The BEAS-2B cell line was derived from normal human bronchial epithelial cells immortalized using an SV40-adenovirus 12 hybrid virus [8]. The expression of mucin gene in the middle ear effusion was not much different from that in the bronchus. The major difference was the presence of MUC6 expression in middle ear effusions. MUC6 has been believed to be an intestinal type of mucin and it was agreed that MUC6 is not expressed in the normal bronchial and nasal tissue [4]. However, recent immunohistochemistry report using MUC6 antibody revealed that MUC6 mucin is detected in the distal, peritumoral and tumoral bronchial epithelial from patients with carcinoma [15].

MUC9, MUC11 and MUC12 are new genes and their expression in the ear and airway has not been known. MUC9 was known as oviduct-specific mucin [16]. Since MUC9 was expressed both in the middle ear effusion and in the bronchus, its expression may be wider than expected earlier. MUC11 is known to have wide epithelial distribution [2] and both middle ear effusion and bronchus expressed MUC11. MUC12 was not detected in the present study and this is compatible with the previous report [2] that MUC12 was restricted to the colon and the pancreas.

Recent advancement of mucin gene studies has clarified that some mucin gene products are membrane-bound and others are secretary [1], [2]. Moreover, signal transduction from certain stimuli to specific mucin gene expression has recently been reported [17], [18]. Thus, accumulation of information and characterization about each mucin gene is being made. Studies of mucin gene expression in the middle ear effusions may give us various information concerning conditions in the middle ear cavities.

In conclusion, we successfully amplified mucin gene mRNA from middle ear effusions. Thus, middle ear effusion can give us valuable information on mucin gene expression in the middle ear. RT-PCR revealed that MUC1, MUC4, MUC5AC, MUC6, MUC7, MUC8, MUC9, MUC11 and MUC12 are expressed in the middle ear effusions. Moreover, there is similarity between mucin gene expression in the middle ear effusion and that in the bronchial epithelia.