Expression of MUC5AC and MUC5B in human sinus mucosa by reverse transcription–polymerase chain reaction (RT-PCR). Ethidium bromide–stained agarose gel shows the presence of 409–base pair (bp) and 415-bp RT-PCR product using specific primer for MUC5AC and MUC5B. Shown are examples of RT-PCR products of MUC5AC, MUC5B, and GAPDH in 2% agarose gel with ethidium bromide stain. The plus sign indicates positive control; minus sign, negative control.
Comparison of the MUC5AC/GAPDH and MUC5B/GAPDH messenger RNA (mRNA) ratios between the tissue samples of normal sinus mucosa and chronic rhinosinusitis. Expression was significantly increased in the chronic rhinosinusitis mucosa compared with that in the normal sinus mucosa (P<.05).
Cellular localization of MUC5AC immunoreactivity in human sinus mucosa. MUC5AC immunoreactivity is present in sinus epithelial goblet cells but not in submucosal glands. There are increased numbers of goblet cells positive for MUC5AC in chronic rhinosinusitis compared with normal sinus mucosa (immunohistochemical stain, original magnification ×400).
Cellular localization of MUC5B immunoreactivity in human sinus mucosa. MUC5B immunoreactivity is localized to the goblet cells and mucous cells of submucosal glands. There are increased numbers of goblet cells and mucous cells of submucosal glands positive for MUC5AC in chronic rhinosinusitis mucosa compared with normal sinus mucosa (ABC method; original magnification ×400).
From the Division of Brain Korea 21 Program for Biomedical Science (Mr Kim, Ms J. Y. Lee, and Dr H.-M. Lee) and the Department of Otorhinolaryngology–Head and Neck Surgery and the Communication Disorders Institute of Medical Science Research Center (Drs Chu, Hwang, S. H. Lee, and H.-M. Lee), Korea University College of Medicine, Seoul, South Korea. The authors have no relevant financial interest in this article.
Background Excess mucus production and hypersecretion characterize upper airway diseases. The primary mechanisms leading to mucus hypersecretion in chronic rhinosinus inflammation are not well understood. Mucus hypersecretion is commonly accompanied by goblet cell and submucosal gland cell hyperplasia. It is important to identify which mucin gene messenger RNAs (mRNAs) are expressed in the sinus mucosa.
Objectives To investigate the expression of MUC5AC and MUC5B mRNAs and localization of these proteins in human sinus mucosa and to compare the expression of MUC5AC and MUC5B mRNAs in normal and in chronic sinus mucosa.
Design Twenty chronic maxillary sinusitis mucosa samples and 20 normal maxillary sinus mucosa samples were obtained; RNAs were extracted from sinus mucosa, and semiquantitative reverse transcription–polymerase chain reaction was performed for MUC5AC and MUC5B. Localization of these proteins was sought by using immunohistochemical analysis.
Results The levels of MUC5AC and MUC5B mRNA in chronic rhinosinusitis were significantly increased compared with those in normal sinus mucosa (P = .02). In inflammed sinus mucosa, MUC5AC protein was expressed in the cytoplasm of the goblet cell in the surface epithelium, and MUC5B expression was restricted to mucous cells of the submucosal glands and to the epithelium of sinus mucosa. However, in the normal sinus mucosa, MUC5AC and MUC5B proteins were expressed at low levels in the sinus epithelium and submucosal glands, respectively.
Conclusion These results suggest that up-regulation of MUC5AC and MUC5B, which are major components of respiratory secretion in chronic rhinosinusitis, may play important roles in the pathogenesis of sinus hypersecretion in chronic rhinosinusitis.
Chronic rhinosinusitis has a feature of mucus hypersecretion of the upper respiratory tract. Currently, contrary to the data on mucin gene and protein expression in the lower airway disease, little is known about the regulation of mucins in chronic rhinosinusitis. We postulated that mucus hypersecretion of the upper airway in chronic rhinosinusitis is commonly caused by the up-regulation of mucin expression as is mucus overproduction in the lower respiratory tract. Because mucus hypersecretion is commonly accompanied by goblet cell hyperplasia and submucosal gland hyperplasia, it is important to identify which mucin gene messenger RNAs (mRNAs) and mucin proteins are expressed in the sinus mucosa in the human airway. Herein, we investigate the expression of MUC5AC and MUC5B mRNA and compare our findings with the localization of MUC5AC and MUC5B in sinus mucosa of human chronic rhinosinusitis.
Twenty subjects (10 men and 10 women, aged 28 to 40 years) undergoing surgery for chronic rhinosinusitis and 20 healthy controls (10 men and 10 women, aged 35 to 50 years) were recruited for this study. Inflamed mucosal tissue samples of 1 maxillary sinus were removed during functional endoscopic sinus surgery through the extended middle meatal antrostomy. As controls, nasal maxillary sinus mucosa samples were obtained from 20 patients with blowout fracture who were undergoing endoscopic endonasal reconstruction of the orbital walls. Patients with nasal polyposis or immunodeficiency and individuals who had undergone prior sinus surgery were excluded from the study. No patients had received any antibiotics, steroids, antihistamines, or mucokinetic agents in the previous month. The study protocols was approved by the Review Board for Human Studies of the Korea University College of Medicine, and informed written consent was obtained from the subjects or their surrogates as required by the institutional review board.
Tissue samples were cut into 2 portions. One portion was immediately flash frozen in liquid nitrogen and stored at −70°C for subsequent RNA studies. Another portion was fixed with 4% paraformaldehyde in 0.1M phosphate-buffered saline (pH, 7.4) and stored overnight at 4°C and then embedded in paraffin for immunohistochemical staining.
Total RNAs from sinus mucosa specimens were prepared using TRIzol (Gibco BRL, Gaithersburg, Md) according to the manufacturer's suggestion. Total RNA from each sample was reverse-transcribed in 20 mL of reaction mixture containing 2.5 U of Moloney murine leukemia virus reverse transcriptase (RT) (Gibco BRL) and 50 pM of random hexanucleotides at 42°C for 60 minutes. Based on the published sequences, oligonucleotides for polymerase chain reaction (PCR) were synthesized commercially at Bioneer Company (Daejon, South Korea).
For MUC5AC, the forward primer is 5′-TGA TCA TCC AGC AGC AGG GCT-3′, and the reverse primer is 5′ CCG AGC TCA GAG GAC ATA TGGG-3′ (nucleotide positions 2897-2917 and 3284-3305, respectively; accession No. AJ001402). For MUC5B, the forward primer is 5′ CTG CGA GAC CGA GGT CAA CATC-3′, and the reverse primer is 5′-TGG GCA GCA GGA GCA CGG AG-3′ (nucleotide positions 9057-9078 and 10108-10127, respectively; accession No. Y09788). The primers 5′-GTG GAT ATT GTT GCC ATC AAT GAC C-3′ and 5′-GCC CCA GCC TTC ATG GTG GT-3′ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were run simultaneously as an internal control.
The PCR product was analyzed by gel electrophoresis. MUC5AC, MUC5B, and GAPDH PCR product sizes are 409, 415, and 271 base pairs (bp), respectively. Amplification of the complementary DNA was carried out using 35 cycles at 94°C for 45 seconds, 55°C for 30 seconds, and 72°C for 1 minute followed by a final extension cycle of 72°C for 7 minutes. As negative controls, MUC5AC and MUC5B primers or RTs were omitted from some RT-PCR reactions.
The PCR products obtained from sinus samples were cut out from the agarose gel, subcloned with the TA Cloning Kit (Invitrogen, Carlsbad, Calif), and sequenced. To analyze semiquantitatively the result of RT-PCR, we scanned the gel images and measured the intensity of the PCR product through use of NIH Image software (National Institutes of Health, Bethesda, Md). We determined the relative intensity of individual bands on a gel image as the ratio of the intensity of MUC5AC and MUC5B to the intensity of GAPDH. The negatives were scanned with a Molecular Dynamics Densitometer (Sunnyvale, Calif), and the signal was analyzed using ImageQuant software (Amersham Biosciences, Sunnyvale, Calif).
Immunohistochemical analysis was performed by the avidin-biotin-peroxidase method. Paraffin-embedded 6-µm sections were deparaffinized through 100% xylene and rehydrated. For immunostainings of MUC5AC and MUC5B, we used monoclonal antihuman MUC5AC antibody (Neo Markers, Union City, Calif) and monoclonal MUC5B antibody (Biotechnology Inc, Santa Cruz, Calif). Distribution of peroxidase was revealed by incubating the sections in a solution containing 3,3′-diaminobezidine tetrahydrochloride (Sigma, St Louis, Mo). For the negative control, nonimmune serum IgG or a phosphate-buffered saline, instead of primary antibody, was used. The slides were counterstained with hematoxylin and mounted. The immunoreactivity within the different cells was scored for immunoreactivity as strongly positive (+++), moderately positive (++), weakly positive (+), or negative (0).
The Mann-Whitney U test was used to test for differences in each of MUC5AC/GAPDH and MUC5B/GAPDH ratios between the 2 groups. A probability of less than .05 was accepted as a statistically significant difference.
The RT-PCR examination showed that the sinus mucosa specimens contained mRNA encoding for MUC5AC and MUC5B. On RT-PCR screening, MUC5AC and MUC5B mRNAs were amplified weekly in normal sinus mucosa (Figure 1). However, the inflamed sinus mucosa showed strong expression of MUC5AC and MUC5B mRNA. MUC5AC/GAPDH and MUC5B/GAPDH mRNA ratios in chronic rhinosinusitis were significantly increased compared with those in normal sinus mucosa (P <.05) (Figure 2). The PCR products extracted from the chronic rhinosinusitis mucosa were 409 bp for MUC5AC/GAPDH and 415 bp for MUC5B/GAPDH, which was expected, given the selected primers. There was no significant difference in the amount of MUC5AC and MUC5B expression between the 20 chronic sinusitis tissue samples examined. For the negative control RT-PCR, MUC5AC and MUC5B mRNA were not expressed.
The tissue distribution of MUC5AC and MUC5B proteins was analyzed by immunohistochemical staining. Positively immunostained cells appeared brown. In sections of chronic rhinosinusitis tissues, staining for MUC5AC immunoreactivity was abundant in goblet cells of the surface epithelium, while there was no staining of submucosal glandular cells (Table 1; Figure 3). There was scanty MUC5AC immunoreactivity in normal sinus mucosa. Immunohistochemical analysis for MUC5B in sections of sinus mucosa showed abundant signaling in chronic rhinosinusitis (Table 1). Next to staining of epithelial goblet cells, submucosal gland exhibited MUC5B immunoreactivity (Figure 4). Within the glandular structures, MUC5B immunoreactivity was restricted to mucous cells. No specific localization occurred with negative controls, which confirmed the specificity of the MUC5AC and MUC5B antibodies.
Chronic inflammatory upper airway diseases such as allergic rhinitis and chronic rhinosinusitis have the common pathologic findings of secretory cell hyperplasia and mucus hypersecretion. Causes of these pathogenic events in chronic rhinosinusitis are not well understood. One possible explanation for this cascade of events is metaplasia or hyperplasia of the 2 mucin-producing types of cells, goblet cells and glandular mucous cells, by increased or altered expression of mucin genes. Thus, an important issue to be resolved is the level of expression of mucin genes and proteins in normal and chronic rhinosinusitis mucosae.
MUC5B is one of the tracheobronchial mucin genes, and its expression was localized to the mucous cells from submucosal glands, which suggests that MUC5B is a major mucin of submucosal glands.9,18 The MUC5B mucin gene and its product were detected in the middle ear secretory cells of patients with chronic otitis media, which suggests that inflammatory cell products are involved in the production of MUC5B.19 Sinus mucus contains MUC5B as its major mucin, which indicates an up-regulation of this mucin in inflammatory disease of the sinuses.20 MUC5AC and MUC5B mucin have been identified in secretions from patients with asthma21 and in even mild cases of asthma, goblet cell hyperplasia and increased expression of MUC5AC are detected.22 Jung et al23 reported that MUC4, MUC5AC, MUC5B, and MUC8 mRNA are main mucins in the ethmoid mucosa and are up-regulated by chronic inflammation.
In summary, the present study indicates that sinus mucosal cells express MUC5AC and MUC5B proteins and genes, which suggests the potential for mucus production in sinus epithelium. Increased expression of MUC5AC and MUC5B are associated with secretory cell hyperplasia and metaplasia in the sinus. Further studies of other mucins, as well as MUC5AC and MUC5B, involved in upper airway diseases may help to define their role in mucus production in response to toxins, infection, and inflammation. The study of mucin gene and protein expression in chronic rhinosinusitis can enhance the understanding of the pathologic processes underlying this disease.
Submitted for publication August 13, 2003; final revision received November 13, 2003; accepted November 26, 2003.
This work was supported by the Brain Korea 21 Project of the Ministry of Education and Human Resources Development, Republic of Korea, 2002.