Secretory cell differentiation and mucus secretion in cultures of human nasal epithelial cells: use of a monoclonal antibody to study human nasal mucin

We have developed an air-liquid interface culture system for human nasal epithelial cells that differentiate into mucociliary phenotypes in a defined serum-free medium. Dissociated cells obtained from nasal polyps were cultured on a collagen gel substrate. At confluence, the cells lost characteristics of differentiated cells, and secretory cell and ciliated cell differentiation appeared after 7 days in an air-liquid interface. After 21 days, about half of the epithelial cells were stained with Alcian blue-periodic acid-Schiff stain or monoclonal antibody HCS18, which was directed against human nasal mucin specific for epithelial secretory (goblet) cells. The quantitative examination using the antibody HCS18 revealed that the antibody-reactive nasal mucin was secreted only on the apical side of the cultures, and interleukin-1beta and tumor necrosis factor alpha stimulated these mucus secretions. The culture system with an antimucin monoclonal antibody developed in this study should be useful for studying polarized mucus secretion from human nasal epithelial cells.     

Monoclonal antibody-detectable carbohydrate epitopes of human nasal secretions are differentially expressed in tissue and diseases

To study the differential carbohydrate expression of airway secretions, we have produced a series of monoclonal antibodies that recognize human nasal secretory cell products. Mice were immunized with purified nasal secretion from patients with chronic sinusitis (CS) and hybridomas were selected by ELISA and immunohistochemical staining of the maxillary sinus mucosa from patients with CS. Eighteen antibodies were obtained. Antibody HCS 18 reacted with epithelial goblet cells, antibody HCS 4, 5, 6, and 16 stained submucosal gland cells, and antibody HCS 13 and 15 reacted with epithelial goblet cells, submucosal gland cells, and endothelial cells of vessels. The other eleven antibodies recognized epithelial goblet cells and submucosal gland cells. Cross-reactivity of these antibodies with secretory cells in other organs and in other species was determined and the different staining pattern was observed between upper and lower airway tissue, suggesting that secretory products from upper and lower airways may be different. Reactivity of the antibodies with nasal secretory cells was also examined in patients with perennial allergic rhinitis (AR) and normal subjects. Antibody HCS 18 weakly reacted with nasal glands in the tissue from CS and AR patients, but minimally reacted with gland cells in normal tissue. Antibody HCS 1 and 7 partially lost their reactivity with nasal epithelium of inferior turbinate from normal subjects and AR patients. These antibodies may be useful to study nasal secretions.     

Secretory IgA and serum type IgA in nasal secretion and antibody activity against the M protein

We studied IgA immunoglobulins in nasal secretions in order to clarify mucosal immunity of the nasal cavity and paranasal sinuses during chronic nasal infection. Secretory IgA and serum type IgA of 165 samples of nasal secretions were analyzed quantitatively by use of electroimmunodiffusion techniques, and the specific antibody activity of secretory IgA against the M protein of Streptococcus pyogenes was investigated by use of enzyme-linked immunosorbent assay. Results show that although the secretory IgA content in nasal secretions was elevated in chronic sinusitis, its specific antibody activity against the M protein was lower than that in normal subjects. This evidence suggests that nonspecific secretory IgA antibodies are predominantly produced in chronic sinusitis, and that mucosal immunity preventing the adherence of bacteria is impaired in the diseased mucosa.     

Expression and localization of TRPV1 in human nasal mucosa

Capsaicin is the pungent principle in chili peppers and previous studies reported that topical application of capsaicin to patients with allergic and non-allergic rhinitis produced significant and long-lasting relief of symptoms. The capsaicin receptor (TRPV1, VR1) is a nociceptive transducer and the existence of TRPV1 in non-neuronal cells as well as neuronal cells has been reported. In order to clarify the role of TRPV1 on the upper airway, we examined the localization and the expression of TRPV1 in human nasal mucosa. Surgically obtained human nasal specimens were processed for immunohistochemistry with commercial anti-TRPV1 antibody. We also performed immunofluorescence with anti-TRPV1 antibody and anti-neurofilament antibody or anti-CD31 antibody. Epithelial cells and vascular endothelial cells were cultured from nasal turbinates, respectively. For RT-PCR analysis, total RNA was isolated, and then RT-PCR was performed. Immunohistochemical studies revealed that TRPV1 positive cells were found on epithelial cells, vascular endothelial cells, submucosal glands and nerves in human nasal mucosa. By RT-PCR analysis, the mRNA expression of TRPV1 was confirmed in human nasal mucosa. These results suggest that capsaicin can directly influence the epithelial secretory and various functions via TRPV1 as well as the activation of the sensory neurons.     

Expression of non-steroidal anti-inflammatory drug-activated gene-1 in human nasal mucosa and cultured nasal epithelial cells: a preliminary investigation

OBJECTIVE: Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a recently discovered transforming growth factor-beta superfamily cytokine. The localization and functions of NAG-1 have not been thoroughly studied. The aims of this study were to investigate its expression and localization in human nasal mucosa and also the change in NAG-1 expression as a function of mucociliary and squamous differentiation. MATERIAL AND METHODS: Anterior and middle portions of human inferior turbinate were used and immunohistochemical studies were performed using NAG-1 antibody. Passage-2 normal human nasal epithelial (NHNE) cell culture was performed for 14 days using the air-liquid interface method and the cells were divided into retinoic acid (RA)-sufficient and -deficient groups. Each group of cells was stained with hematoxylin-eosin to study the degree of differentiation. Western blot analysis for NAG-1 expression was performed in each group after 0, 7 and 14 days. RESULTS: NAG-1 expression in mucociliated epithelium was noted in ciliated cells and serous acini, but was not found in goblet cells or mucous acini. In squamous epithelium, NAG-1 expression was weaker than that in mucociliated epithelium. In RA-sufficient culture, NHNE cells were differentiated into ciliated epithelium, but in RA-deficient culture, keratinizing squamous epithelium was noted. Western blot analysis showed that NAG-1 expression was significantly higher in RA-sufficient than -deficient culture (three-fold difference) and this expression was time-dependent. CONCLUSIONS: NAG-1 may be involved in differentiation and apoptotic processes of nasal epithelial cells. However, it is still unclear whether NAG-1 is an inducer or a byproduct of differentiation or apoptosis. The role of NAG-1 protein remains to be solved.     

A study on collagenase production of nasal polyp-derived fibroblasts stimulated by nasal secretions of chronic sinusitis

The collagenase produced by mesenchymal cells has been thought to have a great importance in the pathophysiology of connective tissue metabolism and prolongation of chronic inflammation. The factors, such as IL-1 and PMN factor, released by inflammatory cells have been known to induce mesenchymal cells to produce collagenase. In the present study, the collagenase activity of the nasal secretions were estimated using FITC-labelled collagens as substrates. The factor, enhancing the fibroblasts to produce collagenase, was also isolated from nasal secretions and partially characterized. The fibroblasts used in the present study were cultured with explant of the sections of nasal polyp obtained from a patient with chronic sinusitis. The collagenase activity in nasal secretions from patients with chronic sinusitis was high, whereas that of allergic nasal secretions was extremely low. Furthermore, the collagenase productions of nasal polyp-derived fibroblasts were enhanced by the extracts of nasal secretions from patients with chronic sinusitis. Crude extracts of nasal secretions were fractionated by ammonium sulfate precipitation. The active materials precipitated by 50% to 80% ammonium sulfate were further purified by Sephadex G-75 gel chromatography. The molecular weight determination of the active fraction checked by HPLC utilizing for TSK 2,000 SW gel column indicates 20,000 daltons for the active materials. However, the collagenase production of human microvascular endothelial cells derived from nasal mucosa was not enhanced by this factor. Although either the origin or the nature was not confirmed, the factor was considered to relate to the prolongation of chronic inflammation in the nasal and paranasal sinus pathology. Analysis of these factors will expected to establish methods for new therapeutics in chronic inflammation.     

Tenascin在鼻息肉组织中的表达及其临床意义

目的：探讨Tenascin(TN)在鼻息肉组织中的表达和分布特征及其在鼻息肉发生中的可能作用。方法：采用免疫组化链霉菌抗生物素蛋白—过氧化物酶(SP)法检测24例鼻息肉标本(鼻息肉组)和15例慢性肥厚性鼻炎下鼻甲标本(下鼻甲组)中TN的表达，并以5例健康者(对照组)下鼻甲黏膜作对照。结果：鼻息肉组和下鼻甲组黏膜上皮细胞及腺上皮细胞均表达TN；鼻息肉组TN的黏膜上皮阳性细胞表达的吸光度值显著高于下鼻甲组(P＜0．01)；鼻息肉组TN的腺上皮阳性细胞表达的吸光度值显著高于下鼻甲组(P＜0．01)；对照组下鼻甲组织中黏膜上皮及腺体几乎检测不到TN的表达；鼻息肉组腺体TN阳性率明显高于下鼻甲组(P＜0．05)。结论：TN在鼻息肉组织中的高表达与鼻息肉的发生、发展相关；TN在鼻腔内的表达细胞是黏膜上皮细胞和浆液性腺上皮细胞。     

The origin and functions of nasal mucus

Using histochemical techniques the source and nature of nasal mucus and secretions has been investigated. The secretions from the serous and mucous glands in the stroma consist of acid mucopolysaccharides and mucoprotein. The mucins from the goblet glands are similar but sulfated. The large ducts have a secretory function also. The capillary net lies in the epithelium, allowing rapid transudation to take place. The basement membrane is PAS negative during early life and is permeable. It becomes progressively more PAS positive with age because of this. A comparative study of the uses of mucus has shown that its physio-chemical characteristics are the most prominent. Mucus may act as a retainer for substances, exhibit surface electrical activity, have permeability and act as a mesh. It is an adhesive, protects the mucosa and transports particulate matter. It allows heat transfer and has water holding properties. It is suggested that mucus has been utilized throughout evolution because of its protean physiochemical properties.     

Role of angiotensin-converting enzyme on bradykinin nasal provocation: comparison of normal volunteers and allergic subjects

Nasal allergy is the most common type I allergic disease. During allergic reaction, chemical mediators may be released from residual cell, and thus, attract additional inflammatory cells. One mediator implicated in this response is bradykinin (BK), a potent proinflammatory nonapeptide. This study was designed to investigate the effects of BK on nasal mucosa, and to determine the role of angiotensin-converting enzyme (ACE) in BK-induced nasal responses. BK nasal provocation (100 micrograms) was studied in 7 normal volunteers and 7 patients with perennial allergic rhinitis. After provoking of BK response, nasal secretions and saline lavage fluids were collected for analysis of total protein (a protein secretion marker), albumin (a vascular permeability marker), and lysozyme (a serous cell marker). In addition, after administering of Captopril 50 mg, a specific ACE inhibitor, the same protocol was performed. In both groups, BK induced plasma exudation and serous gland secretion. Premedication with captopril did not alter BK-induced responses in normal individuals. In allergic patients, captopril enhanced BK-induced vascular permeability, but not glandular secretion. These results indicate that allergic subjects have nasal hyperresponsiveness to BK, and that ACE predominantly modulates the vascular permeability of allergic nasal mucosa. It seems likely that BK may contribute to the expression of nasal allergic symptoms, and that inhibition of ACE may lead to increased nasal responsiveness.     

Secretory differentiation of serially passaged normal human nasal epithelial cells by retinoic acid: expression of mucin and lysozyme

The purpose of this study was to subculture normal human nasal epithelial (NHNE) cells without compromising their ability to differentiate into secretory and ciliated cells and to study the effect of retinoic acid on mucous and serous secretions in passaged cells and to compare the expression of mucin and lysozyme in cultured cells with those in in vivo nasal epithelium. The subcultured cells were tested after every passage for secretory differentiation in air-liquid interface cultures. The cultured NHNE cells secreted mucin and lysozyme. The cells became squamous and mucin secretion decreased when retinoic acid was deleted from the culture media. Cells from passage 1 through passage 2 remained able to differentiate into mucous or squamous cells. Mucin gene 4 (MUC4), MUC5AC, MUC7, MUC8, and lysozyme messenger RNAs were expressed in passage 2 NHNE cells. In conclusion, passage 2 NHNE cell cultures retain features of normal epithelium and are suitable for many studies of upper airway cell biology.     

Ultrastructural findings of the nasal mucosa of "ozaena" in atrophic rhinitis

Alterations in the nasal mucous layer of atrophic rhinitis "Ozaena" patients have been investigated. The vast majority (99%) of these patients were women. Morphological findings in healthy nasal mucosa demonstrated the different functional stages of the glandular tissue cells with the healthy epithelium. On the other hand, nasal mucosal material taken from the patients displayed prominent epithelial deterioration, decrease in and loss of cilia, increase in goblet cells with squamous epithelial metaplasia. A decrease in secretory granules and membrane deterioration in the apical region of the secretory cells was also clearly visible. It is possible to postulate that the ultrastructural changes seen in the secretory and storage cycles of glandular tissue of the nasal mucosa from patients seem to arise as a reaction to the superficial epithelial deterioration of the nasal mucosa together with the resulting deterioration of physiological conditions.     

Collection of nasal secretions for immunological analysis

The biochemical analysis of nasal secretions has become essential in the study of nasal or sinus diseases and the monitoring of medical and surgical treatment. The nasal fluid greatly reflects the inflammatory activity within the nasal mucosa. This paper discusses techniques for nasal fluid collection described before and proposes a new approach for the collection and calculation of nasal secretions based on sinus packs. The method is non-invasive, well standardized and reproducible and therefore may serve as a valid tool for future investigations.Drs. Watelet and Gevaert contributed equally to this work.     

Beta adrenoceptors in human nasal mucosa.

The significance of the beta adrenergic system in the nasal mucosa is unclear. The authors have used the technique of autoradiography to localize and classify beta adrenoceptors in human nasal mucosa. The receptors have been found to be exclusively of the beta-2 subtype and the highest density is found in the glandular ducts. It is suggested that the beta-adrenergic system may have a physiologically important role in controlling the electrolyte composition of nasal secretions.     

人鼻粘膜腺体细胞胶原蛋白凝胶内培养模型的建立

目的建立人鼻粘膜腺体细胞的胶原蛋白凝胶内培养模型。方法采用胶原蛋白凝胶内培养。结果包埋于胶原蛋白凝胶内的腺体细胞呈三维生长，一周后可见管腔结构形成，透射电镜下可见管腔侧细胞表面布满微绒毛，细胞膜之间形成明显的紧密连接，细胞内充满分泌颗粒，具有典型的腺上皮特征。经爱先蓝ＰＡＳ染色，证明有粘液糖蛋白分泌，而经抗溶菌酶多克隆抗体免疫组化染色，９０％以上细胞呈阳性反应。结论胶原蛋白凝胶内培养的鼻粘膜腺体细胞改变了原来的分化表型，同一细胞具有粘液腺和浆液腺两种分化特征。     

人鼻粘膜腺体细胞胶原蛋白凝胶内培养模型的建立

建立人鼻粘膜腺体细胞的胶原蛋白凝胶内培养模型。方法 采用胶原蛋白凝胶内培养。结果包埋于胶的蛋白凝胶内的腺体细胞呈三维生长，一周后可见管腔结构形成，透射电镜下可见管腔侧细胞表面布满微绒毛，细胞膜之间形成明显的紧密连接，细胞内充满分泌颗闰，具有典型的腺上皮特征。     

Compositional difference in middle ear effusion: mucous versus serous.

OBJECTIVES: Serous otitis media is usually responsive to medical treatment, whereas mucoid otitis media is not. The present study was undertaken to elucidate the compositional difference between serous and mucoid effusion and to investigate whether MUC5AC acts as a major mucin in the middle ear mucosa with mucoid otitis media. STUDY DESIGN: This study involved a chemical analysis of middle ear effusion and immunostaining of the middle ear mucosa. METHODS: Middle ear effusion samples were collected from 27 patients with mucoid otitis media and 18 patients with serous otitis media. The levels of mucin, lysozyme, secretory immunoglobulin A, and interleukin-8 were measured by dot blotting or enzyme-linked immunosorbent assay. Periodic acid-Schiff and immunohistochemical staining with monoclonal anti-MUC5AC antibody were performed on the serial sections of middle ear mucosa with mucoid otitis media. RESULTS: Mucoid effusions contained higher levels of mucin, lysozyme, secretory immunoglobulin A, and interleukin-8 than did serous effusions. Immunohistological study revealed that MUC5AC mucin was expressed in only a small portion of the goblet cells of middle ear mucosa with mucoid otitis media. CONCLUSIONS: The study suggests that both serous secretions and mucin might make the middle ear effusion more viscous and that mucins other than MUC5AC might have a major role in the viscosity of middle ear effusion. Further study is necessary to identify the major mucins in the middle ear effusion of otitis media with effusion.     

Immunohistochemical study of m1-m5 muscarinic receptors and nNOS in human inferior turbinate mucosa

CONCLUSION: This study suggested that nitric oxide (NO) takes part in the parasympathetic nerve control functions mainly through m3 receptors and subsequently through m1 receptors. OBJECTIVES: The regulation of glandular secretions and vasomotor tone in human nasal mucosa implicates muscarinic receptors. There are five recognized classes (m1-m5) of muscarinic receptor subtypes. NO is a free radical gas that has been found to be produced in neuronal cells by the action of enzyme neural nitric oxide synthase (nNOS). The aim of this study was to identify the colocalization of muscarinic receptor subtypes (m1-m5) and nNOS in the human inferior turbinate mucosa. MATERIALS AND METHODS: Human inferior turbinate mucosa was stained by using the antibody of muscarine receptor subtypes and nNOS in an immunohistochemical double-staining method. RESULTS: The colocalization of m3 receptors and nNOS-immunoreactive nerve fibers was most extensively distributed on glands, vessels, and epithelium in all muscarinic receptor subtypes. These findings lead us to propose that NO also acts as a neurotransmitter in glands, vessels, and epithelium of the human nasal mucosa and can be assigned to parasympathetic nerve structures through m3 receptors mainly and m1 receptors subsequently.