Alternative Method for Primary Nasal Epithelial Cell Culture Using Intranasal Brushing and Feasibility for the Study of Epithelial Functions in Allergic Rhinitis

PURPOSE: Although differentiated normal human nasal epithelial (NHNE) cells can be used to study the role of human nasal epithelium, there is a need for effective culture models of nasal epithelium in sinonasal disease status, including allergic rhinitis (AR). We aimed to examine the feasibility of intranasal brushing for culture of nasal epithelial cells in AR patients and to verify the hypothesis that allergic nasal epithelial (ARNE) cells differ in histologic and physiologic characteristics. METHODS: We established a system for isolating (via intranasal brushing) and culturing (with air-liquid interface, ALI) nasal epithelial cells from healthy volunteers (n=8) and AR patients (n=8). We used this system to compare the histologic findings and physiologic characteristics of NHNE and ARNE. RESULTS: The histology results showed that fully differentiated ALI culture was obtained at least 14 days after confluence and that both ciliated and secretory cells were well differentiated in ALI culture using nasal brushing. The histology results of ARNE culture were significantly different from NHNE. The number of ciliated cells was lower, and secretory cells were more dominant in ARNE cell culture compared to NHNE cells. We also observed, by electron microscopy, loose tight junctions and short cilia in cultured ARNE cells. In addition, the mRNA level of TSLP which was one of the epithelial-derived allergic cytokines was significantly higher, and the expressions of genes involved in ciliogenesis were lower in cultured ARNE cells without allergen stimulation. CONCLUSIONS: Our findings suggest that ALI culture of ARNE cells using intranasal brushing may be an alternative method for epithelial cell culture in AR patients and that cultured ARNE cells will be useful for in vitro studies of the mechanisms at play during AR because they maintain unique allergic characteristics.

TMEM16A-Mediated Mucin Secretion in IL-13-Induced Nasal Epithelial Cells From Chronic Rhinosinusitis Patients

PURPOSE: Chronic rhinosinusitis with nasal polyps (CRSwNP), a mainly Th2 cytokine-mediated disease, often involves mucus secretion. Recent evidence suggests that transmembrane protein 16A (TMEM16A), a calcium-activated Cl- channel (CaCC), can regulate mucus secretion from airway epithelium by transepithelial electrolyte transport and hydration. However, the role of TMEM16A in mucin production/secretion in the airway epithelium is not clear. This study was conducted to determine the role of TMEM16A in mediating mucin secretion in human nasal polyp epithelial cells (HNPECs) induced by IL-13. METHODS: Human sinonasal mucosa tissue and dissociated sinonasal epithelium from control subjects and patients with CRSwNP were assessed for the expression of TMEM16A and the secretion of human mucin 5AC (MUC5AC) by immunohistochemistry, Western blot analysis, and enzyme-linked immuno-sorbent assay (ELISA). A model of the Th2 inflammatory environment was created by exposure of primary air-liquid interface (ALI)-cultured HNPECs to interleukin-13 (IL-13) for 14 days, with subsequent assessment of TMEM16A expression in cell lysates by Western blotting and MUC5AC secretion in apical washings of cells by ELISA. RESULTS: The expressions of TMEM16A and MUC5AC were increased in human nasal polyp tissue and dissociated nasal polyp epithelium. TMEM16A was detected in IL-13-treated HNPECs, specifically in MUC5AC-positive cells but not in ciliated cells. IL-13 treatment increased percentages of TMEM16A-positive cells, MUC5AC-positive cells, and cells coexpressing TMEM16A/MUC5AC, the expression of TMEM16A protein, and the secretion of MUC5AC. T16Ainh-A01, a TMEM16A inhibitor, attenuated these IL-13-induced effects. CONCLUSIONS: The expression of TMEM16A and MUC5AC are increased in CRSwNP, which might be a direct effect of Th2 cytokines present in the sinonasal mucosa in CRSwNP. Down-regulation of TMEM16A expression and MUC5AC secretion in HNPECs by T16Ainh-A01 indicates that TMEM16A might play an important role in mucin secretion in upper airway inflammatory diseases.

IL-1beta Induces Lysozyme Overexpression through a Mechanism Involving ERK/p38 Mitogen Activated Protein Kinase Activation in Human Airway Epithelial Cells

BACKGROUND AND OBJECTIVES: Lysozyme, a major serous component of airway epithelial secretions, plays an important role in airway defense. However, little is understood about the regulation of its expression and the associated signaling pathway. The object of this study is to investigate the regulation of lysozyme expression, the downstream signaling pathway of lysozyme expression, and the related protein kinases under inflammatory conditions using the IL-1beta, which acts as a significant cytokine in many airway inflammations. MATERIALS AND METHODS: After the IL-1beta treatment of normal human nasal epithelial cells (NHNE), lysozyme mRNA expression was determined by RT-PCR. Expressed levels of ERK/p38 kinase were determined by Western blot analysis. RESULTS: IL-1beta treated NHNEcells had over-expressed lysozyme compared to the control group. Activated ERK/p38 kinase level showed marked increment by treating NHNE with IL-1beta. Lysozyme expression and ERK/p38 kinase levels decreased when inhibitors of ERK/p38 MAP kinases were added to the IL-1beta treated cells. Finally, expression of lysozyme and activated level of ERK/p38 MAP kinases decreased in a dominant-negative cell line even when treated with IL-1beta. CONCLUSION: From these results, we concluded that IL-1beta induces over-expression of lysozyme via ERK/p38 MAP kinase signaling pathways in airway epithelial cells.

Expression and regulation of MUC8 & MUC5AC by various cytokines in normal human nasal epithelial cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Sinusitis is one of the most commonly reported diseases in the world. A network of inflammatory mediators is known to be involved in the pathogenesis of chronic sinusitis and nasal mucus secretion may also be under the control of an inflammatory mediator network. To date, 12 human mucin genes have been identified; however, the regulation of MUC8 has not yet been found out. In this study, we described the regulation of the MUC8 mRNA expression by inflammatory mediators and investigated its cellular location. MATERIALS AND METHOD: MUC8 mRNA and MUC5AC mRNA were detected in culture using passage-2 normal human nasal epithelial (NHNE) cells after the treatment with a mixture of following inflammatory mediators; TNF-alpha, IL-1beta, LPS, IL-4, PAF. The translocation of MUC8 mRNA from the nucleus to the cytoplasm was investigated by treating the inflammatory mediators with in situ hybridization. RESULTS: We found that a mixture of inflammatory mediators increased the MUC8 mRNA expression but decreased the MUC5AC mRNA expression in cultured normal human nasal epithelial cells. Among the inflammatory mediators, Interleukin-4 was responsible for the decrease in the MUC5AC mRNA expression and the MUC5AC mucin secretion. We also found that MUC8 mRNA resides in the nucleus of goblet cells and is transported into the cytoplasm following the treatment with inflammatory mediators. CONCLUSION: These results indicate that MUC8 may play an important role in the pathogenesis of mucus hypersecretion in chronic sinusitis.

Serially Passaged Normal Human Nasal Epithelial Cells: Morphology and Mucous Secretory Differentiation

BACKGROUND AND OBJECTIVES: The purpose of this study was to develop a subculturing technique that allows the formation of large amounts of normal human nasal epithelial (NHNE) cells without compromising the cells' ability to differentiate into secretory and ciliated cells. MATERIALS AND METHODS: Freshly isolated nasal epithelial cells, collected from normal inferior turbinates, were subcultured repeatedly in a serum-free medium on plastic culture dishes. The subcultured cells were tested for growth curve and electromicroscopic characteristics in air-liquid interface (ALI) cultures and for mucous secretory differentiation. RESULTS: The cultures grew rapidly during the first three passages, demonstrating a 20- to 40-fold expansion with each subculture. Ciliogenesis usually started on day 12 and the cultured cells formed cellular sheets exhibiting microvilli in the apical membrane, intracytoplasmic secretory granules, electron lucent, scant endoplasmic reticulum and complex tight junctions on the basolateral side. Passage-1 (P-1) and passage-2 (P-2) cells maintained their potential to differentiate into mucin-secretory and ciliated epithelial cells, and this potential was confirmed by immunocytochemistry with H6C5 and transmission electron microscopy. Although the number of NHNE cells on day 16 of culture decreased as the passage progressed from P-1 to P-3, the relative number of secretory cells did not significantly change. CONCLUSION: In conclusion, P-2 NHNE cell cultures retain the features of normal epithelium and are suitable for conducting many studies on upper airway cell biology.

Comparison of Mucin and Lysozyme Expression between Human in vivo Nasal Epithelial Cells and Cultured Nasal Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The purpose of this study was to compare the expression of mucin and lysozyme in passage-2 normal human nasal epithelial (NHNE) cells with those in human in vivo nasal epithelium and human tracheal RNA. MATERIALS AND METHODS: Cell lysates and total RNA from passage-2 NHNE cells, and human in vivo nasal epithelial cells were obtained. The amount of mucin and lysozyme protein was measured by immunoblotting. and qualitative RT-PCR was done to investigate the expression of mucin mRNAs and lysozyme mRNA. RESULTS: Passage-2 NHNE cells contained 16% of mucin and 76% of lysozyme when compared to the amount of intracellular mucin and lysozyme of normal in vivo nasal epithelial cells. MUC4, MUC5AC, MUC7, MUC8 and lysozyme mRNAs were expressed in passage-2 NHNE cells. However, MUC2 and MUC5B mRNAs were not expressed. CONCLUSION: Passage-2 NHNE cells contain enough amount of mucin and lysozyme protein and express most mRNAs of secretory genes which are known to be expressed in the human airway. Thus, we find passage-2 NHNE cells to be suitable for conducting studies on secretions in the human upper airway.

Changes Produced by Different Concentrations of Inflammatory Cytokines in the Proliferation and Ciliary Movement of Human Respiratory Epithelial Cells

The aim of this study was to determine the effects of cytokines IL-1beta, TNF-alpha, and TGF-beta on the proliferation and ciliary beat frequency (CBF) of human nasal epithelial cells (HNECs) in vitro. Subcultured HNECs were incubated in a medium containing recombinant human (rh) cytokines rhIL-1beta rhTNF-alpha and rhTGF-beta at concentrations of 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml, and 100 ng/ml. After a two-day incubation with these cytokines, daily cell proliferation was measured by MTT assay for six days. The CBF was measured at concentrations of 1 ng/ml of rhIL-1beta 10 ng/ml of TNF-alpha and 1 ng/ml of TGF-beta solutions. While rhIL-1beta inhibited proliferation of HNECs in concentration-dependent and time-dependent manners, rhTNF-alpha stimulated HNEC growth at concentrations ranging from 0.01 ng/ml to 10 ng/ml in concentration-dependent and time-dependent manners. In contrast, rhTGF-beta inhibited HNEC growth irrespective of concentration and incubation time. The CBF of the human nasal ciliated epithelial cells increased after the addition of rhIL-1beta and rhTNF-alpha The CBF increased progressively for four hours after the addition of rhIL-1beta and rhTNF-alpha The increased CBF continued for 24 hours and decreased after two days. However, no variation of the CBF was observed after the addition of rhTGF-beta regardless of concentration or incubation time. The results of this study suggest that during acute inflammation, IL-1beta TNF-alpha and TGF-beta may have important roles in the repair and defense mechanism of the human nasal epithelium by regulating the proliferation and CBF of nasal epithelial cells.

In Vitro Culture of Human Nasal Epithelial Cells by Monolayer Culture of Dissociated Cells / 영남의대학술지

Different techniques for culturing respiratory epithelial cells have been developed to overcome the limitations of studies on in vivo and on bioptic material. Traditionally, culture systems are divided into organ cultures, explant cultures and dissociated cell cultures. The first two contain both epithelial and non-epithelial cells. However, in monolayer cultures of dissociated cells only epithelial cells are present, the effects observed are caused by a pure epithelial responses. The purpose of this study is to establish primary culture method of human nasal epithelium (HNEC) by monolayer culture of dissociated cells to evaluate the role of the epithelial cells in the allergic and non-allergic nasal inflammatory reactions. HNEC was prepared by primary culture method of monolayer culture of dissociated cells from human inferior nasal turbinate mucosa of septal deviation patients. Primary cultured cells were characterized by indirect immunofluorescence assay and transmission electron microscopy. The immunoreactivities of cytokeratin-pan and cytokeratin No. 8 were observed in cultured HNEC. However, the immnoreactivities of vimentin and von Willebrand factor were not observed in cultured HNEC. The tonofilaments and desmosome were observed in cultured HNEC. The cultured epithelial cells were identified to be pure nasal epithelial cells. The monolayer culture of dissociated cells could successfully be employed for further study to investigate the role of the epithelial cells in allergic or non-allergic nasal inflammatory diseases.

Secretory Differentiation of Serially Passaged Normal Human Nasal Epithelial (NHNE) Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: 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 (RA)on mucous and serous secretions in each passaged cells. MATERIALS AND METHOD: Freshly isolated nasal epithelial cells from normal inferior turbinates were subcultured repeatedly in serum-free medium on plastic tissue culture dishes. The subcultured cells were tested after every passage for secretory differentiation in air-liquid interface (ALI) cultures. The apical secretion of cultured NHNE cells was characterized by immunoblotting and Western blotting. RESULTS: Cultured NHNE cells secreted mucin and lysozyme. RA was essential for mucociliary and secretory differentiation. The epithelium became squamous and mucin secretion decreased when RA was deleted from the culture media. Cells from passage 1(P-1) through passage-2 (P-2) remained competent to differentiate into mucous and squamous cells when grown in air-liquid interface culture. CONCLUSION: P-2 NHNE cell cultures retained many important features of normal epithelium and were suitable for conducting many studies of upper airway cell biology with an expanded cell pool.

Effect of Cytokines on Expression of Intercellular Adhesion Molecule - 1 in Nasal Epithelial Cells / 대한면역학회지

Intexcellular adhesion molecule-10CAM- 1) is an important moleade in aehating immune and inflammatory responses. It is found on the surface of hematopoietic and nonhematopoietic cells. It can act as an adhesive ligand for integrins such as LFA-1 (CD1&/CD11a) and MAC-1 (CD18/ CD11b). ICAM-1 is basally expressed in sigaificant amount on a limited number of cell types, including monocytes and endothelial ceRs. But it is inducible or upregulated by INF-r, IL-1p and TNF-a on many cell types. IL-4, a pleiotropic cytokine and mast cell differentiation factor, is upregulated in human allergic disease and stimulates expression of vascular adhesion molecule-1 (VCAM-1) in endothelial cells. IL-4 also promotes expression of surhce ICAM-1 in human mast cells and dermal fidroblasts. So in allergic rhinitis and asthma, IL-4 may be an important cytokine implicated in the pathogenesis of inflammation. We studied the effect of INF-r and IL-4 on expression of ICAM-1 in human nasal epithelial cells (HNEC). HNEC were prepared by primary culture method of monolayer culture of dissociated cells hom human inferior nasal turbinate mucosa. Nasal mucosa were obtained by partial turbinectomy of septal deviation patients. Primary cultured cells were charaterized as an epithelial cell type by indirect immuno-fluorescence assay using antilmdies against cytokeratin-pan, cytokeratin No. 8, vimentin and von Willebrand factor. Using fluorescence activated cell sorter (Coulter EI1TE), we analyzed the quantitative expression of ICAM-1 on HNEC. Treatment of HNEC with IFN-r (1ng/ml) for 24 hours caused about 8 fold increase of the surface ICAM-1 compared with constitutive expression by mean fluorescence intensity (MIF) but IL-4 had little effect. Theses foundings suggest that IFN-r is a potent ICAM-1 inducer in HNEC and further studies are necessary for the role of IL-4 on HNEC.

Expression of Sialic Acids according to the Differentiation of Cultured Human Nasal Epithelial Cells / 대한이비인후과학회지

BACKGROUND: Sialic acid residues are known to play a key role in the normal function of the glycoconjugates. Recently, with the development of specific sialic acid binding lectins such as Maackia seed agglutinin(MAA) and Sambucus nigra agglutinin(SNA), it has made easier to localize the sialic acid residues by the histochemical staining methods. OBJECTIVES: We were to observe the expression of sialic acids according to the differentiation of cultured human nasal epithelial cells by the immunohistochemistry method using Wheat germ agglutinin(WGA), MAA, and SNA. MATERIALS AND METHODS: Human nasal epithelial cell culture was done as floating method for the induction of differentiation. The cultured cells were fixed with 2.5% glutaraldehyde and the epon 812 was used as embedding material. The immunohistochemistry was done as Lim's method. RESULTS: The WGA and MAA positive reactions were noted from the floating zero day through the fourteenth day. The reactions were positive to the squamous-like cells and differentiating cells(ciliated and secretory epithelial cells). The WGA binding patterns were homogeneous but MAA binding patterns were inhomogeneous. The SNA positive reaction was noted only in the fourteenth day and the reaction was inhomogeneous. These results meant that N-acetyl glucosamine and N-acetyl neuraminic acid(alpha 2,3) galactose were expressed from the floating zero day and N-acetyl neuraminic acid(alpha 2,6) galactose was expressed from the floating fourteenth day. CONCLUSION: N-acetyl neuraminic acid(alpha 2,3) galactose may be more important to the primary defence of human nasal epithelial cell. Due to the inhomogeneity of the reaction, the further study using Lowicryl K4M will be needed.