Effects of Fungi and Eosinophils on Mucin Gene Expression in Rhinovirus-Infected Nasal Epithelial Cells

PURPOSE: Fungi, rhinoviruses (RVs), and eosinophils are associated with upper respiratory diseases. We evaluated the effects of fungal stimulation and eosinophil co-culture on the expression of mucin genes in RV-infected nasal polyp epithelial cells. METHODS: Nasal polyp epithelial cells were obtained from chronic rhinosinusitis patients. Cultured epithelial cells were stimulated with Alternaria and Aspergillus with or without RV-16 infection. The epithelial cells were co-cultured with eosinophils for 16 h. MUC4, MUC5AC, MUC5B, and MUC8 mRNA expressions in the epithelial cells were quantified using real-time RT-PCR. To determine the underlying mechanism, nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and mitogen-activated protein kinase (MAPK) inhibitors were used to inhibit mucin gene expression. RESULTS: Fungi and RV-16 induced mucin gene expression in nasal polyp epithelial cells. However, there was no synergistic increase in mucin gene expression, with the exception of MUC4 mRNA expression stimulated by 25 microg/mL Aspergillus. When RV-16-infected epithelial cells were stimulated with fungi and then co-cultured with eosinophils, MUC4, MUC5B, and MUC8 mRNA expressions increased. Mucin gene expression was inhibited by NF-kappaB inhibitors. CONCLUSIONS: RV-16, airborne fungi, and eosinophils may exacerbate the inflammatory process in nasal mucosal diseases by enhancing mucin gene expression.

Th2 Responses Elicited by Nasal Epithelial Cells Exposed to House Dust Mite Extract

OBJECTIVES: Respiratory epithelial cells are the first site of interaction of allergens with the immune system. The aim of this study was to examine the effect of epithelial cells, which were stimulated with house dust mite (HDM) extracts, on the immune response of peripheral blood mononuclear cells (PBMCs). METHODS: Primary nasal polyp epithelial cells were exposed to dermatophagoides pteronyssinus and dermatophagoides farina for 48 hr, and then the supernatant and cells were collected. After stimulation with HDM extract, the epithelial cells were co-cultured with PBMCs for 72 hr and then the supernatant was collected. We measured the interleukin (IL)-8 and granulocyte-macrophage colony stimulating factor to determine the activation of the epithelial cells. The tumor necrosis factor (TNF)-alpha, IL-5 and interferon-gamma were measured to evaluate the interaction between the epithelial cells and the PBMCs. The mRNA expression of intercellular adhesion molecule 1 (ICAM-1) was assessed using the anti-ICAM-1 antibody. RESULTS: The HDM extracts activated the nasal epithelial cells and enhanced the expression of ICAM-1 mRNA and cell membrane ICAM-1. When the activated epithelial cells were co-cultured with PBMCs, the PBMCs produced lager amounts of TNF-alpha and IL-5. However the cytokine production was not inhibited by pretreatment with ICAM-1 antibody. CONCLUSION: HDM allergens induce allergic inflammation by activating nasal epithelial cells, yet the interaction of the epitheila cells and the PBMCs may not be associated with an ICAM-1 medicated mechanism.

MUC8 as a Ciliated Cell Marker in Human Nasal Epithelium / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: To examine the MUC8 mRNA expression patterns according to the mucociliary differentiation of the normal human nasal epithelial (NHNE) cells, and to investigate the localization of the MUC8 proteins in the nasal polyps. MATERIALS AND METHODS: The passage-2 NHNE cells were cultured using an air-liquid interface technique and nasal polyp specimens. On the 2, 7, 14, and 28 days after confluence, the ciliated cells were counted using cytospin slide immunostaining using H6C5 and beta-tubulin, and the MUC8 mRNA levels were determined using real-time quantitative PCR. After synthesizing the polyclonal anti-MUC8 peptide antibodies, MUC8 immunostaining was preformed using the nasal polyps. The MUC8 mRNA and protein levels were determined with the NHNE cells treated with IL-1beta (10 ng/ml for 24 hours) using RT-PCR and Western blot analysis. RESULTS: The increasing pattern of the number of ciliated cells as well as the MUC8 gene expression level with increasing culture time in the NHNE cells was quite similar. MUC8 was expressed in the ciliated cells of the human nasal polyps. The MUC8 protein level as well as the mRNA level was up-regulated as a result of the IL-1beta treatment. CONCLUSIONS: This study indicates that the MUC8 protein is expressed in ciliated cells from the human nasal epithelial cells and is up-regulated by the IL-1beta treatment. These results suggest that the MUC8 gene and protein expression levels might be used as a ciliated cell marker in the human nasal epithelium.

Ultra-Structures And 14C-mannitol Transport Study of Human Nasal Epithelial Cells using ALI Culture Technique / 결핵

BACKGROUND: The information on nasal transport and the metabolism of peptides have been obtained from pharmacokinetic investigations in experimental animals. However, there are no transport and metabolic studies of human nasal epithelial cells. In this study, the permeability characteristics and the metabolic properties of in vitro human nasal cell monolayers were investigated. Material and METHODS: Normal human inferior nasal conchal tissue samples were obtained from patients undergoing endoscopic nasal cavitary surgery. The specimens were cultured in a transwell using an air-liquid interface (ALI) culture, and the transepithelial electrical resistance (TEER) value of the blank filter and confluent cell monolayers were measured. To determine the % leakage of mannitol, 4µmol 14C-labelled mannitol was added and the % leakage was measured every 10 minute for 1 hour. RESULT: Human nasal epithelial cells in the primary culture grew to a confluent monolayer within 7 days and expressed microvilli. The tight junction between the cells was confirmed by transmission electron microscopy. The TEER value of the blank filter, fifth day and seventh day reached 108.5 ohm.cm2, 141 ohm.cm2 and 177.5 ohm.cm2, respectively. Transcellular % leakage of the 14C-mannitol at 10, 20, 30, 40, 50 and 60 minutes was 35.67±5.43, 34.42±5.60, 32.75±5.71, 31.76±4.22, 30.96±3.49 and 29.60±3.68 %, respectively. CONCLUSION: The human nasal epithelial monolayer using ALI using techniques is suitable for a transcellular permeability study. The data suggests that human nasal epithelial cells in as ALI culture technique shows some promise for a nasal transport and metabolism study.

Changes of the Secretion and Morphology on Normal Human Nasal Epithelial Cells by Interleukin-1beta / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES:Interleukin (IL)-lbeta is one of proinflammatory cytokines with an active role in acute nd chronic inflammation of airway mucosa. The present study was undertaken to find out the changes in the amount of released mucin and lysozyme by immunoblot assay which are the main constituents of airway secretion, to see if there were any changes in the mRNA expression of mucin and lysozyme genes by reverse transcription-polymerase chain reaction, and also to observe the morphologic changes of IL-1beta treated the normal human nasal epithelial (NHNE) cells by scanning and transmission electron microscopy. MATERIALS AND METHODS: NHNE cells were cultured using air-liquid interface technique and on the plating day 12, IL-lbeta 10 ng/ml was added and the cells were cultured for 24 hours. Afterwards the media and cells in the insert were harvested. RESULTS:The secretion of mucin and lysozyme didn't change significantly after the stimulation with IL-1beta and there was no significant difference between the control group and IL-1beta treated group in mRNA expression of MUC2, 5AC, 5B and lysozyme genes whereas MUC8 mRNA was significantly increased by IL-1beta stimulation. Electron microscopic findings revealed no significant differences between these two groups. CONCLUSION:The stimulation with IL-1beta alone in the differentiation stage couldn't induce changes in the secretory function and morphology of NHNE cells, but could enhance the expression of MUC8 mRNA. Therefore, the further study on the pathologic changes of NHNE cells by the combined actions of various inflammatory mediators should be followed.

Morphologic Changes in Cultured Normal Human Nasal Epithelial Cells Following Treatment with Histamine

The aims of this study are to observe morphologic changes in normal human nasal epithelial (NHNE) cells caused by varying concentrations of histamine, to evaluate the changes in relation to the degree of epithelial differentiation, and to examine whether these changes are caused by the proper action of histamine or are general inflammatory processes represented by lipopolysaccharide (LPS). Cultured NHNE cells were treated with histamine diphosphate and LPS 0111 : B4 at different concentrations : 20 ng/ml, 200 ng/ml, 2 microgram/ml and 20 microgram/ml. The timing of the treatment was conducted in one of two ways : a duration of 48 hours on floating day 12 or a duration of seven days on floating day seven. On floating day 14, all specimens were collected, and hematoxylin & eosin staining and scanning electron microscopy (SEM) were conducted. The areas of ciliated and secretory cell were calculated with the Optimas program. In SEM of specimens that were given 48-hour treatments of histamine and LPS at 20 microgram/ml dosages, coverage by secretory cells had increased and damaged cilia were noted. In SEM of specimens given the seven-day treatment, enlargement of the secretory cell area and damaged cilia were observed after a treatment of 20 microgram/ml LPS, but in specimens treated with histamine, findings were normal. Morphologic changes caused by histamine treatment may be a nonspecific finding observed in inflammation, and the changes can vary according to the differentiation of the epithelium.

Ultrastructural Changes of Cultured Human Nasal Epithelial Cells by Tumor Necrosis Factor-alpha / 대한이비인후과학회지

BACKGROUND: Tumor necrosis factor(TNF)-alpha plays a critical role in normal host resistance to infections and to the growth of malignant tumor. Among its actions, the induction of the cytokines and the involved factors in the inflammatory reaction is the most important action of TNF-alpha. Until now, the functional role of TNF-alpha has been intensively studied, but the morphological effects on epithelial cells by it was not. OBJECTIVES: The aim of this study was to observe the ultrastructural changes of cultured human nasal epithelial cell(HNEC) by TNF-alpha. MATERIALS AND METHODS: The HNEC culture was done as floating method and the epithelial cells on the floating 14th day were cultured in the culture media containing TNF-alpha(0.1, 1, 10, 100ng/ml) for 48 hours. The observation was done with scanning electron microscopy(SEM) and transmission electron microscopy. For the quantitation of area of ciliated and secretory epithelial cells, SEM photo(1000 magnification) was taken and each area per 60nm 2 was calculated. RESULTS: The ultrastructural changes were observed from 1 ng/ml through 100 ng/ml TNF-alpha and the changes(damage of cilia, increase of mitochondria, intracellular vacuole, increase of intercellular space and the increase of secretory epithelial cell area) were similar to the inflammatory changes in vivo. CONCLUSION: These results suggest that the ultrastructural changes of culture HNECs are induced by TNF-alpha. The study on the reversibility of the changes and the estimation of size and numbers of cells with be required.