Cultures Of Human Nasal Epithelial Cells Research Articles

Culture of human nasal polyp epithelial cells at an air-liquid surface

To study the human nasal ciliated epithelial cells at an air-liquid surface (ALI) so as to establish a reliable cell culture model for nasal mucociliary transport study. The human nasal ciliated epithelial cells were cultured by low-temperature enzymatic digestion method at an air-liquid surface, the cell growth behavior was observed under the inverted phase-contrast microscope, the proliferation, confluence and differentiation of cultured cells were examined by scanning electron microscope and immunocytochemistry, the basal and stimulated ciliary beat frequency (CBF) of cultured epithelial cells were measured by using high-speed digital microscopic imaging system. Prism 4.0 software was used to analyze the data. (1) Under microscope, the cells on transwell membrane adhered well at 24 h and locked tightly to display with a cobblestone-like appearance; the monolayer cells got confluence to 80%-90% after one-week submersion culture, and thereafter exposed to air-liquid interface. (2) Under scanning electron microscope, the cilia and also the small microvilli could be observed to protrude from the cell's surface at ALI day 7; the ciliated cells differentiated well and distributed in cluster; goblet cells and nonciliated columnar cells distributed between ciliated cells. (3) Immunocytochemistry of β-tubulin IV and zona occludens-1 showed a good confluence and differentiation of cilia in cultured epithelial cells at ALI day 14, and the percentage of ciliated epithelial cells was 50%-60 %. (4) The basal CBF of cultured epithelial cells was (8.42 ± 1.24), (8.71 ± 1.11), (9.17 ± 1.11), (8.89 ± 0.91), (8.99 ± 0.91) Hz at ALI day 7, 14 , 21, 28, 35, respectively, no significant difference was found among them(F = 1.451, P > 0.05). (5) At the concentration of 100 µmol/L ATP, an exogenous stimulating agent, significantly increased the CBF of cultured epithelial cells. Air-liquid interface cultured human nasal epithelial cells by enzymatic digestion method manifest with good confluence and differentiation status; the cells could maintain differentiated morphology and physiological function close to in vivo epithelium for a long term, therefore, it may serve as an ideal cell model for mucociliary transport study.

Highlights from the Literature

Highlights from the Literature

Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells

BackgroundHyperactivity of the epithelial sodium (Na+) channel (ENaC) and increased Na+ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na+ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.MethodsWe evaluated by short-circuit current (Isc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.ResultsNeither hNE nor EPI-hNE4 treatments did modify Isc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased Isc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate Isc, an effect which was blocked by EPI-hNE4.ConclusionsThese results indicate that hNE does activate ENaC and transepithelial Na+ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.

High-speed digital imaging method for ciliary beat frequency measurement

The aim of this study was to develop a high-speed digital imaging system and related software for ciliary beat frequency (CFB) analysis in order to establish an automated and reliable method that is observer independent and faster compared to the conventional computerized microscope photometry method. Using primary human nasal epithelial cell cultures, the CBF was recorded with a computerized microscope photometry system and a high-speed digital imaging system. To obtain a wide range of frequencies, glycocholate (0.5%) and chlorocresol (0.005%) were used as ciliostimulatory and cilio-inhibitory reference compounds, respectively. The mean values in hertz (+/- s.d.) obtained with the photometry and high-speed digital imaging systems were: controls 8.2 +/- 0.9 and 7.9 +/- 1.1; chlorocresol 5.0 +/- 0.9 and 5.1 +/- 1.1; glycocholate 9.8 +/- 1.0 and 9.7 +/- 0.8. A similar increase (by 20 and 24%) and decrease (by 38 and 35%) in CBF was determined by the two methods after glycocholate and chlorocresol treatment, respectively. The mean difference between the photometry and high-speed digital imaging methods was 0.2 +/- 0.6 Hz, and the Bland-Altman limits of agreement were from -1.0 to +1.4 Hz, suggesting that the results obtained by these two methods could be used interchangeably. These results show the reliability of the high-speed digital imaging system and the software developed for in-vitro CBF measurements. The advantages of the system include: (i) fast data acquisition and calculation, (ii) whole field automated CBF analysis and (iii) reduction in selection bias.

Immunomodulation of Nasal Epithelial Cells by Staphylococcus aureus-Derived Serine Proteases

The impact of Staphylococcus aureus in the pathogenesis of chronic rhinosinusitis is not well understood. Therefore, we investigated primary human nasal epithelial cell cultures for their ability to produce IL-8, growth-related oncogene-alpha, and IL-6 via stimulation with trypsin and culture supernatants of different S. aureus strains and phenotypes. Inhibition of cytokine synthesis was performed using a glucocorticoid, a serine protease inhibitor, and a cysteine protease inhibitor. Finally, signal transduction pathways were analyzed by quantifying phosphorylated forms of MAPKs (PI3K, ERK, and p38) and DNA-binding assays that quantified NF-kappaB and its inhibition using BAY11-7085. In vitro studies showed that the induction of IL-8, growth-related oncogene-alpha, and IL-6 by S. aureus culture supernatants was significantly inhibited by the serine protease inhibitor. In contrast, steroids and the cysteine protease inhibitor had little effect. Activation of NF-kappaB was observed after cell treatment with trypsin and bacterial supernatants, and was inhibited by BAY11-7085 and the serine protease inhibitor. S. aureus serine proteases were identified to modulate chemokine synthesis and activate NF-kappaB in nasal epithelial cells, and may therefore be relevant for the pathophysiology of chronic rhinosinusitis.

Muco‐ciliary differentiation of nasal epithelial cells is decreased after wound healing in vitro

Epithelial damage and modifications of cell differentiation are frequent in airway diseases with chronic inflammation, in which transforming growth factor-beta1 (TGF-beta1) plays an important role. The aim of this study was to evaluate the differentiation of human nasal epithelial cells (HNEC) after wound healing and the potential effects of TGF-beta1. Basal, mucus, and ciliated cells were characterized by cytokeratin-14, MUC5AC, and betaIV tubulin immunodetection, respectively. Their expression was evaluated in situ in nasal polyps and in an in vitro model of wound healing in primary cultures of HNEC after wound closure, under basal conditions and after TGF-beta1 supplementation. Using RT-PCR, the effects of TGF-beta1 on MUC5AC and DNAI1 genes, specifically transcribed in mucus and ciliated cells, were evaluated. In situ, high TGF-beta1 expression was associated with low MUC5AC and betaIV tubulin expression. In vitro, under basal conditions, MUC5AC expression remained stable, cytokeratin-14 expression was strong and decreased with time, while betaIV tubulin expression increased. Transforming growth factor-beta1 supplementation downregulated MUC5AC and betaIV tubulin expression as well as MUC5AC and DNAI1 transcripts. After a wound, differentiation into mucus and ciliated cells was possible and partially inhibited in vitro by TGF-beta1, a cytokine that may be involved in epithelial remodeling observed in chronic airway diseases.

In vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects

Human rhinoviruses (HRVs) characteristically cause upper respiratory tract infection, but they also infect the lower airways, causing acute bronchitis and exacerbating asthma. Our purpose was to study ex vivo the differences in the response to HRV infection of nasal and bronchial epithelial cultures from the same healthy and asthmatic individuals using conditions favoring development of fully differentiated, pseudostratified mucociliary epithelium. Cells from the inferior turbinates and bronchial tree of 5 healthy and 6 asthmatic individuals were cultured at an air-liquid interface. Cultures were infected with HRV-16, and after 48 hours, the degree of infection was measured. Baseline median transepithelial resistance was lower in human bronchial epithelial (HBE) cell cultures than in human nasal epithelial (HNE) cell cultures (195 Omega.cm2 [95% CI, 164-252] vs 366 Omega.cm2 [95% CI, 234-408], respectively; P < .01). Virus replicated more easily in HBE cells than in HNE cells based on virus shedding in apical wash (log tissue culture infective dose of 50%/0.1 mL = 2.0 [95% CI, 1.0-2.5] vs 0.5 [95% CI, 0.5-1.5], P < .01) and on a 20- to 30-fold greater viral load and number of infected cells in HBE cell cultures than in HNE cell cultures. The increases in expression of RANTES and double-stranded RNA-dependent protein kinase were greater in HBE cell cultures than in HNE cell cultures, as were the concentrations of IL-8, IL-1alpha, RANTES, and IP-10 in basolateral medium. However, no significant differences between asthmatic and healthy subjects (including IFN-beta1 expression) were found. Differentiated nasal epithelial cells might have mechanisms of increased resistance to rhinovirus infection compared with bronchial epithelial cells. We could not confirm previous reports of increased susceptibility to HRV infection in epithelial cells from asthmatic subjects.

Modulation of Epithelial Sodium Channel Trafficking and Function by Sodium 4-Phenylbutyrate in Human Nasal Epithelial Cells

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

Effect of preservatives on ciliary beat frequency in human nasal epithelial cell culture: Single versus multiple exposure

As preservatives may impair mucociliary clearance, we wanted to systematically study their time-dependent effect on the ciliary beat frequency (CBF) in human nasal epithelial cells (HNEC). CBF was determined using a high-speed digital imaging method. Five preservatives were selected including benzalkonium chloride, phenylethyl alcohol, methylparaben, propylparaben and chlorbutol. We were interested in the effect of these preservatives on CBF after single and repetitive exposure. Methylparaben (0.0033%), propylparaben (0.0017%) and chlorbutol (0.005%) did not impair CBF, neither after a single short-term exposure period, nor after a single long-term exposure period. Long-term exposure to benzalkonium chloride (0.001%), phenylethyl alcohol (0.125%) and a combination of methyl- and propylparaben (0.0033 and 0.0017%) significantly decreased CBF. After a short-term exposure period, CBF recovered for phenylethyl alcohol and the combination of methyl- and propylparaben. Benzalkonium chloride decreased CBF non-reversibly. For two compounds, the effect on CBF was evaluated after repetitive exposure during 15 min for 5 consecutive days. Benzalkonium chloride resulted in ciliostasis for all concentrations tested after 5 days. Phenylethyl alcohol revealed a concentration-dependent effect on CBF, but no ciliostasis was observed. In conclusion, methylparaben, propylparaben and chlorbutol can be considered as cilio-friendly. Repetitive exposure revealed a cumulative effect on CBF for benzalkonium chloride and phenylethyl alcohol.

Stable ciliary activity in human nasal epithelial cells grown in a perfusion system

Purpose: Explore the usefulness of a perfusion system in order to establish human nasal epithelial cell cultures suitable for long-term in vitro ciliary beat frequency (CBF) and cilio-toxicity studies. Methods: The cells were obtained by protease digestion of nasal biopsy material. The cells were plated at a density of 0.8–1 × 10 6/cm 2 on Vitrogen-coated polyethylene terephthalate membranes, and cultured under submerged conditions in a CO 2 incubator or in a perfusion system (initiated on days 8–9 after plating). The CBF was determined at 24.1 ± 0.8 °C by a computerized microscope photometry system. The morphology of the cultured cells was characterized by transmission electron microscopy (TEM). Results: Under CO 2 incubator culture conditions, stable ciliary activity was expressed and maintained from day 2 to day 24. Under perfusion system culture conditions, the CBF (mean ± S.D., n = 4) amounted to 8.4 ± 0.9 and 8.8 ± 0.4 Hz on days 7 and 14, respectively. These values were lower as compared to the corresponding CBF obtained in the CO 2 incubator cultures (9.5 ± 0.6 and 9.9 ± 1.0 Hz, respectively). Reference cilio-stimulatory (glycocholate) and cilio-inhibitory (chlorocresol) compounds were used to assess CBF reactivity. In the CO 2 incubator and 7- and 14-days perfusion system cultures, glycocholate (0.5%) showed a reversible cilio-stimulatory effect of 23, 26 and 21%, respectively, while chlorocresol (0.005%) exerted a reversible cilio-inhibitory effect of 36, 40 and 36%, respectively. TEM revealed polarized cuboidal to columnar epithelial morphology, with well-differentiated ciliated cells under CO 2 and perfusion system conditions (up to day 23). Conclusion: Culturing human nasal epithelial cells on Vitrogen-coated polyethylene terephthalate membranes in submerged conditions in a CO 2 incubator and in a perfusion system offers the possibility for long-term preservation (up to 22–24 days) of stable and reactive CBF in vitro.

The use of human nasal in vitro cell systems during drug discovery and development

The nasal route is widely used for the administration of drugs for both topical and systemic action. At an early stage in drug discovery and during the development process, it is essential to gain a thorough insight of the nasal absorption potential, metabolism and toxicity of the active compound and the components of the drug formulation. Human nasal epithelial cell cultures may provide a reliable screening tool for pharmaco-toxicological assessment of potential nasal drug formulations. The aim of this review is to give an overview of the information relevant for the development of a human nasal epithelial cell culture model useful during drug discovery and development. A primary goal in the development of in vitro cell culture systems is to maintain differentiated morphology and biochemical features, resembling the original tissue as closely as possible. The potential and limitations of the existing in vitro human nasal models are summarized. The following topics related to cell culture methodology are discussed: (i) primary cultures versus cell lines; (ii) cell-support substrate; (iii) medium and medium supplements; and (iv) the air–liquid interface model versus liquid–liquid. Several considerations with respect to the use of in vitro systems for pharmaceutical applications (transport, metabolism, assessment of ciliary toxicity) are also discussed.

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 muscociliary and squamous differentiation.Materials 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 dividied 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 expresion 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.

Regulation of E-cadherin expression by dexamethasone and tumour necrosis factor-alpha in nasal epithelium.

Asthma and rhinitis often coexist and share many clinical features. The extent of epithelial alteration in nasal inflammation is controversial. Cell-cell adhesion plays an important role in tissue morphogenesis and homeostasis and is mediated by the cadherin family. In human bronchial epithelial cells the authors have shown that tumour necrosis factor (TNF)-alpha induced a significant decrease of E-cadherin and beta-catenin expression. The addition of dexamethasone inhibited this decrease. The aim of the present study was to investigate the effect of TNF-alpha and dexamethasone on the regulation of E-cadherin, gamma-catenin and beta-catenin in human nasal epithelial cells (HNEC). A primary culture of HNEC, obtained from human nasal turbinates after surgery, was used. The quantitative and qualitative modulation of E-cadherin, gamma-catenin and beta-catenin expression was assessed by Western blot and immunofluorescence analysis. In order to assess the TNF-alpha-induced activation of HNEC, interleukin-8 and RANTES (regulated on activation, normal T-cell expressed and secreted) release was assessed by enzyme-linked immunosorbent assay. The results showed that TNF-alpha induced, a decrease in gamma-catenin and beta-catenin expression, but had no effect on E-cadherin expression. Immunofluorescence showed that TNF-alpha induced cytoplasmic localisation of E-cadherin, gamma-catenin and beta-catenin. Dexamethasone inhibited the effect of TNF-alpha and induced a three-fold increase in E-cadherin expression. These results suggest that the difference in nasal and bronchial epithelial cohesion may be due to the differential effect of tumour necrosis factor-alpha and dexamethasone on E-cadherin expression.

Aspergillus fumigatus causes in vitro electrophysiological and morphological modifications in human nasal epithelial cells.

The role of the airway epithelium in the development of invasive aspergillosis in immunocompromised hosts has rarely been studied although patients at risk for this infection frequently have epithelial damage. We developed an in vitro model of primary culture of human nasal epithelial cells (HNEC) in air-liquid interface, which allows epithelial cell differentiation and mimics in vivo airway epithelium. We subsequently tested 7-day and 24-hour Aspergillus fumigatus filtrates on the apical side of HNEC to know whether A. fumigatus, the main species responsible for invasive aspergillosis, produces specific damage to the epithelial cells. The results were compared with those obtained with non-pathogenic filamentous fungi. Seven-day culture filtrates of A. fumigatus and Penicillium chrysogenum induced electrophysiological modifications whatever the fungus tested. In contrast, only 24-hour A. fumigatus filtrates induced a specific decrease in transepithelial resistance, hyperpolarization of the epithelium, and cytoplasmic vacuolization of HNEC compared with both A. niger and Penicillium chrysogenum. The inhibition of the A. fumigatus effects with amiloride suggests that the 24-hour fungal filtrate acts through sodium channels of HNEC. These early modifications of the epithelial cells could facilitate colonization of the airways by A. fumigatus. To know whether the molecules involved are specific to A. fumigatus or simply produced more rapidly than by other filamentous fungi warrants further investigation. In this perspective, the primary culture of HNEC represents a suitable model to study the interactions between airway epithelial cells and A. fumigatus.

Mucociliary differentiation according to time in human nasal epithelial cell culture

Knowledge of the state of differentiation, cell phenotype, and expression of genes for mucus production at the time of study is important because these may vary at different times during the culture period. The primary purpose of this study was to determine whether the number of ciliated cells increases as a function of differentiation in NHNE cells. If we observed an increase in the number of ciliated cells, the composition ratio of ciliated and secretory cells according to the culture duration was determined. The levels of mucin and lysozyme secretion and their gene expression at this time were also examined. The presence of ciliated cells was not evident up to 2 days after confluence. However, 3.1 ± 0.2 %, 7.4 ± 0.5 %, and 14.5 ± 0.6 % of the cells were ciliated on the 7th, the 14th, and the 28th day after confluence, respectively. Meanwhile, the percentage of secretory cells were 35.6 ± 2.8 %, 32.8 ± 2.5 %, 32.8 ± 2.5 %, and 49.4 ± 1.4 % on the 2nd, the 7th, 14th, and 28th day after confluence. The amount of secreted mucin showed an abruptly increasing pattern by the 14th day after confluence but showed no significant changes thereafter. The amount of secreted lysozyme increased as a function of differentiation. MUC5AC and MUC5B mRNA were mainly expressed between the 7th and the 14th day after confluence with relatively weak MUC8 and lysozyme expression. By the 28th day after confluence however, as the MUC5AC mRNA expression became weaker, MUC5B, MUC8, and lysozyme mRNA expression became stronger. In conclusion, we speculate that in in vitro studies with NHNE cells, the time point of treatment should vary according to the purpose of the study. In addition, the MUC5B and MUC8 gene may play an important role in mucin secretion in fully differentiated human nasal epithelial cells.

Toxicological investigations of the effects carboxymethylcellulose on ciliary beat frequency of human nasal epithelial cells in primary suspension culture and in vivo on rabbit nasal mucosa

The objective of this study was to investigate the safety of a mucoadhesive carboxymethylcellulose (CMC) formulation for intranasal administration of apomorphine. The effect of different concentrations of CMC on ciliary beat frequency (CBF) was studied using a human nasal epithelial suspension cell culture system. The CBF was determined by computerized microscope photometry. The in vivo rabbit nasal mucosal tolerance of the mucoadhesive polymer was investigated using light microscopy. Twice daily, six rabbits received CMC powder in one nostril and CMC/apomorphine powder in the alternate nostril for 4 weeks. Two control rabbits received air puffs in one nostril and nothing in the alternate nostril. The rabbits were subsequently sacrificed and the stained nasal sections examined microscopically. CMC showed both concentration- and time-dependent inhibitory effects on the CBF. Only mild-to-moderate cilio-inhibition was recorded with the different concentrations of the polymer. CMC (both with and without apomorphine) caused mild-to-moderate inflammation after 4 weeks. Necrosis, squamous metaplasia or ciliary degeneration was not observed. Based on: (1) the mild-to-moderate cilio-inhibition induced by different concentrations of CMC; and (2) the mild-to-moderate nasal mucosal inflammation caused by CMC with and without apomorphine, we conclude that this polymer can be considered as a safe carrier for short-term intranasal administration. However, further investigations are required for its use in the treatment of chronic diseases such as with apomorphine in Parkinson's disease.

Effect of fexofenadine on eosinophil-induced changes in epithelial permeability and cytokine release from nasal epithelial cells of patients with seasonal allergic rhinitis

Recent studies have suggested that antihistamines, widely used in the treatment of symptoms of patients with allergic rhinitis, may also possess antiinflammatory properties. The mechanisms underlying this property, however, are not clearly understood. We have cultured epithelial cells from nasal biopsy specimens from patients with seasonal allergic rhinitis outside the pollen season and studied the effect of 0 to 10 -3 mol/L fexofenadine, the main active metabolite of terfenadine, on eosinophil-induced changes in electrical resistance (measure of permeability) and release of proinflammatory mediators from these cells. Additionally, we have studied the effect of this drug on eosinophil chemotaxis and adherence to endothelial cells induced by conditioned medium from these human nasal epithelial cell (HNEC) cultures. Incubation of HNEC in the presence of eosinophils treated with opsonized latex beads significantly decreased the electrical resistance of these cultures, an effect that was abrogated by treatment of the cultures with 10 -9 to 10 -3 mol/L fexofenadine. Similarly, incubation of HNEC in the presence of eosinophils treated with latex beads also significantly increased the basal release of the chemokine “regulated upon activation, normal T cell expressed and secreted” (RANTES) (from 96.0 to 613.0 fg/μg cellular protein; p < 0.05), IL-8 (from 42.0 to 198.5 pg/μg cellular protein; p < 0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF) (from 0.54 to 3.4 pg/μg cellular protein; p < 0.05), and soluble intercellular adhesion molecule-1 (sICAM-1) (from 7.8 to 18.4 pg/μg cellular protein; p < 0.05) from HNEC. The eosinophil-induced release of IL-8, GM-CSF, and sICAM-1 from the HNEC was significantly attenuated by treatment with fexofenadine. Analysis of the effects of conditioned medium from HNEC demonstrated that this significantly increased both eosinophil chemotaxis and adherence to endothelial cells. Addition of 10 -6 to 10 -3 mol/L fexofenadine to the conditioned medium significantly attenuated eosinophil chemotaxis and adherence to endothelial cells. These results suggest that fexofenadine may reduce nasal inflammation by modulating the release of proinflammatory mediators and adhesion molecules from HNEC. (J Allergy Clin Immunol 1998;101:410-20.)

Development of a human nasal epithelial cell culture model and its suitability for transport and metabolism studies under in vitro conditions.

A human nasal epithelial cell culture model has been adapted to observe transport and metabolism of drugs, e.g., peptides. Human nasal epithelial cells, isolated by protease treatment of human nasal conchae, grew to confluency after 6-8 days using DMEM supplemented with 1% nonessential amino acids, 1% glutamine, 10% FCS and 1% antibiotics. These cultures expressed microvilli and actively beating cilia as documented by light microscopy and scanning electron microscopy (SEM). Tight junctions were confirmed by dome formation and positive actin staining using FITC-labelled phalloidin. Preliminary transport studies, carried out with FITC-labelled Dextran (FD 4, MW 4400) and Sulforhodamine (SR 101, MW 607), demonstrated the intact barrier function of the cultured monolayer, grown on filter membranes. In addition, the cultured cells metabolized Leu-Enkephalin to Des-Tyr-Leu-Enkephalin demonstrating the presence of aminopeptidase, a naturally occurring enzyme in the human nasal mucosa.

Synthesis of interleukin-1α, interleukin-6, and interleukin-8 by cultured human nasal epithelial cells

Nasal epithelium forms the initial barrier between the environment and the respiratory system and may be a potential source of proinflammatory interleukins, which contribute to the pathophysiology of allergic and nonallergic rhinitis. To explore this possibility, epithelium and cultured human nasal epithelial cells from nasal turbinates of patients undergoing surgery for treatment of upper airway obstruction were examined for the spontaneous expression of interleukin (IL)-1α, IL-1β, IL-6, and IL-8. Human nasal epithelial cell lysates and culture supernatants were assayed by two-site ELISAs specific for IL-1α, IL-1β, IL-6, or IL-8. Maximum concentrations of these cytokines in supernatants ranged from approximately 0.2 to 2 ng/ml for IL-1α, 1.5 to 7 ng/ml for IL-6, and 100 to 3000 ng/ml for IL-8. IL-1α was predominantly cell-associated, whereas most of the IL-8 and all of the IL-6 were detected in the supernatant. Little or no IL-1β was detected by ELISA in the supernatants or cell lysates. Whole tissue turbinates and isolated epithelium were also examined for IL-1β, IL-6, and IL-8 mRNA expression by Northern blot analysis. IL-6 and IL-8 mRNAs were detected, whereas IL-1β mRNA was not. Furthermore, IL-6 and IL-8 release from human nasal epithelial cell cultures was enhanced by addition to the cultures of lipopolysaccharide, and IL-6 release was inhibited by polymyxin B. Thus human nasal epithelium may be a major source of IL-1α, IL-6, and IL-8 in allergic and nonallergic rhinitis. Production of those proinflammatory cytokines by epithelial cells of the nasal and sinus mucosa may contribute to the pathologic and clinical events that occur in these diseases. (J A LLERGY C LIN I MMUNOL 1994;93:1060-7.)