Nasal Polyp Epithelial Cells Research Articles

Expression of the glucocorticoid receptor alpha and beta isoforms in human nasal mucosa and polyp epithelial cells

The lower sensitivity of the inflamed nasal mucosa to glucocorticoids might be related to an increased expression of the glucocorticoid receptor (GR) beta isoform. We investigated GR α and GR β mRNA expression in epithelial cells from nasal mucosa and nasal polyps. GR α mRNA was at least 1000 times more expressed than GR β mRNA in both tissues. GR β expression (mean± SEM of 10 3 cDNA copies/μg of total RNA) was higher in nasal polyps (1.15±0.19; n=27; P<0.01) than in nasal mucosa (0.62±0.10; n=32). Nasal polyps with>3% of inflammatory cells had higher GR β levels (1.40±0.29; n=16) than both nasal mucosa ( P<0.01) and polyps with ≤3% of inflammatory cells (0.80±0.18; n=11; P<0.05). No difference in GR β expression was found between nasal mucosa and polyps with ≤ 3% of inflammatory cells. GR β expression correlated with the inflammatory cell number, especially with mast cells ( r=0.50, P<0.0001). There was no difference in GR α mRNA expression between nasal mucosa and nasal polyps. In summary, GR α is far more expressed than GR β in both tissues. The increased expression of GR β may be related to the presence of inflammatory cells.

Early mitochondrial dysfunction, superoxide anion production, and DNA degradation are associated with non-apoptotic death of human airway epithelial cells induced by Pseudomonas aeruginosa exotoxin A.

It has been shown that bacterial exoproducts may induce airway epithelium injury. During the epithelial repair process, the respiratory epithelial cells no more establish tight junctional intercellular complexes and may be particularly susceptible to bacterial virulence factors. In this study, we analyzed the effect of Pseudomonas aeruginosa exotoxin A (ETA) at different periods of time and concentrations on 16 HBE 14o(-) human bronchial epithelial cells in culture conditions inducing a phenotype of repairing cells. ETA treatment for 24 and 48 h led to the killing of 40.0 +/- 5.7% and 79.0 +/- 1.4% of the cells, respectively, as determined by the dimethylthiazole 2,5 diphenyl tetrazolium bromide assay. At 1,000 ng/ml, ETA led to the killing of 25.2 +/- 6.6, 59.4 +/- 5.9, and 82.3 +/- 3.7% of the cells, after treatment periods of 7, 24, and 48 h, respectively. Cell death could not be inhibited by z-VAD-fmk, a broad spectrum caspase inhibitor. By transmission electron microscopy, ultrastructural characteristics described in apoptosis were not detected in ETA-treated cells. Instead, the mitochondria of cells treated for 24 and 48 h with ETA at 100 and 1,000 ng/ml were highly condensed. Human nasal polyp epithelial cells in primary culture exposed to ETA at 1,000 ng/ml did not exhibit characteristic features of apoptotic cells either. Cytofluorometric analysis of ETA-treated 16 HBE 14o(-) cells labeled with DiOC(6)(3) and hydroethidine showed a time- and dose-dependent reduction of the mitochondrial transmembrane potential, detected 7 h after ETA treatment, and an increase in superoxide production, detected at 24 h, respectively. By a photometric assay, DNA degradation was also detected 7 h after cell treatment with ETA at 100 and 1,000 ng/ml. Taken together, our results show that ETA-induced death of epithelial respiratory cells was preceded by early mitochondrial dysfunction and superoxide anion production, but was not followed by the classically described apoptotic pathways.

Effect of budesonide and nedocromil sodium on IL-6 and IL-8 release from human nasal mucosa and polyp epithelial cells

We investigated the effect of budesonide and nedocromil sodium on the secretion of IL-6 and IL-8 by cultured epithelial cells from healthy nasal mucosa and nasal polyps. Human epithelial cell conditioned media was generated with fetal calf serum (FCS) in the presence or absence of budesonide and/or nedocromil sodium. Budesonide inhibited FCS-induced IL-6 and IL-8 release in a dose-dependent manner. The IC25(25% inhibitory concentration) of budesonide on IL-6 release was higher in nasal polyp than in nasal mucosa epithelial cells (34 n Mvs. 200 p M). The IC25of budesonide on IL-8 release was higher in nasal mucosa than in nasal polyps (145 p Mvs. 4 pM ). Nedocromil sodium caused a dose-related inhibitory effect on IL-8 release from nasal mucosa (IC25, 207 nM ), while it only had a significant effect in nasal polyps at 10−5M. Nedocromil sodium had no effect on IL-6 release. The inhibitory effect of budesonide was higher than that of nedocromil sodium on both nasal polyps and nasal mucosa. Budesonide and nedocromil sodium may exert their anti-inflammatory action in the respiratory mucosa by modulating the secretion of IL-6 and IL-8. The different effect of budesonide and nedocromil sodium on IL-6 and IL-8 release may be explained by differences in the mechanisms which regulate the upregulation of these cytokines in inflammatory responses.

Localization of Cystic Fibrosis Transmembrane Conductance Regulator in Epithelial Cells of Nasal Polyps and Postoperative Polypoid Mucosae

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel protein that plays an important role in electrolyte and water transport through the respiratory epithelial cells. In order to understand the possible role of CFTR in the pathogenesis of nasal polyps and postoperative polypoid mucosae, we aimed to characterize the localization of CFTR in the epithelia of nasal polyps and postoperative polypoid mucosae of subjects who did not manifest the phenotypic expression of cystic fibrosis. Immunohistochemical staining for CFTR, using monoclonal mouse anti-human CFTR, was performed on tissue sections of 4 normal turbinates and nasal polyps and postoperative polypoid mucosae from 10 patients who underwent endoscopic intranasal operations. CFTR showed a typical apical distribution in the normal turbinate mucosae whereas, in the nasal polyps, CFTR demonstrated a heterogenous pattern of localization comprising diffuse or scattered cytoplasmic labelling, very low to undetectable labelling, intense perinuclear staining and intermingled typical apical location. In postoperative polypoid mucosae, the pattern of CFTR localization was less heterogeneous than in the nasal polyp epithelial cells and showed a more prominent feature of diffuse cytoplasmic staining. These results suggest that an altered localization of the CFTR may have a role in the pathogenesis of nasal polyps and postoperative polypoid mucosa.

Differential metabolism of arachidonic acid in nasal polyp epithelial cells cultured from aspirin-sensitive and aspirin-tolerant patients.

The mechanism of aspirin (acetylsalicylic acid [ASA]) sensitivity associated with severe asthma and chronic rhinosinusitis with nasal polyps ("aspirin triad") has been attributed to arachidonic metabolism alternations, although the putative biochemical defects have not been elucidated. The aim of this study was assessment of the hypothesis that local production of eicosanoids in the respiratory epithelium in patients with ASA-sensitive asthma/rhinosinusitis (ASARS) differs from that of ASA-tolerant patients with rhinosinusitis (ATRS). Nasal polyps were obtained from 10 patients with ASARS and 15 with ATRS during routine polypectomies, and epithelial cells (ECs) were cultured on bovine collagen type I matrix (Vitrogen 100), in medium supplemented with growth factors. The generation of eicosanoids in supernatants of confluent ECs (6 to 8 d of culture; purity > 98%) was quantified by immunoassays. Unstimulated ECs from ASARS patients generated significantly less prostaglandin E(2)(PGE(2)) compared with ATRS (0.8 +/- 0.3 versus 2. 4 +/- 0.5 ng/microg double-stranded deoxyribonucleic acid [dsDNA], respectively), although a similar relative increase in response to calcium ionophore and inhibition with ASA was observed in both groups. Basal levels of 15-hydroxyeicosatetraenoic acid (15-HETE) were not different between groups, and calcium ionophore enhanced its production to a similar extent. However, cells incubation with 200 microM ASA for 60 min resulted in a significant increase (mean +359%) in 15-HETE generation only in ASARS patients, whereas no effect of ASA on 15-HETE generation in ATRS patients was observed. PGF(2alpha) generation was similar in both groups, and no significant generation of PGD(2) or leukotriene C(4) (LTC(4)) was observed in epithelial cell cultures from either group. Our results indicate that nasal polyps ECs from ASA-sensitive patients have significant abnormality in basal and ASA-induced generation of eicosanoids which may be causally related to the mechanism of ASA sensitivity.

PKC signaling in CF/T43 cell line: regulation of NKCC1 by PKC-δ isotype

Cystic fibrosis (CF) airway epithelial cells have a reduced mass of ether-linked diacylglycerols which might alter protein kinase C (PKC)-regulated Cl secretion. PKC regulation of basolateral Na-K-2Cl cotransport (NKCC1) was investigated in CF nasal polyp epithelial cells and a CF/T43 cell line to ascertain whether PKC signaling was altered in CF. NKCC1 was detected as bumetanide-sensitive 86Rb influx. Methoxamine, a α 1-adrenergic agonist, increased PKC activity in cytosol and a particulate fraction for a prolonged time period, as predicted from previous studies on the generation of diglycerides induced with methoxamine. Short-term stimulation of CF/T43 cells for 40 s promoted a shift in PKC-δ and -ζ to a particulate fraction, increased activity of immune complexes of cytosolic PKC-δ and of particulate PKC-ζ and increased activity of NKCC1. Pretreatment with antisense oligonucleotide to PKC-δ blocked methoxamine-stimulated PKC-δ activity, reduced PKC-δ mass by 61.4%, and prevented methoxamine-stimulated activity of NKCC1. Sense and missense oligonucleotide to PKC-δ and antisense oligonucleotide to PKC-ζ did not alter expression of PKC-δ or the effects of methoxamine. These results demonstrate that PKC-δ-dependent activation of NKCC1 is preserved in CF cells and suggest that regulation of NKCC1 is independent of low ether-linked diglyceride mass.

HLA class II antigens and T lymphocytes in human nasal epithelial cells. Modulation of the HLA class II gene transcripts by gamma interferon

Nasal polyps are characterized by a proliferation of the epithelial layer of the mucosa, cellular infiltrates and other pathological changes; however the mechanisms involved in polyp pathogenesis remain largely unclear. We have taken two different approaches to study the cellular events involved in nasal polyposis. First, through use of immunohistochemical methods, we have studied the expression of HLA class II antigens in epithelial cells of nasal polyps and the distribution of lymphocytes in the epithelium and in the subepithelial layer in patients with clinical conditions, such as asthma, atopy, aspirin intolerance or cystic fibrosis, and in subjects with an absence of concomitant diseases. Second, in order to investigate whether HLA class II expression is controlled at the pre- or post-transcriptional level, we studied the effect of interferon gamma (INF gamma) on epithelial cells in primary culture, which were derived from HLA class II negative and HLA class II positive nasal polyps. Total RNA was extracted from the cells and reverse-transcribed, and the c-DNA corresponding to DR, DP, DQ loci was amplified by PCR. Expression of HLA class II antigens by the epithelia of nasal polyps was more common in the presence rather than in the absence of concomitant asthma, atopy or cystic fibrosis (59% versus 40%). HLA-DR was the only HLA class II antigen expressed in the seven polyps taken from cystic fibrosis patients. The number of CD8+ cells was significantly higher in polyps associated with known clinical conditions and HLA class II antigen expression than it was in 'isolated' polyps and in HLA class II negative polyps. RNA transcripts for at least one or all three HLA-DR, DP and DQ antigens were detected in 10 cultures of the II HLA class II positive polyps. Conversely, 8 of 10 cultures derived from HLA class II negative polyps did not express HLA class II transcripts in the absence of INF gamma. Adding INF gamma (100 U/ml) to the latter cell cultures caused expression of transcripts of one or more HLA class II genes. We have shown that HLA class II antigens were more frequently detected in polyps of patients with an identified clinical syndrome than in those of asymptomatic subjects. Our results also suggest that IFN gamma regulates expression of HLA class II antigens in airway epithelial cells of the nasal polyps at the transcriptional level, and that cultured cells from nasal polyps represent a suitable model to investigate immune mechanisms involved in diseases such as atopy, asthma and cystic fibrosis.

Specific inhibition of beta-tryptase expression in a human mast cell line by granulocyte-macrophage colony-stimulating factor produced by airways structural cells.

The growth and differentiation of mast cells are regulated by cytokines produced in tissue microenvironments. We previously reported that mast cells isolated from the epithelial compartment of nasal polyp tissue contain significantly less tryptase when compared with mast cells isolated from the stroma of the same tissue. In an attempt to explore this finding, we analyzed the ability of supernatants obtained from cultured nasal polyp epithelial cells (NP-EpCM) or nasal polyp fibroblasts (NP-FbCM) to regulate the tryptase content of the immature human mast cell line HMC-1. HMC-1 cells were cultured for 7 days in Iscove's modified Dulbecco's medium (IMDM) with 30% of either NP-FbCM or NP-EpCM or 20% MoCM (supernatant of a leukemic T cell line). As assessed by radioimmunoassay and test for enzymatic activity, all three conditioned media were shown to significantly decrease tryptase protein expression in HMC-1, when compared with cultures performed with IMDM alone (NP-EpCM P < 0.001; NP-FbCM P < 0.04; MoCM P < 0.004). In addition, Northern blot analysis demonstrated lower tryptase mRNA levels upon exposure to all three conditioned media tested, suggesting that tryptase downregulation occurs at the transcriptional level. In further studies we found that preincubation of MoCM with anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) completely blocked the observed downregulation of tryptase expression mediated by this conditioned medium. The findings suggest that GM-CSF has a suppressive effect on expression of protease in mast cells, and may thus play a modulatory role in determining the extent of tissue inflammation in allergic airways disease.

Identification and function of A1 adenosine receptors in normal and cystic fibrosis human airway epithelial cells

A role for adenosine in the regulation of ion transport in pulmonary epithelial cells has recently been proposed. Although evidence exists documenting the presence and function of adenosine A2 receptors in airway epithelia, the presence of adenosine A1 receptors remains controversial. The present study used reverse transcriptase-polymerase chain reaction (PCR) and whole cell patch-clamp analysis to investigate A1 receptor presence and function in normal and cystic fibrosis (CF) human airway epithelial cells. Oligonucleotide primers complementary to the human brain A1 receptor sequence generated a PCR product of the predicted size (311 bp) in normal tracheal (9HTEo-) and CF submucosal (2CFSMEo-) airway cell lines and in primary cultures of CF nasal polyp epithelial cells. An oligonucleotide probe internal to the PCR primers hybridized with the 311-bp cDNAs by Southern blot analysis. cDNA sequencing demonstrated that the normal and CF airway cell PCR products are 100% identical to the corresponding sequence of the human brain adenosine A1 receptor. Northern blot analysis of 9HTEo-and 2CFSMEo- poly(A)+ RNA revealed the presence of two bands of approximately 3.0 and approximately 5.5 kb corresponding to the A1 receptor. Whole cell patch-clamp analyses demonstrated that 8-cyclopentyl-1,3-dipropylxanthine, a specific A1 receptor antagonist, increases adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance in 9HTEo-airway cells and allows cAMP to increase Cl- conductance in 2CFSMEo- CF airway cells and CF nasal polyp epithelial cells in primary culture. These results provide evidence for the presence and function of A1 receptors in normal and CF airway epithelial cells and provide support for a role of adenosine A1 receptors in modulating airway epithelial cell Cl- transport.

The role of protein kinase C in alpha-adrenergic regulation of NaCl(K) cotransport in human airway epithelial cells

alpha 1-Adrenergic (alpha 1-AR) agents stimulate NaCl(K) cotransport and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]-specific phospholipase C in human trachea and nasal polyp epithelial cells. One second messenger generated by PtdIns(4,5)P2 degradation is inositol trisphosphate. We now show that diglycerides (DG) are also generated during alpha 1-AR stimulation. In cells prelabeled with [3H]arachidonic acid, alpha 1-AR agents produced a biphasic DG generation in normal and cystic fibrosis (CF) cells that is blocked by pertussis toxin. The early DG peak closely paralleled PtdIns(4,5)P2 degradation, stimulation of cotransport by phorbol 12-myristate 13-acetate (PMA), and inhibition of cotransport by the protein kinase C (PKC) inhibitor staurosporine. This suggests that cotransporter activation requires PKC-protein phosphorylation. This possibility was tested using the protein phosphatase inhibitor okadaic acid. Okadaic acid elevated bumetanide-sensitive Cl efflux. Staurosporine also blocked > 63% of okadaic-acid-stimulated Cl transport. The late DG peak did not support hormone-stimulated cotransport. The results demonstrate that DGs are a pivotal link between alpha 1-AR stimulation and NaCl(K) cotransport activation with a role for PKC and protein phosphorylation. alpha 1-AR intracellular signaling mechanisms apparently operate normally in CF cells.

Increased ion transport in cultured nasal polyp epithelial cells.

To measure transepithelial bioelectric properties of cultured human nasal polyp and turbinate epithelial cells to test the hypothesis that nasal polyps have increased rates of ion transport. Cohort study. Private referral center. Individuals undergoing surgery for symptomatic nasal obstruction due to polyps caused by cystic fibrosis, nonatopic rhinosinusitis, or allergic rhinosinusitis. Epithelial cells were removed from separated polyp and turbinate samples by protease disaggregation and cultured on permeable collagen matrix supports. Transepithelial potential difference and resistance were measured daily. At the time of maximal transepithelial potential difference, the cultures were mounted in modified Ussing chambers and exposed to a sodium-positive channel blocker (amiloride hydrochloride) and to selected chloride-negative channel agonists (isoproterenol bitartrate, adenosine triphosphate). Maximal transepithelial potential difference, resistance, and equivalent short-circuit current. Bioelectric responses to amiloride, isoproterenol, and adenosine triphosphate. Polyp cultures had higher transepithelial potential difference and equivalent short-circuit current than turbinate cultures. The mediator responses were greater for polyp than for turbinate cultures. Polyp epithelia have increased Na+ absorption and Cl- permeability relative to turbinate epithelia. These results are consistent with the hypothesis that increased epithelial fluid absorption contributes to the development of nasal polyps.

Comparison of the role of nasal polyp and normal nasal mucosal epithelial cells on in vitro eosinophil survival. Mediation by GM–CSF and inhibition by dexamethasone

Eosinophilic infiltration of the respiratory mucosa is considered an inflammatory hallmark of allergic rhinitis, bronchial asthma and nasal polyposis. However, the mechanisms involved in this infiltration have not yet been totally elucidated. The objective of this study was to investigate and compare the influence of epithelial cell secretions from both nasal polyps (NP) and normal nasal mucosa (NM) on in vitro eosinophil survival. Epithelial cells were identified by microscopy and immunohistochemistry, cultured to confluence, and human epithelial cell conditioned media (HECM) was generated from cultures. Eosinophils were isolated at high viability and purity (> 90%) from peripheral blood and incubated with HECM. HECM from both NM and NP increased eosinophil survival in a dose-dependent manner, this effect being maximal at a concentration of 25% for NM (73.4% +/- 5.5%, n = 26, P < 0.001) and of 10% for NP (74.5% +/- 8.4%, n = 18, P < 0.001). Incubation of monoclonal antibody to human GM-CSF with HECM, neutralized the induction of eosinophil survival by HECM from both NM and NP. HECM from NP contained higher concentrations of GM-CSF (111 +/- 25.4 pg/ml, n = 17) than HECM from NM (97.1 +/- 15.2 pg/ml, n = 8), without reaching statistical significance. Pre-incubation of dexamethasone with eosinophils also blocked HECM-induced eosinophil survival from both NM (10(-8)-10(-5) M; IC50 = 9.5 nM) and NP (10(-7)-10(-5) M; IC50 = 83 nM). These results suggest that: firstly eosinophil infiltration into the respiratory mucosa during allergic reaction and nasal polyposis may be modulated at least in part by GM-CSF from epithelial cells; and secondly epithelial cells from NP might have a more potent effect on inducing eosinophil infiltration of the respiratory mucosa than epithelial cells from NM. Finally, we may consider this as a reliable in vitro model to compare the role of epithelial cells from inflammatory (NP) and non-inflammatory (NM) tissue in respiratory inflammation.

Characterization of immortal cystic fibrosis tracheobronchial gland epithelial cells.

Tracheobronchial glands were isolated and cultured from a patient with cystic fibrosis (CF). Cultured epithelial cells were transformed with pSVori-. All transformed cell lines express cytokeratin filaments and at early passages express the junctional complex molecule cell CAM 120/80, indicating their epithelial origin. Several gland cell lines express antigens that localize to secretory cells in vivo. Cl- transport measured by 36Cl efflux shows that CF gland epithelial cells, like CF surface airway and nasal polyp epithelial cells, are unable to respond to increases in intracellular cAMP. However, they do produce an increase in intracellular cAMP after treatment with isoproterenol or forskolin. One CF gland cell line shows increased intracellular calcium in response to a number of agents and increased Cl- efflux comparable to that observed in a non-CF airway surface epithelial cell line after addition of calcium ionophore. All cell lines express CF transmembrane conductance regulator mRNA, as measured by PCR amplification of first-strand cDNA. The CF tracheobronchial gland cell lines described here are compound heterozygotes, having a single copy of the delta F508 mutation.

Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions

The understanding of pathways associated with differentiated function in human epithelial cells has been enhanced by the development of methods for the short-term culture of human epithelial cells. In general these methods involve the use of serum. The subculture and maintenance of epithelial cells in long-term culture has been more problematic. A serum-free medium developed for human bronchial epithelial cells was slightly modified and found to be useful for the subculture and long-term maintenance of not only bronchial epithelial cells, but also tracheal, nasal polyp, and sweat gland epithelial cells from either normal or cystic fibrosis individuals. The cells maintained epithelial-specific characteristics after multiple subcultures. Monolayers of epithelial cells showed junctional complex formation, the presence of keratin, and micro villi. Functional studies with Ussing chambers showed short circuit current (Isc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells.

Bioelectric Properties of Cultured Nasal Polyp and Turbinate Epithelial Cells

Epithelial cells isolated from nasal polyps and inferior turbinates from patients with cystic fibrosis (CF) and without CF were studied on permeable collagen matrices. Transepithelial potential difference and resistance were measured daily, and the equivalent short-circuit current was calculated from Ohm's law. CF polyp and CF turbinate samples had higher transepithelial potential differences than the corresponding non-CF samples, as has been previously reported. However, polyp-derived epithelium appeared to have a greater rate of transepithelial ion transport than turbinate-derived epithelium in both CF and non-CF patients. This may reflect differences in excision techniques, different distribution of cell types, or persistent effects of inflammatory mediators in polyp samples. It is suggested that mediators of inflammation that are known to be present in nasal polyps may have an effect on regulating ion transport in nasal polyp epithelium. This, in turn, could have an effect on movement of water into the cell and into the interstitial tissue, causing edema, which is the most prominent histopathological finding in nasal polyps.

Chloride transport in cultured nasal epithelium of cystic fibrosis patients

In this study, nasal polyp epithelial cells from control and cystic fibrosis (CF) patients were cultured using a method which allows multiple passages. The cells were tested in Ussing chamber experiments to study transcellular ion transport. Cultured CF nasal polyp cells did not exhibit spontaneous transcellular chloride transport in the presence of amiloride, in contrast to normal cells. Forskolin increased the short circuit current (Isc) in control but not CF cells. Forskolin and isoproterenol increased the cAMP levels in control and CF cells. Histamine, bradykinin and isoproterenol transiently increased the intracellular calcium level and caused a parallel increase of the transcellular chloride current in both normal and CF cells. The transient effects of isoproterenol were not sensitive to the beta blocker atenol and could not be mimicked by forskolin. We conclude that in cultured nasal polyp cells a difference in chloride transport activity between CF and control cells is retained following multiple passages. Our results suggest that the active state of chloride channels in nasal polyp cells does not require activation of a second messenger pathway. This apparently spontaneous activity appears to be reduced in CF cells. The calcium- but not the cAMP-dependent activation of transepithelial chloride secretion is at least partially preserved in cultured CF airway epithelium.

Hematopoietic growth factor production by cultured cells of human nasal polyp epithelial scrapings: Kinetics, cell source, and relationship to clinical status

The conditions and cell sources for colony stimulating activity (CSA) production by nasal polyp epithelial scrapings were examined. Epithelial scrapings removed from patients were grown to confluence during 7 days as monolayers of epithelial cells in media supplemented with fetal calf serum (FCS) on collagen-coated microwell plates. Growth kinetics of nasal polyp epithelial cells (NPECs) were determined, and CSA in NPEC conditioned medium (CM) was assessed with density-gradient separated, nonadherent peripheral blood mononuclear cells in standard 14-day methylcellulose assays. Nasal polyp cultures in the presence of 5% or 15% FCS (vol/vol) demonstrated significantly more epithelial cell proliferation than cultures at 0% and 1% FCS. There were comparable metachromatic cell counts in polyp epithelial scrapings from allergic and nonallergic donors. Similarly, NPEC CM from allergic and nonallergic donors had equivalent CSA for basophill mast cell (BMC) and eosinophil (EO) lineages, respectively. CSA production was enhanced under conditions of higher FCS concentration and NPEC proliferation. These studies confirm an epithelial cell origin of BMC and EO growth and differentiation factors derived from nasal polyps and point to the existence of a unique microenvironment for BMC and EO development provided by polyp epithelium that appears to be independent of the presence of an allergic diathesis.

Immortalization of nasal polyp epithelial cells from cystic fibrosis patients

We have developed immortalized epithelial cystic fibrosis (CF) cell lines by infecting cultured nasal polyp cells with a SV40/Adenol2 hybrid virus. The cell lines obtained are epithelial in nature as shown by cytokeratin production and morphology, although cytokeratins 4 and 13 typical of primary nasal polyp cells are produced at a much reduced rate. Ussing chamber experiments showed that the precrisis CF cell line NCF3 was able to perform trans-cellular chloride transport when activated by agents which elevate intracellular calcium. cAMP agonists had no effect on chloride flux in NCF3 as expected for CF cells. The apical chloride channels found with the patch clamp technique in NCF3 and in the postcrisis cell line NCF3A have a conductance similar to that of chloride channels found earlier in normal and CF epithelial cells. The channels show a delay in the onset of activity in off-cell patches and are not activated by increased cAMP levels in the cell. This indicates that immortalized CF epithelial cells will provide a useful model for the study of cystic fibrosis.