Human Nasal Polyp Epithelial Cells Research Articles

Involvement of the extracellular matrix proteins periostin and tenascin C in nasal polyp remodeling by regulating the expression of MMPs.

BackgroundTissue remodeling caused by increased MMPs is involved in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). We previously found higher levels of periostin and tenascin C in CRSwNPs, but whether they are associated with the dysregulation of MMPs is unknown. Therefore, the present study aimed to investigate the regulatory roles of these two ECM proteins in the expression of MMPs in nasal polyps.MethodsThe concentrations of MMP‐2, MMP‐3, MMP‐7, MMP‐8, MMP‐9, MMP‐12, MMP‐13, TIMP‐1, TIMP‐2, TIMP‐3, TIMP‐4, periostin, and tenascin C in tissue homogenates of 51 patients with chronic rhinosinusitis with and without nasal polyps and 15 control subjects were measured and were analyzed by adjusted logistic regression and spearman correlation test. Primary human nasal polyp fibroblasts and epithelial cells were stimulated ex vivo with periostin and tenascin C and the gene expression of MMPs and TIMPs was determined by means of real‐time PCR.ResultsThe protein levels of MMP‐3, MMP‐7, MMP‐8, MMP‐9, TIMP‐1, TIMP‐2, periostin, and tenascin C were significantly higher in patients with CRSwNPs than in healthy control subjects. The adjusted logistic regression analyses showed that MMP‐3, MMP‐7, MMP‐8, MMP‐9, TIMP‐2, periostin, and tenascin C were related to the occurrence of CRSwNP. Spearman correlation test showed periostin was positively correlated with MMP‐3 and TIMP‐2, and tenascin C was positively correlated with MMP‐3, MMP‐7, MMP‐8, MMP‐9, and TIMP‐2. Periostin stimulated the gene expression of MMP‐3, MMP‐7, MMP‐8, and MMP‐9 in fibroblasts and MMP‐9 in epithelial cells ex vivo. Tenascin C stimulated the expression of MMP‐3, MMP‐7, MMP‐8, and MMP‐9 in epithelial cells. The expression of TIMPs in fibroblasts and epithelial cells was affected by neither periostin nor tenascin C.ConclusionsPeriostin and tenascin C might be involved in the remodeling of nasal polyps by regulating the expression of different MMPs in epithelial cells and fibroblasts. Our findings have the potential to identify key factors of tissue remodeling in CRSwNPs.

Compressive stress induces collective migration through cytoskeletal remodelling in nasal polyp epithelium.

Nasal polyps in the nasal cavity and mucous discharge inside the maxillary sinus exhibit compressive stress on the nasal mucosal epithelium. However, there have been only a few studies on how compressive stress impacts the human nasal mucosal epithelium. We investigated the effect of compressive stress on collective migration, junctional proteins, transepithelial electri- cal resistance, epithelial permeability, and gene expression in well-differentiated normal human nasal epithelial (NHNE) cells and human nasal polyp epithelial (HNPE) cells. NHNE cells barely showed collective migration at compressive stress up to 150 mmH20. However, HNPE cells showed much greater degree of collective migration at a lower compressive stress of 100 mmH20. The cell migration of HNPE cells sub- jected to 100 mmH2O compression was significantly decreased at day 3 and was recovered to the status prior to the compressive stress by day 7, indicating that HNPE cells are relatively more sensitive to mechanical pressure than NHNE cells. Compressive stress also increased transepithelial electrical resistance and decreased epithelial permeability, indicating that the compressive stress disturbed the structural organization rather than physical interactions between cells. In addition, we found that compressive stress induced gene expressions relevant to airway inflammation and tissue remodelling in HNPE cells. Taken together, these findings demonstrate that compressive stress on nasal polyp epithelium is capable of inducing collective migration and induce increased expression of genes related to airway inflammation, innate immunity, and polyp remo- delling, even in the absence of inflammatory mediators.

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

PurposeChronic 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.MethodsHuman 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.ResultsThe 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.ConclusionsThe 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.

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.

132 Characterization of 2 Epithelial Cell Air-Liquid Interface (ALI) Culture Models for Human Healthy Nasal Mucosa and Nasal Polyps

BackgroundPrimary human airway epithelial cells, when submerged in culture, undergo a dedifferentiation with loss of many features of the in vivo airway epithelium. However, when cultured in an air-liquid interface (ALI), cells develop a well-differentiated, polarized, and pseudostratified epithelium. The aim of the current study was to characterize the mucociliary differentiation of human nasal mucosa and polyp epithelial cells cultured using an ALI system.MethodsNasal mucosa (NM, n = 3) and nasal polyps (NP, n = 3) were obtained from patients undergoing nasal corrective surgery and endoscopic sinus surgery, respectively. Epithelial cells were obtained from the explant method, and differentiated in ALI culture during 28 days. Cultures were studied at different time points (0, 7, 14, 21, and 28 days): tissue ultrastructure by scanning electron microscopy (SEM) and transmission electron microscopy (TEM); mucous (MUC5AC, MUC5B) and serous (lactoferrin) cell secretion by ELISA; and cytokeratin 18 (epithelial marker), β-tubulin IV (cilia marker), MUC5AC (goblet cell marker), and p63 (basal cell marker) expression by immunocytochemistry.ResultsIn both NM and NP ALI cultures and at days 14 and 28, a pseudostratified epithelium with ciliated, mucus-secreting and basal cells was observed, and expression of cytokeratin 18, b-tubulin IV, MUC5AC and p63 was detected. In NP cultures, both MUC5AC (day 14: 2.2 ± 0.1-folds; day 28: 3.6-fold ± 0.7-fold) and MUC5B (day 14: 3.2-fold ± 0.6-fold; day 28: 3.1-fold ± 1-fold) increased over time compared to day 0 (P < 0.05). In NM cultures, only MUC5B (day 14: 3.9-fold ± 0.9-fold; day 28: 3.4-fold ± 0.4-fold; P < 0.05) but not MUC5AC increased over time compared to day 0 (P < 0.05). Secretion of lactoferrin was present but showed no changes over time in either NM or NP ALI cultures.ConclusionsEpithelial cell ALI cultures provide a well-differentiated human nasal mucosa and polyp tissues that may be used as an in vitro model to study mucin regulation, inflammatory mechanisms of upper airways, and their regulation by antiinflammatory drugs.

Expression of Membrane-Bound Mucins in Human Nasal Mucosa

To acquire basic information concerning the function of the membrane-bound mucin MUC16 in nasal mucosa compared with the best-characterized membrane-bound mucin, MUC4. In vitro study using semiquantatitive reverse transcription-polymerase chain reaction analysis and immunoassay. Yeungnam University College of Medicine. We examined the nasal polyps obtained during endoscopic sinus surgery in 10 patients, the normal ethmoid sinus mucosa obtained from 10 patients, and human nasal polyp epithelial (HNPE) cells. Gene expression of MUC4 and MUC16 in nasal polyps and normal nasal mucosa. In addition, we evaluated the effect of 4 physiologically relevant agents, including retinoic acid, interleukin 1beta, phorbol 12-myristate 13-acetate (PMA), and dexamethasone, on the expression of MUC4 and MUC16 in HNPE cells at the gene and protein levels. In nasal polyps, MUC4 was upregulated compared with normal nasal mucosa (P = .009), whereas MUC16 expression did not differ between nasal polyps and normal nasal mucosa. Retinoic acid and interleukin 1beta increased MUC4 expression at the gene and protein level in HNPE cells, whereas MUC16 expression was not affected. Unlike retinoic acid and interleukin 1beta, PMA and dexamethasone increased MUC16 expression, whereas they had no significant effect on MUC4 expression. Expression of MUC4 and MUC16 are regulated differently in nasal mucosa. Dexamethasone and PMA are potent mediators for the expression of MUC16 in nasal polyps.

AP2α is essential for MUC8 gene expression in human airway epithelial cells

AbstractMucins are high molecular weight proteins that make up the major components of mucus. Hypersecretion of mucus is a feature of several chronic inflammatory airway diseases. MUC8 is an important component of airway mucus, and its gene expression is upregulated in nasal polyp epithelium. Little is known about the molecular mechanisms of MUC8 gene expression. We first observed overexpression of activator protein‐2alpha (AP2α) in human nasal polyp epithelium. We hypothesized that AP2α overexpression in nasal polyp epithelium correlates closely with MUC8 gene expression. We demonstrated that phorbol 12‐myristate 13‐acetate (PMA) treatment of the airway epithelial cell line NCI‐H292 increases MUC8 gene and AP2α expression. In this study, we sought to determine which signal pathway is involved in PMA‐induced MUC8 gene expression. The results show that the protein kinase C and mitogen‐activating protein/ERK kinase (MAPK) pathways modulate MUC8 gene expression. PD98059 or ERK1/2 siRNA and RO‐31‐8220 or PKC siRNA significantly suppress AP2α as well as MUC8 gene expression in PMA‐treated cells. To verify the role of AP2α, we specifically knocked down AP2α expression with siRNA. A significant AP2α knock‐down inhibited PMA‐induced MUC8 gene expression. While dominant negative AP2α decreased PMA‐induced MUC8 gene expression, overexpressing wildtype AP2α increased MUC8 gene expression. Furthermore, using lentiviral vectors for RNA interference in human nasal polyp epithelial cells, we confirmed an essential role for AP2α in MUC8 gene expression. From these results, we concluded that PMA induces MUC8 gene expression through a mechanism involving PKC, ERK1/2, and AP2α activation in human airway epithelial cells. J. Cell. Biochem. 110: 1386–1398, 2010. © 2010 Wiley‐Liss, Inc.

Expression of leptin receptor in nasal polyps: Leptin as a mucosecretagogue

Leptin is a pleiotropic hormone that regulates food intake and metabolic and endocrine functions. Serum leptin levels have been reported to be increased in patients with allergic rhinitis and nasal polyposis; however, the explanation for this is unclear. We aimed to demonstrate the differential expression of leptin receptors in normal human nasal mucosa and nasal polyps, and to elucidate the effects of leptin on mucin gene expression in human nasal polyp epithelial cells. Case-control and in vitro study. Normal ethmoid sinus mucosa was obtained from 10 subjects and used as a control; nasal polyps were obtained from 10 patients. Leptin receptor expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Leptin-induced expression of major respiratory mucins (MUC5AC and MUC5B) in the human nasal polyp epithelial cells was determined using RT-PCR and enzyme-linked immunosorbent assay. The leptin receptor expression was stronger in the nasal polyps than in the normal nasal mucosa. In human nasal polyp epithelial cells, leptin increased the expression of MUC5AC and MUC5B, in a dose- and time-dependent manner, at the gene and protein levels. Leptin-induced mucin expression was inhibited by the leptin receptor antagonist. The increased expression of leptin receptors in nasal polyps implies leptin has a certain role in nasal polyposis. In addition, leptin appears to induce the expression of MUC5AC and MUC5B through leptin receptors in the human nasal polyp epithelial cells.

Effects of double-stranded RNA poly(I:C) on matrix metalloproteinase mRNA expression in human nasal polyp epithelial cells

Chronic rhinosinusitis was often exacerbated by viral infection. A disruption of the mechanisms that regulate the activity of matrix metalloproteinases (MMPs) during viral infection was one possible mechanism responsible for the exacerbation. The purpose of study was to achieve a better understanding of MMP expression in nasal epithelial cells after viral infection. Human nasal epithelial cells were isolated from nasal polyp specimens obtained during endoscopic endonasal surgery in chronic rhinosinusitis patients. The expression of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-1 mRNA in primary human nasal polyp epithelial cells after double stranded RNA (ds RNA) stimulation were investigated. Among the genes whose expression was evaluated, only expression of MMP-9 mRNA increased significantly after dsRNA stimulation (22.61 +/- 5.47 fold increase, Z = -2.52, P = 0.012). The significant up-regulation of MMP-9 mRNA, which was not modulated by TIMP-1, was an additional source of increased proteolytic activity in virus-infected upper airways that might contribute to the exacerbation of chronic rhinosinusitis with nasal polyps.

Expression of SPLUNC1 Protein in Nasal Polyp Epithelial Cells in Air–Liquid Interface Culture Treated with IL-13

Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is an airway epithelial cell-derived molecule exerting host defense against pathogen. However, the function and regulation of SPLUNC1 in nasal epithelial cells are still unclear. Chronic rhinosinusitis with nasal polyps (CRSwNPs) is a disorder characterized by eosinophilic Th2 inflammation and frequent microbial colonization. The pathogenesis has been postulated as a disturbed mucosal immune response. This study investigates the SPLUNC1 expression of nasal polyp epithelial cells in air-liquid interface (ALI) culture and after treating with Th2 inflammatory cytokines IL-13. Human nasal polyp epithelial cells isolated from patients with CRSwNPs were put in different cell culture models at days 0 and 21 and were assessed for expression of SPLUNC1 by microarray. Cultured cells in ALI plus retinoic acid (ALI + RA) model were then incubated with 0, 1, 10, and 100 ng/mL human recombinant IL-13 for up to 5 days. The expression of SPLUNC1 was assessed by real-time quantitative polymerase chain reaction (RT-Q-PCR), reverse-transcriptase PCR (RT-PCR) and Western blot analysis. ALI + RA culture model harvesting ciliary differentiated nasal epithelial cells constitutively expressed high levels of SPLUNC1. In contrast, SPLUNC1 is reduced under classic submerged single layer culture. SPLUNC1 is also dose-responsively down-regulated after incubation with IL-13. A microenvironmental milieu containing IL-13 may be detrimental to the host innate immunity response, at least in part, through the inhibition of SPLUNC1 production.

Double-stranded RNA poly(I:C) enhances matrix metalloproteinase mRNA expression in human nasal polyp epithelial cells

Conclusion: The significant up-regulation of matrix metalloproteinase (MMP)-9 mRNA, which is not modulated by tissue inhibitor of metalloproteinase (TIMP)-1, is an additional source of increased proteolytic activity in virus-infected upper airways that might contribute to the exacerbation of chronic rhinosinusitis with nasal polyps. Objectives: Chronic rhinosinusitis is often exacerbated by viral infection. We hypothesized that a disruption of the mechanisms that regulate the activity of MMPs during viral infection is one possible mechanism responsible for the exacerbation. In the present study we attempted to achieve a better understanding of MMP expression in nasal epithelial cells after viral infection. Materials and methods: Human nasal epithelial cells were isolated from nasal polyp specimens obtained during endoscopic endonasal surgery in chronic rhinosinusitis patients. We investigated the expression of MMP-2, MMP-9, and TIMP-1 mRNA in primary human nasal polyp epithelial cells after dsRNA stimulation. Results: Among the genes whose expression was evaluated, only expression of MMP-9 mRNA increased significantly after dsRNA stimulation.

Involvement of Toll-like receptors in the immune response of nasal polyp epithelial cells

Recognition systems employed by airway epithelial cells to respond to microbial exposure include the action of Toll-like receptors (TLRs). We investigated the presence and function of TLR2, 3, and 4 in primary cultures of human nasal polyp epithelial cells. dsRNA stimulation significantly enhanced the expression and secretion of RANTES, IP-10, IL-8, and GM-CSF. LPS also exhibited stimulatory action, but it was much weaker than dsRNA. Peptidoglycan had no significant stimulatory action on the genes. Flow cytometry showed that the nasal polyp epithelial cell mainly expressed TLR3 in an intracellular compartment, but expression of TLR2 and TLR4 was very low on both the cell surface and in the cell. The immune response of primary nasal polyp epithelial cells induced by TLR3 could not be blocked by anti-TLR3 antibody. Among the TLR ligands evaluated, dsRNA, the ligand for TLR3, mediated the strongest pro-inflammatory effects in primary nasal polyp epithelial cells.

Association of stem cell factor expression in nasal polyp epithelial cells with aspirin sensitivity and asthma

Mast cells constitute a significant proportion of cells infiltrating nasal polyp tissue, and epithelial cells may release stem cell factor (SCF), a cytokine with chemotactic and survival activity for mast cells. We aimed to assess the expression of SCF in human nasal polyp epithelial cells (NPECs) as related to patients' clinical phenotypes. Nasal polyp tissues were obtained from 29 patients [including nine with aspirin (ASA)-hypersensitivity and 12 with bronchial asthma] undergoing polypectomy for nasal obstruction. Epithelial cells were obtained following 6-week culture of nasal polyps explants. The SCF released into the culture supernatant was assessed by enzyme-linked immunosorbent assay (ELISA) and total SCF mRNA in the polyp tissue was determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). For the whole group of patients, the number of polypectomies correlated with expression of SCF mRNA (r = 0.62; P < 0.005), SCF protein in the NPECs supernatants (r = 0.39; P < 0.05) and with density of mast cells in epithelial layer (r = 0.37; P < 0.05) and stromal layer (r = 0.5; P < 0.01) of nasal polyps. The SCF/beta-actin mRNA ratios were significantly higher in ASA-hypersensitive (AH) asthmatics (median 0.97, range: 0.8-1.5) when compared with ASA-tolerant (AT) patients (median 0.5, range: 0.1-0.7; P < 0.001). The SCF protein concentration in NPEC supernatants was also significantly higher in AH asthmatics (median 1.10 pg/microg DNA, range: 0.4-1.9) when compared with AT patients (median 0.1 pg/microg DNA, range: 0.02-1.2; P < 0.001). In the subpopulation of ASA-sensitive asthmatics the number of polypectomies correlated also with the density of mast cells and eosinophils in the polyp tissue.

Extracellular signal-regulated kinase is involved in tumor necrosis factor-α-induced MUC5AC gene expression in cultured human nasal polyp epithelial cells

Objectives—Mucus secretion and inflammatory cytokines are increased in cases of chronic sinusitis with polyp. In this study we initially investigated the localization of MUC5AC protein in human nasal polyps. We then examined whether tumor necrosis factor-α (TNF-α) induces MUC5AC gene expression and whether extracellular signal-regulated kinase (ERK) plays a role in TNF-α-induced MUC5AC gene expression in cultured human nasal polyp epithelial cells. Finally, we investigated whether MUC5AC and activated ERK are co-expressed in goblet cells.Material and Methods—Human nasal polyp epithelial cells were isolated and subcultured. Passage-2 polyp epithelial cells were treated with 10 ng/ml TNF-α. ERK involvement was determined by Western blotting and MUC5AC gene expression by reverse transcriptase polymerase chain reaction. An ERK kinase assay and an inhibitor study using PD98059 were also performed. Immunohistochemical staining using polyclonal MUC5AC antibody and a phospho-specific ERK antibody was done in nasal polyps.Results—MUC5AC was found to be expressed in the majority of goblet cells in human nasal polyp epithelium. TNF-α induced MUC5AC gene expression, and ERK activation was found to be essential for TNF-α-induced MUC5AC gene expression in cultured human nasal polyp epithelial cells. In addition, both MUC5AC and activated ERK were co-expressed in goblet cells of human nasal polyp epithelium.Conclusions—TNF-α induces MUC5AC gene expression via ERK in cultured human nasal polyp epithelial cells.

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.

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.