Guinea Pig Tracheal Epithelium Research Articles

Lipopolysaccharide increases Na+,K+-pump, but not ENaC, expression in guinea-pig airway epithelium

Earlier, we found in functional experiments that lipopolysaccharide (LPS; 4mg/kg; i.p.) hyperpolarized the epithelium by stimulating the transepithelial transport of Na+ in guinea-pig tracheal epithelium. Epithelial sodium channel (ENaC) activity and Na+,K+-pump activity were increased. In this study, we hypothesized that LPS increases the expression of ENaC and the Na+,K+-pump in the epithelium and investigated the levels of transcription and protein abundance. Using qPCR, the effects of LPS on the transcription of αENaC, α1 Na+,K+-pump, COX-2, eNOS, iNOS, IL-1β, and TNF-α were measured at 3 and 18h. In the epithelium, LPS increased the transcription of COX-2, IL-1β, and, to a nonsignificant extent, TNF-α at 3h, but not at 18h. In alveolar macrophages, TNF-α, and, to a nonsignificant extent, COX-2 and IL-1β were up-regulated at 3h, but not at 18h. Even though LPS stimulated the transcription of some genes, αENaC and α1 Na+,K+-ATPase transcription were not affected. The expressions of α-, β-, and γ-ENaC and α1 Na+,K+-pump from the tracheal epithelium and kidney cortex/medulla were investigated by western blotting. All three ENaC subunits were detected as cleavage fragments, yet LPS had no effect on their expression. LPS increased the expression of the α1 subunit and the α1, α2, and α3 subunits, collectively, of the Na+,K+-pump. Taken together, these data indicate that LPS increases Na+ transport downstream of the genetic level, in part, by stimulating the expression of the Na+,K+-pump.

W1575 Effect of Eosinophil Granule Proteins On P27 Expression in Esophageal Epithelial Cells

INTRODUCTION: p27, a tumor suppressor that regulates the G1 to S phase transition of the cell cycle, is reduced or mislocalized in Barrett's associated adenocarcinoma (BAA). Mislocalization of p27 from the nucleus to cytoplasm disrupts its normal function, and has been associated with poor prognosis in BAA. We have shown that bile acids and hydrochloric acid (HCl) cause mislocalization of p27 from the nucleus to the cytoplasm in normal squamous esophageal epithelial cells (HET-1A) in culture, without affecting total cell p27 levels. Both bile and acid reflux have been associated with an increased risk for development of Barrett's esophagus (BE), but not every patient with BE has a history of GERD. Many patients with symptoms mimicking GERD are later found to have eosinophilic esophagitis (EoE). Eosinophil granule proteins implicated in EoE [major basic protein (MBP) and eosinophil peroxidase (EPO)], have been shown to damage human pneumocytes and guinea pig tracheal epithelium. We sought to determine whether MBP or EPO affects p27 expression in HET-1A cells similar to bile acids and HCl. METHODS: HET-1A cells were incubated with MBP at 5-100μg/ml or EPO at 0.8-80μg/ml for 24 hours. Cells were harvested for cytotoxicity studies and immunoblot analysis to determine p27 expression. Indirect immunofluorescence (IF) was performed to determine the subcellular localization of p27. Flow cytometric analysis was used to study theMBP and EPO effects on cell proliferation. RESULTS: Exposure of HET-1A cells to MBP or EPO at various concentrations did not affect total cell p27 levels. When HET-1A cells were treated with either MBP or EPO, increased p27 was detected in the cytoplasm, along with reduced but not absent p27 nuclear staining. Flow cytometric profiles were similar for untreated cells and cells treated with MBP or EPO. CONCLUSIONS: 1) Exposure of HET-1A cells toMBP or EPO results in increased cytoplasmic p27. 2) The presence of p27 in the cytoplasm may disrupt its normal cell cycle inhibitory function and be an important early marker of dysplasia.

W1576 Changes of Growth Factors' Expression in Subepithelial Follicles in Ulcerative Colitis: the Severity of Inflammation Is Accompanied By Epithelial Growth Factor Receptor and Hepatocyte-Derived Growth Factor Receptor Overexpression

INTRODUCTION: p27, a tumor suppressor that regulates the G1 to S phase transition of the cell cycle, is reduced or mislocalized in Barrett's associated adenocarcinoma (BAA). Mislocalization of p27 from the nucleus to cytoplasm disrupts its normal function, and has been associated with poor prognosis in BAA. We have shown that bile acids and hydrochloric acid (HCl) cause mislocalization of p27 from the nucleus to the cytoplasm in normal squamous esophageal epithelial cells (HET-1A) in culture, without affecting total cell p27 levels. Both bile and acid reflux have been associated with an increased risk for development of Barrett's esophagus (BE), but not every patient with BE has a history of GERD. Many patients with symptoms mimicking GERD are later found to have eosinophilic esophagitis (EoE). Eosinophil granule proteins implicated in EoE [major basic protein (MBP) and eosinophil peroxidase (EPO)], have been shown to damage human pneumocytes and guinea pig tracheal epithelium. We sought to determine whether MBP or EPO affects p27 expression in HET-1A cells similar to bile acids and HCl. METHODS: HET-1A cells were incubated with MBP at 5-100μg/ml or EPO at 0.8-80μg/ml for 24 hours. Cells were harvested for cytotoxicity studies and immunoblot analysis to determine p27 expression. Indirect immunofluorescence (IF) was performed to determine the subcellular localization of p27. Flow cytometric analysis was used to study theMBP and EPO effects on cell proliferation. RESULTS: Exposure of HET-1A cells to MBP or EPO at various concentrations did not affect total cell p27 levels. When HET-1A cells were treated with either MBP or EPO, increased p27 was detected in the cytoplasm, along with reduced but not absent p27 nuclear staining. Flow cytometric profiles were similar for untreated cells and cells treated with MBP or EPO. CONCLUSIONS: 1) Exposure of HET-1A cells toMBP or EPO results in increased cytoplasmic p27. 2) The presence of p27 in the cytoplasm may disrupt its normal cell cycle inhibitory function and be an important early marker of dysplasia.

Altered ion transport and responsiveness to methacholine and hyperosmolarity in air interface-cultured guinea-pig tracheal epithelium

Introduction: Challenge of guinea-pig tracheal epithelium with hyperosmolar solution alters ion transport and evokes the release of epithelium-derived relaxing factor (EpDRF). Cultured tracheal epithelial cells (CE) offer the potential to examine biochemical pathways related to EpDRF release, but whether the bioelectric properties and responses of fresh, adherent epithelial cells (FE) are modeled by CE has not been established. Methods: Tracheal epithelial cells grown in air-interface culture and fresh tracheal segments were mounted in Ussing chambers to determine short circuit current ( I sc) and transepithelial resistance ( R t) and to compare responses to transport inhibitors, methacholine and hyperosmolarity. Results: Significant differences in basal I sc and R t between FE and CE were observed ( I sc, 41.3 ± 3.5 and 8.5 ± 0.8 μA/cm 2, P < 0.05; R t, 106 ± 7 and 422 ± 4 Ω cm 2, P < 0.05; respectively); basal spontaneous potential difference values were not different (4.2 ± 0.3 and 3.4 ± 0.3 mV, respectively). Amiloride (mucosal, 3 × 10 − 5 M), bumetanide (basolateral, 10 − 5 M) and ouabain (basolateral, 10 − 5 M) reduced I sc equally in FE and CE. In contrast, NPPB (10 − 5 M) in the presence of amiloride had a differential effect, decreasing I sc by 11% in FE and 71% in CE ( P < 0.05). Iberiotoxin (basolateral, 10 − 7 M) was without effect in either preparation. In FE, serosal methacholine (3 × 10 − 5 M) elicited an NPPB-insensitive monotonic increase in I sc, but in CE caused a large, transient, NPPB-inhibitable increase which was followed by an NPPB-resistant plateau. Addition of apical d-mannitol (0.3–267 mosM) to increase osmolarity decreased I sc in FE, whereas in CE d-mannitol initially increased (0.3–84.3 mosM) and then decreased (84.3–267 mosM) I sc. Discussion: Cell culture causes substantial changes in the bioelectric and pharmacological properties of respiratory epithelium. Caution should be exercised when using CE as a substitute for FE in studies of ion transport- and cell volume-dependent processes.

Differential Muc2 and Muc5ac secretion by stimulated guinea pig tracheal epithelial cells in vitro

BackgroundMucus overproduction is a characteristic of inflammatory pulmonary diseases including asthma, chronic bronchitis, and cystic fibrosis. Expression of two mucin genes, MUC2 and MUC5AC, and their protein products (mucins), is modulated in certain disease states. Understanding the signaling mechanisms that regulate the production and secretion of these major mucus components may contribute significantly to development of effective therapies to modify their expression in inflamed airways.MethodsTo study the differential expression of Muc2 and Muc5ac, a novel monoclonal antibody recognizing guinea pig Muc2 and a commercially-available antibody against human MUC5AC were optimized for recognition of specific guinea pig mucins by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). These antibodies were then used to analyze expression of Muc2 and another mucin subtype (likely Muc5ac) in guinea pig tracheal epithelial (GPTE) cells stimulated with a mixture of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interferon- γ (IFN-γ)].ResultsThe anti-Muc2 (C4) and anti-MUC5AC (45M1) monoclonal antibodies specifically recognized proteins located in Muc2-dominant small intestinal and Muc5ac-dominant stomach mucosae, respectively, in both Western and ELISA experimental protocols. IHC protocols confirmed that C4 recognizes murine small intestine mucosal proteins while 45M1 does not react. C4 and 45M1 also stained specific epithelial cells in guinea pig lung sections. In the resting state, Muc2 was recognized as a highly expressed intracellular mucin in GPTE cells in vitro. Following cytokine exposure, secretion of Muc2, but not the mucin recognized by the 45M1 antibody (likely Muc5ac), was increased from the GPTE cells, with a concomitant increase in intracellular expression of both mucins.ConclusionGiven the tissue specificity in IHC and the differential hybridization to high molecular weight proteins by Western blot, we conclude that the antibodies used in this study can recognize specific mucin subtypes in guinea pig airway epithelium and in proteins from GPTE cells. In addition, Muc2 is highly expressed constitutively, modulated by inflammation, and secreted differentially (as compared to Muc5ac) in GPTE cells. This finding contrasts with expression patterns in the airway epithelium of a variety of mammalian species in which only Muc5ac predominates.

Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory system

Electrophysiological studies within the lung have documented the presence of heterogenous groups of afferent fibers composed of Aδ and C-fibers and studies of somatosensory nerves within the skin reveal a complex pattern of distribution of sensory neuropeptides and transient receptor potential vanilloid (TRPV)1 positive nerves. However, the anatomical location of these different subpopulations of nerves within the lung has not been extensively studied. In the present study we have demonstrated that TRPV1 axons represented only a small proportion of the total number of PGP9.5 staining nerves within guinea-pig tracheal epithelium and only half the number of TRPV1 axons was immunopositive for substance P. In contrast, most TRPV1 positive neurones found within guinea-pig intrapulmonary airways were found to co-localize with sensory neuropeptides substance P and calcitonin gene-related peptide within and beneath the epithelium, around blood vessels, within airway smooth muscle and alveoli, indicative of heterogeneity of TRPV1 positive axons throughout the airways. However, in the smooth muscle layer of the trachea there was evidence of substance P and calcitonin gene-related peptide containing nerves that did not stain for TRPV1. We also demonstrated a complete loss of TRVP1 positive axons in the trachea and intrapulmonary airways and associated loss of bronchoconstriction induced by capsaicin, in animals chronically treated with capsaicin. However, some neuropeptide immunoreactive axons remained in the smooth muscle layer of capsaicin-treated animals which could represent the small subset of neuropeptide containing fibers which do not co-localize with TRPV1. We have provided evidence of heterogeneity of TRPV1 positive nerve fibers, including fibers characterized by lack of co-localization with neuropeptides in various regions of the airways and the existence of neuropeptide containing fibers that were not TRPV1 positive in guinea-pigs.

Interleukin-13 induces goblet cell differentiation in primary cell culture from Guinea pig tracheal epithelium.

The Th2 cytokines, interleukin (IL)-4 and IL-13, bind to IL-4Ralpha, and cause goblet cell metaplasia/hyperplasia with increased mucin expression in vivo. However, there is not enough evidence that these cytokines directly induce mucin production in vitro. In this study, primary epithelial cells from guinea pig trachea were cultured at an air-liquid interface, and immediately after achieving confluence at Day 7 they were treated with human recombinant IL-4 or IL-13 for 14 d. IL-13-treated cells consisted of a large number of fully mature goblet cells with a smaller number of ciliated cells. Secretory granules of the goblet cells were positive for both periodic acid-Schiff and toluidine blue, and showed exocytosis. By contrast, IL-4 failed to induce goblet cell differentiation. The electric resistances of IL-13-treated cells were lower than those of IL-4-treated cells and nontreated cells, suggesting leaky epithelia. MUC5AC protein level in cell lysates measured by ELISA was several-fold higher in IL-13-treated cells than in nontreated cells, whereas the level in IL-4-treated cells was not changed. These data suggest that human recombinant IL-13, but not IL-4, can induce differentiation into mature goblet cells that produce MUC5AC protein in guinea pig tracheal epithelial cells in vitro.

Residual Oil Fly Ash Induces Cytotoxicity and Mucin Secretion by Guinea Pig Tracheal Epithelial Cells via an Oxidant-Mediated Mechanism

Inhalation of ambient air particulate matter (PM) is associated with pulmonary injury and inflammation. Using primary cultures of guinea pig tracheal epithelial (GPTE) cells as an in vitro model of airway epithelium, we examined effects of exposure to suspensions of six different emission and ambient air PM samples: residual oil fly ash (ROFA) from an electrical power plant; fly ash from a domestic oil burning furnace (DOFA); ambient air dust from St. Louis (STL), Ottawa (OT), and Washington, DC (WDC); and volcanic ash from the eruption of Mount Saint Helens (MSH) in 1980. Effects of these particulates on cell viability (assessed via LDH assay), secretion of mucin (measured by a monoclonal antibody-based ELISA), and steady-state mRNA levels of the mucin gene MUC2 were determined. ROFA was the most toxic of the dusts tested, as it significantly increased LDH release following a 24-h incubation with 50 μg/cm2 ROFA. ROFA also enhanced MUC2 mRNA after 4-h exposure, and mucin secretion after 8 h. ROFA-induced mucin secretion and cytotoxicity were attenuated by the oxidant scavenger, dimethylthiourea (DMTU). ROFA exposure also depleted cells of glutathione (GSH). Relatedly, depletion of intracellular GSH by treatment of the cells with buthionine sulfoxamine (BSO) also provoked mucin secretion, as well as enhancing the secretory effect of ROFA when the two agents were added together. L-NMA, the nitric oxide synthase (NOS) inhibitor, did not affect ROFA-induced mucin secretion. Of the soluble transition metals in ROFA (nickel, iron, vanadium), only vanadium individually, or combinations of the metals containing vanadium, provoked secretion. The results suggest ROFA enhances mucin secretion and generates toxicity in vitro to airway epithelium via a mechanism(s) involving generation of oxidant stress, perhaps related to depletion of cellular antioxidant capacity. Deleterious effects of inhalation of ROFA in the respiratory tract in vivo may relate to these cellular responses. Vanadium, a component of ROFA, may be important in generating these reactions.

Impairment of epithelium-dependent relaxation in coaxial bioassay by reactive oxygen species

The purpose of the present study was to investigate the effects of reactive oxygen species on the activity of epithelium-derived relaxant factor (EpDRF) released by guinea-pig tracheal epithelium. Reactive oxygen species were generated by the electrolysis of the physiological buffer in which the tissues were bathed. Epithelium-dependent relaxation induced by acetylcholine in precontracted rat anococcygeus muscle that was placed in epithelium-intact guinea-pig trachea (coaxial bioassay system) was significantly attenuated when the tissues were exposed to electrolysis. Impairment of the acetylcholine response was prevented by incubation with free radical scavengers prior to electrolysis. In isolated rings of guinea-pig trachea, the contractile responses elicited by acetylcholine, histamine and 5-hydroxytryptamine were not altered after electrolysis of the bathing solution. The results of the present study suggested that exposure to reactive oxygen species impaired EpDRF release from guinea-pig trachea epithelium but did not alter the contractility of tracheal smooth muscle.

Tumor necrosis factor-alpha stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro.

Tumor necrosis factor (TNF)-alpha, a pluripotent cytokine implicated in the pathogenesis of airway inflammation, has been shown to provoke hypersecretion of mucin by airway epithelial cells in vitro. In this study, we investigated potential signaling pathways mediating TNF-alpha-induced mucin secretion using guinea pig tracheal epithelial (GPTE) cells in air-liquid interface culture. Exogenously applied TNF-alpha (human recombinant) stimulated mucin secretion in a concentration-dependent manner, with maximal effects at 10 to 15 ng/ml (286 to 429 U/ml). The pathway of stimulated secretion appeared to involve generation of intracellular nitric oxide (NO), activation of soluble guanylate cyclase (GC-S), production of cyclic guanosine monophosphate (cGMP), and activation of cGMP-dependent protein kinase (PKG). TNF-alpha increased production of nitrite and nitrate by GPTE cells; both mucin secretion and cGMP production were attenuated by NG-monomethyl-L-arginine (1 mM), a competitive inhibitor of nitric oxide synthase (NOS), or by the GC-S inhibitor LY83583 (50 microM); and mucin secretion in response to TNF-alpha or to the cGMP analogue dibutyryl cGMP (100 and 500 microM) was attenuated by the specific PKG inhibitor KT5823 (1 microM). Increased mucin secretion and increased cGMP production in response to TNF-alpha both appeared to be mediated by a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC), and by protein kinase C (PKC), since both responses were attenuated by either D609 (10 and 20 microg/ml), a specific PC-PLC inhibitor, or by each of three PKC inhibitors: Calphostin C (0.3 and 0.5 microM), bisindoylmaleimide (GF 109203X, Go 6850; 20 nM), or Ro31-8220 (10 microM). Collectively, the results suggest that TNF-alpha stimulates secretion of mucin by GPTE cells via a mechanism(s) dependent on PC-PLC and PKC, and involving activation of NOS, generation of NO, production of cGMP, and activation of PKG.

Concurrent Production of Reactive Oxygen and Nitrogen Species by Airway Epithelial Cells In Vitro

Intracellularly generated reactive species of both oxygen (ROS) and nitrogen (RNS) have been implicated in signaling responses in airway epithelial cells, but these radicals have not been measured directly in such cells. In this study, intracellular production of both ROS and RNS were measured in the same cell lysates of guinea pig tracheal epithelial (GPTE) cells maintained in primary culture. ROS and RNS were quantified under basal (constitutive) conditions and in response to different stimuli: LPS and TNFα [activators of inducible nitric oxide synthase (iNOS)]; several activators of calcium-dependent cNOS (ATP, bradykinin, ionophore A23187, and thapsigargin); and exogenous oxidant stress generated by addition of xanthine oxidase to purine ( p + XO). Studies with LPS and TNFα also were performed using the murine macrophage cell line, RAW 264.7, as a positive control. Intracellular oxidant production was detected from oxidation of dihydrorhodamine to rhodamine. NO x was quantified by either chemiluminescent or fluorescent detection. NOS activity was measured as citrulline production from arginine. Basal production of oxidants by GPTE cells (0.08 + 0.00 nmol rhodamine) was less than 10% that of RAW.267 cells (0.91 + 0.03 nmol rhodamine). TNFα and LPS significantly increased intracellular oxidant production in GPTE cells, as did p + XO, but none of the cNOS activators affected production of oxidants in these cells. Concentrations of NO 2 after 4 h in unstimulated RAW 264.7 and GPTE cells were similar and comprised 63% of total NO x in GPTE and 62% in RAW cells. TNFα and LPS both increased NO 2 in GPTE cells, but none of the Ca ++-mobilizing agents nor p + XO significantly affected intracellular RNS. The results suggest both ROS and RNS can be measured in the same lysates from airway epithelial cells, and that both ROS and RNS are produced in these cells in response to different stimuli.

Proliferation and repair of guinea pig tracheal epithelium after neuropeptide depletion and injury in vivo.

Neuropeptides stimulate airway epithelial cell proliferation and migration in vitro, but the role of neuropeptides in the repair of the epithelium after injury in vivo is not clear. We studied epithelial proliferation and repair in 83 male Hartley guinea pigs. Animals received capsaicin weekly for 3 wk to deplete airway neuropeptides. One week later, the dorsal aspect of the trachea was injured with a metal stylette. Animals were killed 1 h to 1 wk later, after which epithelial cell proliferation was assessed for the presence of proliferating cell nuclear antigen (PCNA). PCNA labeling was < 3% in noninjured animals. PCNA labeling increased substantially in the first 72 h after injury in control animals but was significantly decreased in capsaicin-treated animals within and adjacent to the site of injury. PCNA labeling increased opposite to the injury site in both control and capsaicin animals over the first 72 h. We conclude that neuropeptide depletion significantly attenuates both epithelial cell proliferation and repair in the first 72 h after mechanical injury to the trachea. However, neuropeptide depletion did not slow the ultimate repair of tracheal mucosal injury. Proliferation of epithelial cells in response to injury occurs throughout the airway, even away from the injury site.

Characterization of Endothelin Release from Guinea-pig Tracheal Epithelium: Influence of Proinflammatory Mediators Including Major Basic Protein

The characteristics of endothelin (ET) release from guinea-pig tracheal epithelium were investigated, including examination of the effects of several pro-inflammatory mediators. In confluent cultured guinea-pig tracheal epithelial cells (GPTECs) there was a time-dependent basal release of immunoreactive ET (ir-ET) from 4–48 h. Basal ir-ET release from GPTECs was unaffected by the peptidase inhibitors, thiorphan (10 μm), benzamidine (1 mm), pepstatin-A (30 μm), aprotinin (1 μg/ml), bacitracin (20 μg/ml) or leupeptin (50 μm), but was inhibited by phosphoramidon, the neutral metalloprotease inhibitor (IC50=16.8 μm), or the calcium chelator, EGTA (10 mm). There was little ir-ET release 1 day after placing GPTECs in culture, although appreciable release (>10-fold higher) was detected on days 5 and 7. No significant release of ir-ET was demonstrated from intact guinea-pig trachea. Human thrombin (0.1–10 U/ml), LPS (0.3–10 ng/ml) and the phorbol ester, phorbol 12-myristate-13-acetate (0.1 nm–1 μm), significantly increased ir-ET release, whereas TNF-α (0.1–10 ng/ml), RANTES (0.1–100 nm), IL-1 (0.01–10 ng/ml), bradykinin (1 nm–10 μm), CGRP (0.01 nm–1 μm), PDGF (0.1–3 ng/ml), Sar9,Met(O2)11-Sub P, Nle10-NKA 4–10 and senktide (selective NK-1, NK-2 and NK-3 receptor agonists, respectively; 1 nm–10 μm), LTD4(1 nm–10 μm) or major basic protein (10 nm–1 μm) were without stimulatory effect. The results indicate that the enzyme responsible for the basal release of ET from cultured GPTECs is a Ca2+-dependent, phosphoramidon-sensitive, neutral metalloprotease. Furthermore, normally there is minimal ET release from guinea-pig airway epithelium but this can be increased markedly by culturing the cells to confluence, and by select pro-inflammatory mediators.

Influence of ageing on ciliary beat frequency and on ciliary response to leukotriene D4 in guinea-pig tracheal epithelium.

1. In the present study on guinea-pig tracheal epithelium, the influence of age on ciliary beat frequency (CBF) was investigated. 2. An age-dependent decrease in CBF was seen: as the age of the guinea-pig increased from 0.9 to 16.3 months, CBF decreased from 20.2 to 6.9 Hz. 3. The effect of leukotriene D4 (LTD4) on CBF was also examined using tracheal explants from guinea-pigs of varying ages. The lower CBF of the older animal was increased by LTD4 to a larger extent than was the higher CBF seen in explants taken from younger animals. 4. Ciliary activity and possibly also the sensitivity of the cilia to drugs, seems to be dependent upon the age of the animal, which has to be taken into consideration when comparing the results of different studies on the effects of xenobiotics on mucociliary function.

Oxidant stress stimulates mucin secretion and PLC in airway epithelium via a nitric oxide-dependent mechanism.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide variety of respiratory diseases. We investigated mechanisms of ROS-induced mucin secretion by guinea pig tracheal epithelial (GPTE) cells in primary culture, and ROS-induced activation of the second messenger-producing enzyme phospholipase C (PLC), in GPTE cells and in a virally transformed cell line (BEAS-2B) derived from human bronchial epithelium. Mucin secretion was measured by a monoclonal antibody-based enzyme-linked immunosorbent assay, and PLC activation was assessed by anion exchange chromatography. ROS generated enzymatically by xanthine oxidase (XO, 500 microM) in the presence of purine (500 microM) enhanced release of mucin by GPTE cells and activated PLC in GPTE and BEAS cells. Hypersecretion of mucin and activation of PLC in response to purine + XO appeared to occur via an intracellular pathway(s) dependent on endogenously produced nitric oxide and possibly intracellularly generated oxidants. Both responses could be blocked or attenuated by preincubation of the cells with NG-monomethyl-L-arginine, an inhibitor of the enzyme nitric oxide synthase, or with dimethylthiourea, a compound that can react with a variety of intracellular oxidant species. Reactive nitrogen species generated chemically also stimulated secretion of mucin and activated PLC via a mechanism dependent (at least in part) on intracellular oxidant-mediated process(es). The results suggest that intracellularly generated radical species of nitrogen and oxygen may be important modulators of the response of airway epithelial cells to external oxidant stress.

Damage to respiratory epithelium by guinea‐pig eosinophils stimulated with IgG‐coated Sepharose beads

There are no reports of respiratory epithelial damage induced by immunoglobulin-stimulated eosinophils. We tried to induce damage to respiratory epithelium by guinea-pig (GP) eosinophils stimulated with guinea-pig IgG (GP-IgG)-coated Sepharose 4B beads. GP tracheal epithelium was cultured together with GP eosinophils that had been collected from the peritoneal cavity, purified on a Percoll gradient, and stimulated with GP-IgG-coated beads (GP eosinophils:beads = 20:1). Damage to the epithelium was observed with an inverted microscope. After 24 h of culture with three 10(6) eosinophils, irregularity of the surface of the epithelium, desquamation, shedding of cilia, and abnormal beating of cilia were observed. This damage was first observed after 12 h of incubation, and was more severe at 24 h. No damage was found when beads coated with human serum albumin (HSA) were used. GP eosinophils stimulated with GP-IgG released significantly more EPO than those stimulated with HSA, at 6 and 24 h. Heparin and catalase partially inhibited the epithelial damage. O2-production by eosinophils was also enhanced with GP-IgG-coated beads. Both granule basic proteins and reactive oxygen radical may be responsible for epithelial damage, probably via an EPO + H2O2 + halide system. These results confirmed that stimulated eosinophils can damage respiratory epithelium.

Effects of Some Preservative Agents on Rat and Guinea Pig Tracheal and Human Nasal Ciliary Beat Frequency

Many preservatives commonly included in nasal drops and sprays are known to impair mucociliary clearance. We studied the effects of four frequently used preservatives on ciliary beat frequency (CBF) in respiratory tissue. Sodium metabisulfite and chlorbutol did not change the CBF at concentrations up to 50 mg/L in rat tracheal mucosa. Chlorocresol 25 mg/L and 50 mg/mL reversibly decreased CBF (by 33% and 68%, respectively) in 60 minutes. Benzalkonium chloride impaired CBF irreversibly already at a relatively low concentration (12.5 mg/L). To further clarify these results, we studied the effects of the most ciliotoxic (benzalkonium chloride) and nonciliotoxic (chlorbutol) preservative on guinea pig tracheal epithelium and human nasal mucosa. Although chlorbutol had no effect on the CBF, a dose-dependent decrease on CBF was seen in guinea pig and human ciliated epithelium during their immersion in benzalkonium chloride. At a concentration of 50 mg/L it stopped the ciliary activity in 40 minutes in guinea pig trachea, and in human nasal mucosa, benzalkonium chloride concentrations of 25 and 50 mg/L, decreased the CBF irreversibly (by 28% and 60%, respectively) in 60 minutes. These results suggest that chlorbutol is a safe and well tolerated preservative. Banzalkonium chloride is ciliostatic in vitro to rat, guinea pig, and human respiratory mucosa. Therefore, prolonged clinical use of benzalkonium chloride may impair mucociliary clearance, a major defense respiratory mechanism.

Hypersecretion of mucin in response to inflammatory mediators by guinea pig tracheal epithelial cells in vitro is blocked by inhibition of nitric oxide synthase.

Primary cultures of guinea pig tracheal epithelial cells in air/liquid interface were exposed to one of four agents associated with airway inflammation: the peptide histamine (100 microM), the lipid mediator platelet-activating factor (1 microM), the cytokine tumor necrosis factor-alpha (15 ng/ml; specific activity 2.86 x 10(7) U/mg), or enzymatically generated reactive oxygen species (purine [500 microM]+xanthine oxidase [20 mU/ml]). Effects of each of these substances on release of mucin by guinea pig tracheal epithelial (GPTE) cells were measured using a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Each secretagogue significantly enhanced release of mucin, but the stimulatory effect of each was inhibited by pre-(+)co-incubation of the cells with the competitive inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine (L-NMA), but not by NG-monomethyl-D-arginine (D-NMA), the inactive stereoisomer that does not inhibit nitric oxide synthase. Neither L-NMA nor D-NMA affected mucin secretion by themselves. The results suggest that each of these inflammation-associated mediators provokes airway epithelial mucin secretion via a mechanism involving intracellular production of nitric oxide (NO) as a critical signaling molecule.

Migration and proliferation of guinea pig and human airway epithelial cells in response to tachykinins.

Restoration of the epithelial lining of a damaged airway is a necessary component of airway repair. Tachykinins, including substance P (SP) and neurokinin A (NKA), are localized to sensory nerves within the airway mucosa. These tachykinins regulate several airway functions, but their role in the repair of the epithelium has not been explored. To determine whether tachykinins stimulate migration and proliferation of airway epithelial cells, guinea pig tracheal epithelial (GPTE) and human bronchial epithelial (HBE) cells were grown in primary culture for 4-5 days. Epithelial cell migration was assessed in a blindwell chemotaxis chamber, and proliferation was determined by immunohistochemistry after incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). Both GPTE and HBE cells migrated after stimulation with 10(-11) M NKA [23.0 +/- 3.6 vs. 5.4 +/- 1.2 cells per 10 high-power fields (hpf), P < 0.001, n = 8 for GPTE cells; 18.4 +/- 2.3 vs. 3.8 +/- 0.5 cells per 10 hpf for control, P < 0.001, n = 4 for HBE cells]. Migration was stimulated within 2 h, was maximal after 6 h, and was attenuated substantially by the neurokinin 2 (NKA)-receptor antagonist SR-48968. NKA-stimulated migration was both chemokinetic and chemotactic, and it could be blocked by inhibition of protein synthesis with cyclohexamide, inhibition of microtubular function with colchicine, or inhibition of actin microfilament elongation with cytochalasin D.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation of guinea pig tracheal epithelial cells in coculture with rat dorsal root ganglion neural cells.

Neuropeptides secreted by sensory afferent nerves in airways may modulate growth of airway epithelial cells. To determine whether airway sensory C-fiber nerves secrete neuropeptides that stimulate airway epithelial cell proliferation, we measured S-phase traversal in guinea pig tracheal epithelial (GPTE) cells after coculture with rat dorsal root ganglion (DRG) cells. GPTE cells were grown in subconfluent culture on collagen-coated filters for 2 days. DRG cells were harvested from newborn rat pups and grown in primary culture for 7-10 days in separate wells. GPTE and DRG cells then were cocultured for 48 h, and 10 mM bromodeoxyuridine (BrdU), a thymidine analogue, was added in the final 24 h. Control GPTE cells were grown under similar conditions but without DRG cells. Coculture with DRG cells stimulated GPTE cell traversal of S phase. BrdU labeling in cocultured GPTE cells was 42.8 +/- 5.8 compared with 18.1 +/- 7.2% in control GPTE cells (P < 0.001, n = 6). Coculture in the presence of either the neurokinin (NK)1 receptor antagonists LY-297911 or CP-99,994, the NK2 receptor antagonist SR-48,968, or the calcitonin gene-related peptide (CGRP) receptor antagonist hCGRP-(8-37) (10(-7) M of each) during coculture attenuated proliferation of GPTE cells. Treatment with all three antagonists together during coculture decreased BrdU labeling to 2.4 +/- 0.9% of labeled cells vs. 8.5 +/- 0.5% of labeled cells during coculture without antagonists (n = 4, P < 0.02). DRG cells in coculture secreted substantial concentrations of CGRP [71.0 +/- 11.3 (+/- SE) pmol/ml], substance P (1.26 +/- 0.35 pmol/ml), and neurokinin A (0.45 +/- 0.10 pmol/ml) (n = 19 for each).(ABSTRACT TRUNCATED AT 250 WORDS)