Rat Tracheal Explants Research Articles

Effect of Myrtol Standardized and Other Substances on the Respiratory Tract: Ciliary Beat Frequency and Mucociliary Clearance as Parameters

Myrtol standardized is a phytomedicine obtained by distillation, consisting of many constituents. In vitro and in vivo, the major monterpenes, d-limonene, 1,8-cineole, and alpha-pinene, are used as biological marker substances. Myrtol standardized has secretolytic, secretomotor, and mucolytic effects in addition to anti-inflammatory and antioxidative actions. The aim of the study was to investigate the effects of different concentrations of Myrtol standardized on in vivo mucociliary clearance in mice and the ciliary beat frequency (CBF) in rat tracheal rings. Data regarding the effects of 1,8-cineole and N-acetylcysteine (NAC) were compared. Salbutamol was used as a positive control. CBF was measured using rat tracheal explants and a high-speed video camera linked to a microscope with specific software equipment. Mucociliary clearance was determined using the microdialysis technique, which measured the acceleration of a fluorescent sample in the trachea in vivo. Myrtol standardized accelerated both CBF and mucociliary transport in a concentration-dependent manner. Significant effects were seen at a concentration of 0.01% Myrtol regarding CBF (P<0.01) and 17.1 mg/kg body weight regarding mucociliary clearance experiments (P<0.05) according to doses relevant to humans. The 1,8-cineole dosage relative to humans only accelerated the mucociliary clearance in vivo without having an effect on the CBF. Extremely high doses of Myrtol were not able to additionally increase the CBF effect in comparison to salbutamol. Compared to NAC, also used in a dosage relative to humans, Myrtol standardized showed a tendency to be more effective. In summary, the present data suggest that Myrtol standardized is a pharmacologically important drug which, when used at a dose relative to humans, shows positive effects on both measured parameters, CBF and mucociliary clearance, in vivo.

Modification of the rat airway explant transcriptome by cigarette smoke

Although a number of animal model studies have addressed changes in gene expression in the parenchyma and their relationship to emphysema, much less is known about the pathogenesis of cigarette smoke–induced small airway remodeling. In this study the authors exposed rat tracheal explants, a model of the airway wall, to whole smoke for 15 min, and then cultured the explants in air. The airway transcriptome was evaluated using RAE 230_2 gene chips. By 2 h after starting smoke exposure, expression levels of 502 genes were differentially expressed by more than 1.5 times (p < .01 or less) and by 24 h 1870 genes were significantly changed up or down. These included genes involved in antioxidant protection, epithelial defense and remodeling, inflammatory mediators and transcription factors, and a number of unexpected genes, including the matrix metalloproteinase (MMP)-12 inducer, tachykinin-1 (substance P). Pretreatment of the explants with 1 × 10−7 M dexamethasone reduced the number of significantly changed genes by approximately 47% at 2 h and 68% at 24 h and in almost all instances reduced the magnitude of the smoke-induced changes. The authors conclude that even a very brief exposure to cigarette smoke can lead to rapid changes in the expression of a large number of genes in rat tracheal explants, and that these effects are directly mediated by smoke, without a need for exogenous inflammatory cells. Steroids, contrary to the usual belief, are able to ameliorate many of these changes, at least in this very acute model.

The expectorant activity of naringenin

The expectorant activity of naringenin was studied. Mucus secretion was evaluated in mice by measuring the tracheal output of phenol red. Mucociliary movement function was investigated using a migration method of carbon granules in unanesthetized pigeons. And the effect of naringenin on the secretion of mucin and lysozyme was performed in the rat tracheal ring explants. Naringenin could significantly increase the secretion of phenol red from mouse tracheas at the doses of 30–67 mg/kg (i.g.) ( P<0.05). Naringenin, at the dose of 90 mg/kg, increased the tracheal mucociliary velocity (TMV) to 144.4% of control ( P<0.01). 100 μM naringenin could enhance the basal lysozyme secretion, but had no effect on the basal mucin secretion from the rat tracheal ring explants. Treatment with naringenin at higher concentration (10 μmol/l) could inhibit the 100 ng/ml lipopolysaccharide (LPS)-induced mucin increase. These data suggest, therefore, that naringenin has the expectorant activity.

Cigarette Smoke Causes Small Airway Remodeling by Direct Growth Factor Induction and Release

Small airway remodeling (SAR) is one of the anatomic causes of airflow obstruction in patients with chronic obstructive pulmonary disease. The pathogenesis of SAR is poorly understood, but there are two general theories: (1) SAR is caused by smoke-induced inflammatory cells that damage the airway and evoke a repair process and (2) SAR is caused by direct induction of growth factors by cigarette smoke. To investigate this process, we briefly exposed rat tracheal explants, a model system free of exogenous inflammatory cells, to whole cigarette smoke, and then maintained the explants in organ culture for varying periods. With explants cultured in air, smoke induced up-regulation of both transforming growth factor (TGF)-β1 and procollagen gene expression at 24 h after initial exposure. Increased procollagen gene expression was prevented in a dose–response fashion by the TGF-β antagonist fetuin, indicating that TGF-β was driving fibrosis. Collected supernatant from explants exposed to smoke and cultured in fluid medium showed increased release of TGF-β1, and this was abolished by the oxidant scavenger tetramethylthiourea. Gene expression of connective tissue growth factor (CTGF) was sharply increased 2 h after smoke exposure. Using pure recombinant proteins, smoke-conditioned medium oxidized TGF-β latency-associated peptide and also caused release of active TGF-β1 from latent TGF-β1 . We conclude that cigarette smoke directly induces release of preformed TGF-β1 by oxidizing the latency-associated peptide in rat tracheal explants, and that release must be extremely rapid, since gene expression of CTGF, the downstream fibrogenic driver of TGF-β effects on collagen synthesis, is elevated very shortly after smoke exposure. Although tracheal explants are only an approximate model of small airways, these findings suggest that smoke-induced SAR may reflect direct induction of growth factor release and signaling and does not require smoke-evoked inflammatory cells.

Transforming Growth Factor-β1 Drives Airway Remodeling in Cigarette Smoke–Exposed Tracheal Explants

Small airway remodeling (SAR) is an important cause of airflow obstruction in cigarette smokers, but whether SAR represents a response to smoke-evoked inflammation or is directly mediated by smoke-induced growth factor production is disputed. To examine this process, we exposed rat tracheal explants, a model free of exogenous inflammatory cells, to cigarette smoke in vitro. Cigarette smoke caused release of active transforming growth factor (TGF)-beta1, and this was prevented by the oxidant scavenger tetramethythiourea. Nuclear immunostaining for phospho-Smad2, a TGF-beta downstream signaling molecule, was present in epithelial and interstitial cells within 1 h after exposure. Smoke caused upregulation of gene expression of connective tissue growth factor (CTGF), a mediator of TGF-beta fibrogenic effects, within 2 h, and upregulation of procollagen gene expression at 24 h; both changes could be prevented by the TGF-beta antagonist fetuin (alpha2-HS-glycoprotein). In a cell-free system, recombinant human TGF-beta latency-associated peptide was oxidized by cigarette smoke, and smoke released active TGF-beta1 from recombinant latent TGF-beta1 via an oxidant mechanism. These experiments suggest that SAR in cigarette smokers may be caused by direct, smoke-mediated, oxidant-driven induction of growth factor signaling in the airway wall, and that SAR does not necessarily require exogenous inflammatory cells.

Air pollution particles activate NF-κB on contact with airway epithelial cell surfaces

Air pollution particles (PM) are known to elicit an acute inflammatory response in vivo that is mediated in part through PM-induced activation of the NF-κB signaling pathway. Many of the details of this process and particularly where in the cell it occurs are unclear. To determine whether contact of PM particles with an epithelial cell surface activates NF-κB, rat tracheal explants were exposed to Ottawa Urban Air Particles or iron-loaded fine TiO 2, a model PM particle, for up to 2 h. During this period, there was no evidence of particle entry into the tracheal epithelial cells by light or electron microscopy, but both types of particle activated NF-κB as assayed by gel shifts. NF-κB activation could be inhibited by the active oxygen species scavenger, tetramethylthiourea; the redox-inactive metal chelator, deferoxamine; the Src inhibitor, PP2; and the epidermal growth factor (EGF) receptor inhibitor AG1478. An iron-containing citrate extract of both dusts also produced NF-κB activation. Both dusts and a citrate extract caused phosphorylation of the EGF receptor on tyrosine 845, an indicator of Src activity. We conclude that iron-containing PM particles can activate NF-κB via a pathway involving Src and the EGF receptor. This process does not require entry of particles into the airway epithelial cells but is dependent on the presence of iron and generation of active oxygen species by the dusts. These findings imply that even brief contact of PM with a pulmonary epithelial cell surface may produce deleterious effects in vivo.

Cigarette smoke produces airway wall remodeling in rat tracheal explants.

Small airway remodeling ("small airways disease") is a common finding in cigarette smokers and is an important cause of airflow obstruction. Airway remodeling is usually attributed to the effects of cigarette smoke-induced inflammation in the airway wall, but little is actually known about its pathogenesis. We exposed rat tracheal explants to cigarette smoke and then maintained them in air organ culture. At 24 hours after smoke exposure, there was a dose-dependent increase in gene expression of procollagen and a significant increase in tissue hydroxyproline, a measure of collagen content. Greater increases in procollagen gene expression were found with repeated smoke exposures. Increased procollagen gene expression could be prevented with SN50, a selective inhibitor of nuclear factor-kappaB activation, and superoxide dismutase, catalase, and tetramethylthiourea, scavengers of active oxygen species. AG1478, an inhibitor of epidermal growth factor receptor signaling, also prevented increased procollagen gene expression, but PD98059 and SB203580, inhibitors of mitogen-activated protein kinases, did not. These findings indicate that cigarette smoke can directly induce airway remodeling, specifically airway wall fibrosis, probably through active oxygen species-dependent transactivation of the epidermal growth factor receptor and subsequent nuclear factor-kappaB activation. Smoke-evoked inflammatory cells are not required for this process.

Air pollution particles produce airway wall remodeling in rat tracheal explants.

There is evidence that chronic exposure to high levels of ambient particulate pollutants (PM) is associated with chronic airflow obstruction, but how this occurs is not known. We exposed rat tracheal explants to Ottawa urban air particles (ECH93) or diesel exhaust particles. After 7 d in air organ culture, both types of PM increased explant procollagen and transforming growth factor (TGF)-beta 1 gene expression, and markedly increased tissue hydroxyproline. For both types of particle, nuclear factor-kappa B inhibitor SN50 completely blocked increased gene expression. With EHC93, procollagen expression was inhibited by the oxidant scavenger, tetramethylthiourea, and by the iron chelator, deferoxamine, but TGF-beta1 expression was not inhibited by deferoxamine. Inhibitors of extracellular signal regulated kinase and p38 kinase did not affect EHC93-induced gene expression. With diesel exhaust particles, tetramethylthiourea and deferoxamine had no effect, but extracellular signal regulated kinase and p38 inhibitors completely blocked effects on procollagen and TGF-beta 1. Fetuin, an inhibitor of TGF-beta receptor binding, prevented increases in procollagen gene expression. We conclude that two common types of PM can directly induce expression of genes involved in fibrogenesis and actual airway wall fibrosis through nuclear factor-kappa B- and TGF-beta-mediated mechanisms. PM-induced airway wall remodeling may play an important role in producing airflow obstruction in individuals living in high PM regions.

Airway Wall Remodeling Induced by Occupational Mineral Dusts and Air Pollutant Particles

COPD has been reported in workers exposed to particulates, and there is increasing evidence that high levels of ambient particulate pollutants may also be associated with COPD. The studies here investigate the hypothesis that particulates, including air pollution particles, can induce airway wall fibrosis, a process that can lead to COPD. Rat tracheal explants were exposed to various occupationally encountered dusts, air pollution particles, and model air pollution particles. In some experiments, iron was loaded onto the particle surface. Gene expression and nuclear factor (NF)-kappaB activation were measured after 7 days of air culture. Adhesion to and uptake of dusts by the tracheal epithelium were also evaluated. Known fibrogenic dusts such as amosite asbestos produced increased gene expression of procollagen, transforming growth factor-beta, and platelet-derived growth factor, and increased hydroxyproline in the explants, and the addition of iron increased these effects. The addition of iron also converted nonfibrogenic TiO2 into a fibrogenic dust. Dusts with surface complexed iron activated NF-kappaB via an oxidant mechanism. However, an ultrafine TiO2 with very low iron was also fibrogenic. In separate experiments, exogenous tumor necrosis factor-alpha increased dust adhesion to, and exogenous ozone increased dust uptake by, tracheal epithelial cells. Mineral dusts can directly induce fibrosis in the airway wall. Exogenous inflammatory cells and exogenous agents are not required, but they probably exaggerate the fibrogenic effects. An iron-mediated oxidant mechanism underlies the fibrogenic effects of some, but not all, of these dusts. Particle-induced airway wall fibrosis may lead to COPD.

TNFα Increases Binding of Air Pollutant Particles to Tracheal Epithelial Cells: Possible Role of ICAM-1

Exposure of alveolar macrophages (MAC) to particulates frequently results in the production of tumor necrosis factor α (TNFα). We have previously shown that TNFα increases the binding of amosite asbestos to tracheal epithelial cells and that this process proceeds via an NF-κBdependent mechanism. To examine whether TNFα affects binding of compact particles and air pollutant particles (PM) as well, we exposed rat tracheal explants to various concentrations of TNFα followed by Ottawa PM 10 , ROFA or fine TiO 2 . Using scanning electron microscopy, TNFα increased particle binding of all three species. This process could be partially prevented by cycloheximide, deferoxamine and tetramethylthiourea. The phosphotidyl-choline-related phospholipase C inhibitor, D609 and anti-ICAM antibody completely abolished the increase in binding. We conclude that TNFα is able to increase epithelial binding of PM particles to epithelial cells. Active oxygen species appear to play a role in binding, and TNFα-enhanced binding may be mediated through increases in ICAM-1 cell surface protein. Increased particle binding probably increases particle internalization and adverse molecular responses.

Iron loading makes a nonfibrogenic model air pollutant particle fibrogenic in rat tracheal explants.

To examine the potential role of particle iron in fibrogenicity, we loaded nonfibrogenic fine (0.12micro) TiO(2) with increasing amounts of Fe(II)-Fe(III) chloride. Dusts were applied to rat tracheal explants, which were maintained in air organ culture for 1 wk. Iron-loaded dust increased procollagen gene expression and tissue hydroxyproline. The active oxygen species (AOS) scavenger tetramethylthiourea prevented these effects. Iron loading caused nuclear factor (NF)-kappaB activation, decreased levels of total IkappaBalpha, but relatively increased levels of both IkappaBalpha-phosphoserine 32/36 and IkappaBalpha-phosphotyrosine. A citrate extract of iron-loaded dust increased procollagen expression. Gel shift using a probe consisting of the NF-kappaB consensus sequence from the prolyl-4-hydroxylase promoter and adjacent bases showed increased nuclear binding, and RT-PCR examination showed increased prolyl-hydroxylase alpha-chain gene expression after iron loading. We conclude that addition of surface iron can convert a nonreactive model air pollutant particle into a fibrogenic particle via AOS- and NF-kappaB-dependent pathways, probably through two different NF-kappaB activation pathways in two different anatomic compartments. This process may proceed in vivo through iron extracted from the dust into the cytoplasm. NF-kappaB activation may directly increase expression of prolyl hydroxylase, an enzyme involved in collagen synthesis. These findings suggest that air pollutant particles containing significant quantities of transition metals may produce airway wall fibrosis and lead to chronic obstructive pulmonary disease.

Relationship of fiber surface iron and active oxygen species to expression of procollagen, PDGF-A, and TGF-beta(1) in tracheal explants exposed to amosite asbestos.

To investigate the role of iron and active oxygen species (AOS) in asbestos-induced fibrosis, we loaded increasing amounts of Fe(II)/Fe(III) onto the surface of amosite asbestos fibers and then applied the fibers to rat tracheal explants. Explants were harvested after 7 d in air organ culture. Asbestos by itself doubled procollagen gene expression, and a further increase was seen with increasing iron loading; actual collagen content measured as hydroxyproline was increased in a similar pattern. Iron loading also increased gene expression of platelet-derived growth factor (PDGF)-A and transforming growth factor (TGF)-beta(1). Neither asbestos alone nor iron-loaded asbestos affected gene expression of PDGF-B, tumor necrosis factor-alpha, or TGF-alpha. The AOS scavenger tetramethylthiourea or treatment of fibers with the iron chelator deferoxamine prevented asbestos-induced increases in procollagen, PDGF-A, and TGF-beta gene expression, whereas glutathione had no effect. The proteasome inhibitor MG-132 abolished asbestos-induced increases in procollagen gene expression but did not affect increases in PDGF-A or TGF-beta(1) expression, whereas the extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 had exactly the opposite effect. We conclude that surface iron as well as the iron-catalyzed generation of AOS play a role in asbestos-induced matrix (procollagen) production and that this process is driven in part through oxidant-induced nuclear factor kappa B activation. Surface iron and AOS also play a role in PDGF-A and TGF-beta gene expression, but through an ERK-dependent mechanism.

TNF-alpha increases tracheal epithelial asbestos and fiberglass binding via a NF-kappaB-dependent mechanism.

Tumor necrosis factor (TNF)-alpha is released from alveolar macrophages after phagocytosis of mineral fibers. To determine whether TNF-alpha affects the binding of fibers to epithelial cells, we exposed rat tracheal explants to TNF-alpha or to culture medium alone, followed by a suspension of amosite asbestos or fiberglass (MMVF10). Loosely adherent fibers were removed from the surface with a standardized washing technique, and the number of bound fibers was determined by scanning electron microscopy. Increasing doses of TNF-alpha produced increases in fiber binding. This effect was abolished by an anti-TNF-alpha antibody, the proteasome inhibitor MG-132, and the nuclear factor (NF)-kappaB inhibitor pyrrolidine dithiocarbamate. Gel shift and Western blot analyses confirmed that TNF-alpha activated NF-kappaB and depleted IkappaB in this system and that these effects were prevented by MG-132 and pyrrolidine dithiocarbamate. These observations indicate that TNF-alpha increases epithelial fiber binding by a NF-kappaB-dependent mechanism. They also suggest that mineral particles may cause pathological lesions via an autocrine-like process in which the response evoked by particles, for example, macrophage TNF-alpha production, acts to enhance subsequent interactions of particles with tissue.

Regulation of ciliary beat frequency by the nitric oxide-cyclic guanosine monophosphate signaling pathway in rat airway epithelial cells.

Nitric oxide (NO) upregulates ciliary beat frequency (CBF). The present study evaluates mechanisms of the NO-cyclic guanosine monophosphate (cGMP) pathway regulation of CBF. Rat tracheal explants were loaded with 4,5-diaminofluorescein diacetate for the demonstration of NO production by ciliated epithelial cells after L-arginine (L-Arg) stimulation. CBF was measured using phase contrast microscopy and videotape analysis. The roles of NO, soluble guanylate cyclase (sGC), cGMP-dependent protein kinase (PK) G, and phosphodiesterase (PDE) V in regulation of CBF were evaluated. NO synthase (NOS) was activated with L-Arg or inhibited with N(G)-monomethyl-L-Arg. sGC was stimulated with NO donors 1-hydroxy-2-oxo-3- (N-ethyl-2-aminoethyl)-3-ethyl-1-triazene and S-nitroso-L-glutathione or mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (8-Br-cGMP) and inhibited with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one. The effects of the PKG inhibition with KT5823 and PDE V inhibition with Zaprinast were also examined. The studies demonstrate that ciliated epithelial cells produce NO, which is correlated with CBF stimulation. L-Arg dose- and time-dependently increases CBF, and NO donors, 8-Br-cGMP, and Zaprinast also enhance CBF. Inhibitors of NOS, sGC, and PKG can block the stimulant effect of L-Arg on CBF. Thus, NO is a regulator of CBF acting via sGC and PKG. The NO-cGMP signaling pathway regulates CBF in an autocrine manner in cultured rat ciliated airway epithelium.

Induction of fibrogenic mediators by fine and ultrafine titanium dioxide in rat tracheal explants.

Respirable ambient particles [particulate matter <10 micrometer (PM(10))] are associated with both acute and chronic adverse health effects including chronic airflow obstruction. PM(10) can induce expression of inflammatory and fibrogenic mediators, but there is controversy about the types and/or sizes of particles involved and, in particular, whether ultrafine particles are the major toxic agents. To examine whether particle size affects mediator generation, we exposed rat tracheal explants, an inflammatory cell-free model of the airway wall, to various concentrations up to 500 microgram/cm(2) of fine (0.12 micrometer) or ultrafine (0.021 micrometer) titanium dioxide (anatase), maintained the explants in an organ culture in air for 1-7 days, and used RT-PCR to examine the expression of fibrogenic mediators and procollagen. No increase in gene expression was seen at 1 or 3 days, but at 5 days, ultrafine dust induced a small increase in procollagen. At 7 days, fine titanium dioxide produced significantly greater increases for platelet-derived growth factor (PDGF)-B, transforming growth factor-alpha, and transforming growth factor-beta compared with those by ultrafine dust; both dusts produced similar increases for PDGF-A; and ultrafine dust produced increases in procollagen expression, whereas fine dust had no effect. Expression levels were dose related. Both dusts produced a similar decrease in expression of PDGF receptor-alpha and a similar increase in PDGF receptor-beta. These observations suggest that ultrafine particles are intrinsically able to induce procollagen expression even in the absence of inflammatory cells; that chronic exposure to PM(10) may result in chronic airflow obstruction, in part because of ultrafine particle-mediated increases in airway wall fibrosis; and that chemically identical dusts of differing size can produce quite different patterns of gene expression in the airway wall. Differential upregulation of PDGF receptors does not appear to explain dust-induced fibrosis in this model.

Cigarette smoke increases amosite asbestos fiber binding to the surface of tracheal epithelial cells

Binding of asbestos fibers to the cell surface appears to be important in the initiation of intracellular signaling events as well as in initiation of particle uptake by the cell. We have previously shown that cigarette smoke increases the uptake of asbestos fibers by tracheal epithelial cells in explant culture. Whether smoke acts by increasing surface binding of fibers is not known. In this study, we exposed rat tracheal explants to air or cigarette smoke and then to a suspension of amosite asbestos. Explants were harvested after 1 or 24 h of dust exposure and washed by repeated dips in culture medium to remove loosely bound fibers, and the number of fibers adhering to the apical cell surfaces was determined by scanning electron microscopy. Smoke-exposed explants retained significantly more surface fibers than air-exposed explants. After four washes, binding levels were similar at 1 and 24 h. The smoke effect was still present when incubations were carried out at 4 degrees C, but binding was decreased approximately 25%. Preincubation of the asbestos fibers with iron chloride to increase surface iron increased fiber binding in both air- and smoke-exposed explants, whereas preincubation of the fibers with the iron chelator deferoxamine decreased binding after air exposure and completely eliminated the smoke effect. Inclusion of mannitol or catalase in the medium or preincubation of the explants with GSH produced decreases in binding of 10-25% in air-exposed explants and generally greater decreases in smoke-exposed explants. We conclude that 1) amosite binding is a very rapid process that does not require active cellular metabolism, 2) cigarette smoke increases adhesion of fibers to the epithelial surfaces, and 3) iron on the asbestos fiber appears to play an important role in binding, probably through an active oxygen species-mediated process.

Differentiation of respiratory epithelium: the effects of retinoic acid and carcinogens on the expression of mucociliary vs. squamous phenotype.

Changes in ultrastructural characteristics and mucin gene expression were examined in rat tracheal explants cultured in a synthetic medium +/- retinoic acid (RA), benzo[a]pyrene (B[a]P) and N-methyl-N-nitrosourea (NMNU). In the RA(+) cultures, no changes in either ultrastructural features or mucin gene expression were detected after 48 h incubation. After 96 h incubation, however, the ultrastructural features associated with the squamous phenotype were characteristics of cultures containing the two carcinogens and the mucin gene expression was slightly reduced. Thus, in the presence of retinoic acid, the carcinogen induced changes in cytology to the squamous phenotypes were not matched by a marked loss of mucin gene expression. Explants cultured for 48 h without RA and +/- carcinogens showed none of the cytological changes associated with onset of the squamous phenotype. While mucin mRNA was still detected, it was clearly reduced compared to 48 h cultures in RA(+) medium. However, 48 h later, all explants exhibited pronounced squamous metaplasia and the mucin message decreased to trace levels. Thus, the results of these experiments with B[a]P and NMNU in RA(+) and RA(-) media indicates that at least the early carcinogen induced changes may be distinct from those associated with the retinoid pathway controlling expression of the mucin component of the mucociliary epithelium.

Phosgene effects on F-actin organization and concentration in cells cultured from sheep and rat lung

Pulmonary edema and immunosuppression of the lung are primary causes of debilitation and death from phosgene gas exposure. The pathophysiology that gives rise to these conditions shares a common clinical pathway. However, the target cells and lesions that disrupt normal barrier function and immune response of the lung are complex and poorly understood. Using confocal laser microscopy and FITC-conjugated phalloidin, we have studied the effects of phosgene on F-actin in endothelial cells from sheep pulmonary arteries and epithelial cells from rat tracheal explants. Image analyses from attached culture systems indicate that F-actin was a sensitive target molecule in both species. Exposures ranging from 0.15 to 1.0 x LCt50 for sheep in vivo (3300 ppm.min) produced immediate, dose-dependent decreases in average F-actin content of cultured endothelial cells. Dense peripheral bands and stress fibers were diminished and partially disrupted but were not destroyed by these doses. Changes in ultrastructure and the permeability barrier of endothelial tissues included separation of basal lamina and development of paracellular leakage paths. Phosgene also decreased the F-actin in airway epithelial cells and potentiated phenotypic transformations that gave rise to progeny with dendritic processes. Differences in endothelial and airway epithelial response indicate that the cytoskeletal effects of phosgene were cell-type specific. Disruption of basal lamina, depletion of F-actin, and development of endothelial leakage paths may contribute to decreased barrier function and increased permeability of vascular tissues. Phosgene-induced transformations that involved F-actin reorganization and appearance of dendritic cells among airway epithelial may affect other functions of the lung.

Effect of retinoic acid on mucin gene expression in rat airways in vitro

Ultrastructural examination of rat tracheal explants at various times of culture in a serum-free and hormone-supplemented medium containing retinoic acid showed that the cytological characteristics of the epithelium were well preserved for at least 192 h. Hybridization analyses for mucin core protein mRNA in the explants were performed with a 30-base oligonucleotide probe, the design of which was based on the tandem repeat sequence of the rat intestine mucin core protein. The probe reacted with total RNA prepared from trachea, intestine and colon, but not with total RNA obtained from liver or alveolar region of the lung. Type-I keratin expression was observed in the explant grown at different periods of time in a medium with and without retinoic acid. The hybridization probe gave a prominent reaction with RNA preparations obtained from tracheal explants incubated for as long as 192 h in a medium containing retinoic acid. In the absence of retinoic acid, however, the mucin message was evident at the 24 h time point but thereafter decreased to barely detectable levels. When retinoic acid was added at 96 h to the latter cultures, the mucin mRNA was prominent again after additional incubation for 24 and 48 h. Northern-blot analyses of tracheal RNA showed a diffuse band at approx. 7.5 kb. Addition of a variety of chemical and pharmacological agents to explants cultured in the presence of retinoic acid had no dramatic induction or inhibitory effects on the mucin mRNA. Only the steroid prednisolone had a reproducible inhibitory effect.

Effects of Cigarette Smoke on Epithelial Uptake of Non-Asbestos Mineral Particles in Tracheal Organ Culture

Cigarette smoke is believed to increase the pulmonary retention of many different types of mineral dusts, but the mechanisms of this process are unclear. We have previously shown, using a tracheal organ culture system, that exposure to cigarette smoke directly increases the uptake of asbestos fibers by tracheal epithelial cells, and that this process is mediated by active oxygen species. To determine whether the same effects are seen with other types of mineral dust, we exposed rat tracheal explants to cigarette smoke or air (control) and then to a variety of dusts generally considered "inert" or of low pathogenicity. Explants were maintained in organ culture to allow dust uptake, and segments fixed and prepared for light microscopy at various times up to 1 wk; particle uptake was determined morphometrically. We observed that cigarette smoke significantly increased the epithelial uptake of nonfibrous titanium dioxide and talc, and of fibrous silicon carbide, but not of fibrous or nonfibrous iron oxide, nonfibrous silicon carbide, or fibrous wollastonite. Scavengers of active oxygen species (catalase) or agents that prevent the formation of active oxygen species (deferoxamine) blocked the effects of smoke in enhancing titanium dioxide uptake but did not block the effects of smoke on talc uptake. These observations indicate that cigarette smoke may potentiate the effects of many types of dust, including relatively inert dusts, by directly increasing the numbers of particles entering the tracheobronchial epithelium and eventually reaching the interstitium. However, smoke does not directly enhance the uptake of every dust.(ABSTRACT TRUNCATED AT 250 WORDS)