Ovine Tracheal Epithelial Cells Research Articles

Differentiated ovine tracheal epithelial cells support the colonisation of pathogenic and non-pathogenic strains of Mannheimia haemolytica

Mannheimia haemolytica is the primary bacterial species associated with respiratory disease of ruminants. A lack of cost-effective, reproducible models for the study of M. haemolytica pathogenesis has hampered efforts to better understand the molecular interactions governing disease progression. We employed a highly optimised ovine tracheal epithelial cell model to assess the colonisation of various pathogenic and non-pathogenic M. haemolytica isolates of bovine and ovine origin. Comparison of single representative pathogenic and non-pathogenic ovine isolates over ten time-points by enumeration of tissue-associated bacteria, histology, immunofluorescence microscopy and scanning electron microscopy revealed temporal differences in adhesion, proliferation, bacterial cell physiology and host cell responses. Comparison of eight isolates of bovine and ovine origin at three key time-points (2 h, 48 h and 72 h), revealed that colonisation was not strictly pathogen or serotype specific, with isolates of serotype A1, A2, A6 and A12 being capable of colonising the cell layer regardless of host species or disease status of the host. A trend towards increased proliferative capacity by pathogenic ovine isolates was observed. These results indicate that the host-specific nature of M. haemolytica infection may result at least partially from the colonisation-related processes of adhesion, invasion and proliferation at the epithelial interface.

Optimisation of growth conditions for ovine airway epithelial cell differentiation at an air-liquid interface.

Respiratory tract infections are of significant concern in the agriculture industry. There is a requirement for the development of well-characterised in vitro epithelial cell culture models in order to dissect the diverse molecular interactions occurring at the host-pathogen interface in airway epithelia. We have analysed key factors that influence growth and differentiation of ovine tracheal epithelial cells in an air-liquid interface (ALI) culture system. Cellular differentiation was assessed at 21 days post-ALI, a time-point which we have previously shown to be sufficient for differentiation in standard growth conditions. We identified a dose-dependent response to epidermal growth factor (EGF) in terms of both epithelial thickening and ciliation levels. Maximal ciliation levels were observed with 25 ng ml-1 EGF. We identified a strict requirement for retinoic acid (RA) in epithelial differentiation as RA exclusion resulted in the formation of a stratified squamous epithelium, devoid of cilia. The pore-density of the growth substrate also had an influence on differentiation as high pore-density inserts yielded higher levels of ciliation and more uniform cell layers than low pore-density inserts. Differentiation was also improved by culturing the cells in an atmosphere of sub-ambient oxygen concentration. We compared two submerged growth media and observed differences in the rate of proliferation/expansion, barrier formation and also in terminal differentiation. Taken together, these results indicate important differences between the response of ovine tracheal epithelial cells and other previously described airway epithelial models, to a variety of environmental conditions. These data also indicate that the phenotype of ovine tracheal epithelial cells can be tailored in vitro by precise modulation of growth conditions, thereby yielding a customisable, potential infection model.

Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface.

The respiratory tract and lungs are subject to diverse pathologies with wide-ranging implications for both human and animal welfare. The development and detailed characterization of cell culture models for studying such forms of disease is of critical importance. In recent years the use of air-liquid interface (ALI)-cultured airway epithelial cells has increased markedly, as this method of culture results in the formation of a highly representative, organotypic in vitro model system. In this study we have expanded on previous knowledge of differentiated ovine tracheal epithelial cells by analysing the progression of differentiation over an extensive time course at an ALI. We observed a pseudo-stratified epithelium with ciliation and a concurrent increase in cell layer thickness from 9 days post-ALI with ciliation approaching a maximum level at day 24. A similar pattern was observed with respect to mucus production with intensely stained PAS-positive cells appearing at day 12. Ultrastructural analysis by SEM confirmed the presence of both ciliated cells and mucus globules on the epithelial surface within this time-frame. Trans-epithelial electrical resistance (TEER) peaked at 1049 Ω × cm2 as the cell layer became confluent, followed by a subsequent reduction as differentiation proceeded and stabilization at ~200 Ω × cm2. Importantly, little deterioration or de-differentiation was observed over the 45 day time-course indicating that the model is suitable for long-term experiments.

Growth of differentiated ovine tracheal epithelial cells in vitro.

Culture of ovine tracheal epithelial cells is a useful tool for conducting various in vitro studies. We describe herein an in vitro technique and the conditions for culturing primary epithelial cells derived from tracheas of adult sheep. Ovine tracheas were surgically removed from 2- to 3-month-old healthy sheep and tracheal epithelial cells were isolated by 0.15% pronase digestion. After epithelial cells isolation, a Millicell insert with porous membrane was coated with 0.05% human placental collagen and the epithelial cells were added to the membrane. To create an air-liquid interface environment for the cells, the apical compartment of the membrane containing the tracheal epithelial cells was left exposed to 5% CO(2) at 37 degrees C for 2 days then increased to 9% CO(2) while cells in the basolateral compartment underneath the membrane contained the growth medium necessary for cells nourishment. Pepsin digestion was more effective in reducing the number of fibroblasts than other procedures. Cells were allowed to grow for 6-7 days to form a confluent monolayer and nearly 21 days for cilia formation on the apical surface as determined by light microscopy of haematoxylin and eosin-stained sections of membranes. In order to further confirm the epithelial origin of cells, cells were stained for cytokeratin antigen by immunohistochemistry. Most ciliated epithelial cells were immunoreactive for cytokeratin. This is the first report of differentiated ovine tracheal epithelial cells growth and isolation. This technique can be used in numerous in vitro investigative studies in ovine species as an animal model for human disease.

Effects of albuterol enantiomers on ciliary beat frequency in ovine tracheal epithelial cells.

beta(2)-Adrenergic agonists stimulate ciliary beat frequency (CBF), an integral part of mucociliary clearance. To evaluate the differential effects of albuterol enantiomers and their racemic mixture on ciliary function, CBF and intracellular calcium were measured at room temperature from single ovine airway epithelial cells with use of digital videomicroscopy. Baseline CBF was 7.2 +/- 0.2 (SE) Hz (n = 80 measurements). R-albuterol (10 microM to 1 mM) stimulated CBF in a dose-dependent manner to maximally 24.4 +/- 5.4% above baseline. Racemic albuterol stimulated CBF to maximally 12.8 +/- 3.6% above baseline, a significantly lower increase compared with R-albuterol alone, despite identical R-enantiomer amounts in both groups. Simultaneous recordings of intracellular calcium concentration and CBF from single cells indicated that the CBF increase in response to R-albuterol was mediated through beta-receptors and stimulation of protein kinase A, in a calcium-dependent and -independent fashion. S-albuterol had a negligible effect on CBF and did not change intracellular calcium. Together, these results suggest that R-albuterol is more efficacious than racemic albuterol in stimulating CBF. Thus S-albuterol may interfere with the ability of R-albuterol to increase CBF.

Agonist-stimulated calcium decreases in ovine ciliated airway epithelial cells: role of mitochondria.

1. In ovine ciliated tracheal epithelial cells, acetylcholine (ACh) activates signal transduction pathways that not only transiently increase cytoplasmic Ca2+ ([Ca2+]i) but also actively lower [Ca2+]i. The pathway for decreasing [Ca2+]i is clearly revealed after depletion of intracellular Ca2+ stores by thapsigargin (Tg), 2,5-di-(tert-butyl)-1,4-benzohydroquinone or NiCl2. Measurements with microinjected fura-2 excluded a [Ca2+] measurement artefact. 2. A four-compartment model to simulate calcium transients in non-excitable cells (consisting of a plasma membrane Ca2+ pump and channel; Ca2+ store with pump and channel; and cytosolic Ca2+ buffer) could not account for the observed [Ca2+]i decrease. We therefore explored, by simulation and experimentation, several different mechanisms that could account for it. 3. The ACh-stimulated [Ca2+]i decrease was not due to an inhibition of Ca2+ influx (Ca2+ channel blockers or absence of extracellular calcium had no effect), activation of a plasma membrane Ca2+-ATPase (two inhibitors, vanadate (30 mM) and lanthanum (10 mM), had no effect) or inhibition of the Na+-Ca2+ exchanger (replacing extracellular Na+ with N-methylglucamine had no effect). 4. The application of mitochondrial uncouplers (5 microM CCCP or 5 microM FCCP), eliminated the ACh-induced [Ca2+]i decrease. Addition of CCCP at the nadir of the decrease restored intracellular calcium levels of Tg-treated cells to baseline faster than controls not exposed to mitochondrial uncouplers. CCCP application to naïve cells did not block the ACh-induced transient increase in [Ca2+]i. 5. These data suggest that ACh-induced [Ca2+]i decreases in ciliated cells are caused by stimulated Ca2+ uptake into mitochondria.

Lack of nitric oxide involvement in cholinergic modulation of ovine ciliary beat frequency.

Ciliary beat frequency (CBF) is regulated, at least in part, by the cytoplasmic calcium concentration ([Ca(2+)](i)). Because Ca(2+) can stimulate nitric oxide (NO) production by nitric oxide synthase (NOS) and NO has been implicated in the regulation of CBF in some species, we examined whether NOS is present in cultured ovine ciliated epithelial cells and whether NO plays a role in the Ca(2+)-mediated muscarinic stimulation of CBF. Dissociated ovine tracheal epithelial cells were grown in culture for 2 to 14 days. Frequency from a single cilium was measured by on-line Fourier transform methods using video microscopy. [Ca(2+)](i) was determined with fura-2 using fluorescence ratio imaging from the same single cells. Ciliated cells contained NOS in culture as indicated by NADPH-diaphorase staining. Acetylcholine (ACh) increased CBF and [Ca(2+)](i) transiently as previously shown. Measurements with 2',7'-dichlorofluorescin diacetate indicated that reactive oxygen/nitrogen species were produced in these cells on ACh exposure. NOS inhibitors N(G)-nitro-L-arginine methyl ester (< or =10 mM), N(G)-nitro-L-arginine (< or =10 mM), and 7-nitro indazole (1 microM) were unable to block the CBF or [Ca(2+)](i) response to ACh. Furthermore, the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine (< or =1 mM) did not change CBF or [Ca(2+)](i). Above these concentrations, they both lead to a reversible decrease in CBF. The membrane-permeable cyclic guanosine monophosphate analogue 8-bromo-cyclic guanosine monophosphate had no effect on CBF, whereas 8-bromo-cyclic adenosine monophosphate stimulated CBF. Taken together, these results suggest that NO does not play a role in mediating the ACh-induced increase in CBF through [Ca(2+)](i). The role and targets for NO in ovine ciliated cells remains to be determined.

Mode of Ca2+ action on ciliary beat frequency in single ovine airway epithelial cells.

1. We analysed the kinetics of coupling between cytoplasmic calcium ([Ca2+]i) and ciliary beat frequency (CBF) using simultaneous single cilium recording and single cell [Ca2+]i measurements from cultured ovine tracheal epithelial cells. 2. CBF and [Ca2+]i (indicated by fura-2) were measured at rest and in response to activation of the G-protein coupled M3 muscarinic receptor by 10 microM acetylcholine (ACh). 3. Fourier transform analysis of 3 s data segments of light intensity from phase-contrast microscopy showed no significant delay between changes in [Ca2+]i and CBF during a 2 min exposure to ACh and subsequent washout. 4. CBF time resolution was improved by computing instantaneous beat frequency. This revealed that CBF lagged the rapid increase in [Ca2+]i in response to ACh with a delay of less than 1 beat cycle (143 ms at 7 Hz). When CBF was estimated by an improved Fourier method, this delay was observed to be 70 +/- 30 ms (mean +/- s.e.m.; n = 20 cilia). During the slower return to baseline, a lag of 8 +/- 3.2 s was observed, indicative of hysteresis. 5. While calmodulin inhibitors (calmidazolium and W-7; each n = 5) decreased baseline CBF by an average of 1.1 +/- 0.1 Hz, they did not alter the kinetic relationship between [Ca2+]i and CBF. Similarly, phosphatase inhibitors (okadaic acid and cyclosporin A; each n = 5), changed neither baseline CBF nor the kinetic coupling between [Ca2+]i and CBF. 6. These data suggest that the timing of Ca2+ action on CBF in ovine airway epithelial cells, is unlikely to be determined by phosphorylation reactions involving calmodulin or kinase/phosphatase reactions. 7. A simple model for Ca2+ stimulation of CBF is presented. Fits of the model to the data suggest four or more Ca2+ ions bind cooperatively to speed up CBF.

Regulation of guanosine 3':5'-cyclic monophosphate in ovine tracheal epithelial cells.

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue. 2. We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)). 3. Both 10(-7)-10(-3) M and 10(-7)-10(-3) M SNAP generated a concentration-dependent marked elevation in cyclic GMP production when incubated with 10(-3) M 3-isobutyl-l -methylxanthine (IBMX) (both greater than 25 x baseline values with highest drug concentration). 4. The increase in production of cyclic GMP in response to 10(-6) M SNP and 10(-5) M SNAP was markedly inhibited by both 5 x 10(-5) M haemoglobin (102% and 92% inhibition) and 5 x 10(-5) M methylene blue (82% and 84% inhibition). 5. The increase in cyclic GMP in response to 10(-3) M SNP was measured following co-incubation with the phosphodiesterase inhibitors 10(-7)-10(-3) M IBMX, 10(-7)-10(-4) M milrinone and 10(-7)-10(-4) M SKF 96231. Only 10(-4)-10(-3) M IBMX significantly increased cyclic GMP levels. 6. Cyclic GMP production was also significantly elevated from baseline by 10(-5) M ANP, 10(-5) M BNP, 10(-5) M CNP and 200 iu ml-3 of E. coli STa toxin in the presence of 10(-3) M IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (2-4 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed. 7. These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.

Muscarinic signaling in ciliated tracheal epithelial cells: dual effects on Ca2+ and ciliary beating.

To examine cholinergic signal transduction pathways that modulate ciliary beat frequency (CBF), cultured ovine tracheal epithelial cells were imaged using a combination of phase-contrast (CBF) and fluorescence (Ca2+) microscopy techniques. In single cells, acetylcholine (ACh) transiently increased CBF and intracellular Ca2+ concentration ([Ca2+]i), mainly by Ca2+ release from internal stores, with a small delayed contribution from Ca2+ influx. Nicotinic agonists did not alter CBF or [Ca2+]i, whereas atropine blocked the ACh-stimulated transients, consistent with the involvement of muscarinic receptors. 4-Diphenylacetoxy-N-methylpiperidine methiodide was approximately 100 times more potent than pirenzepine in inhibiting the ACh-induced [Ca2+]i peaks, suggesting that the receptor is a pharmacologically defined (M3) subtype. Interestingly, after depletion of intracellular Ca2+ stores by thapsigargin, ACh caused a rapid transient decrease in both CBF and [Ca2+]i, again with an antagonist profile of M3 receptors. We conclude that activation of M3 muscarinic receptors initiates specific signaling pathways that act simultaneously to increase and decrease [Ca2+]i and CBF.

Molecular Cloning, Functional Expression, and Selective Regulation of Ovine Prostaglandin H Synthase-2

Structural characterization for ovine prostaglandin H synthase-1 (PGHS-1) is extensive, but the corresponding structure for the homologous ovine PGHS-2 isoform is undefined. Accordingly, we isolated a full-length (3.4 kb) ovine PGHS-2 cDNA from a primary-culture cell model (ovine tracheal epithelial cells) originally described as containing both PGHS isoforms. Analysis of ovine PGHS-2 cDNA sequence indicated conservation of critical amino acid residues, but differences in other hydrophilic regions allowed for the development of an anti-peptide antibody highly selective for PGHS-2. Enzymatic activities of the recombinant ovine PGHS isozymes indicated significant differences in response to aspirin-acetylation consistent with the characteristics of endogenous cellular PGHS activities under basal and serum-induced conditions. The results fully account for previous evidence of two distinct PGHS activities in cultured airway epithelial cells and provide for additional definition of PGHS structure–function relationships.

Coupling of [Ca2+]i and ciliary beating in cultured tracheal epithelial cells.

The molecular mechanisms responsible for the regulation of ciliary beating frequency (CBF) are only partially characterized. To determine whether elevation of intracellular Ca2+ ([Ca2+]i) can cause an increase in CBF, we measured CBF and Ca2+ in single cells. Ovine tracheal epithelial cells, obtained by dissociation with protease, were grown in primary culture for 1 to 28 days in a mucus-free system. CBF of a single cilium was measured by digital video phase-contrast microscopy and on-line Fourier-transform analysis. Changes in [Ca2+]i from single cells were determined with fura-2, using ratio imaging video microscopy. Activation of a muscarinic pathway with 10 microM ACh (acetylcholine) increased [Ca2+]i from 53 +/- 9 nM (mean +/- s.e.m.) to 146 +/- 12 nM or to 264 +/- 51% above initial baseline. In the same cells, ACh increased CBF from a baseline of 7 +/- 0.5 Hz to 9 +/- 0.2 Hz or to 31 +/- 2.8% above baseline (n = 14). The elevations of both [Ca2+]i and CBF were transient and relaxed back to an elevated plateau (10/14 cells) as long as ACh was present. To elevate [Ca2+]i by mechanisms independent of a G-protein-coupled receptor, we measured [Ca2+]i and CBF of the same cells in extracellular solutions with either 0 Ca2+ (+ 1 mM EGTA) or 10 mM Ca2+. Both signals rose and fell with similar kinetics in response to changing [Ca2+]0, suggesting that changes in [Ca2+]i alone can modulate CBF. In a second independent manipulation, cells were treated with 1 microM thapsigargin, an irreversible inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. Upon thapsigargin addition, 37 of 42 cells showed a transient [Ca2+]i increase and, as measured in different experiments, 8 of 9 cells showed a transient increase in CBF. Interestingly, application of ACh after cells were treated with thapsigargin produced decreases in both [Ca2+]i and CBF in 8/8 cells. Lastly, after 1-3 days in culture, addition of 10 microM ACh often produced [Ca2+]i oscillations rather than transients in [Ca2+]i. Measurements of CBF in these cells showed frequency modulation of CBF with the same peak-to-peak time interval as the Ca2+ oscillation. These results show that: (1) CBF can be measured from a single cilium and monitored on-line to track changes; (2) CBF and [Ca2+]i can be measured in the same single cell; (3) transient changes in [Ca2+]i (induced by 4 different manipulations) are associated with kinetically similar changes in CBF; and (4) [Ca2+]i oscillations are coupled to frequency modulation of ciliary beating.(ABSTRACT TRUNCATED AT 400 WORDS)

A cryptic, microsomal-type arachidonate 12-lipoxygenase is tonically inactivated by oxidation-reduction conditions in cultured epithelial cells.

Cultured ovine tracheal epithelial cells converted arachidonic acid to prostaglandin E2 (PGE2), but microsome-containing subcellular fractions prepared from these cells under calcium-free conditions converted arachidonic acid to PGE2 and to 12-hydroxyeicosatetraenoic acid (12-HETE) at a high rate (2-4 nmol/mg of protein/15 min). Identification of the membrane-bound 12-HETE-forming activity as a 12-lipoxygenase included 12S-stereospecificity of product formation and trapping of 12-hydroperoxyeicosatetraenoic acid as a reaction product. The 12-lipoxygenase activity was extracted from cell membranes only with detergent (1% Triton X-100), and the activity (membrane-bound or detergent-solubilized) was completely inactivated by mixing with the cytosol-containing subcellular fraction. The inhibitory effect of the cytosolic fraction was reversed by treating the cytosol with GSH-depleting agents (2-cyclohexene-1-one or N-ethylmaleimide) or by mixing it with lipid hydroperoxide (13-hydroperoxyoctadecadienoic acid) at a concentration that had little direct effect on enzyme activity. Inhibition of 12-lipoxygenase activity could also be achieved by treatment of enzyme preparations with GSH at levels (0.1-10 mM) found in epithelial cell cytosol. In addition, treatment of cultured epithelial cells with a GSH-depleting agent (buthionine sulfoximine) and lipid hydroperoxide restored cellular 12-lipoxygenase activity. Little or no detectable 12-lipoxygenase activity was found in freshly isolated ovine tracheal epithelial cells, but the cytosolic 12-lipoxygenase found in freshly isolated bovine tracheal epithelial cells was relatively insensitive to regulation by GSH or lipid hydroperoxide. These observations indicate that a 12-lipoxygenase is expressed in a cryptic, microsomal-type form in primary-culture epithelial cells and that this form of the enzyme may be selectively regulated by changes in cellular oxidation-reduction conditions dependent on cytosolic levels of GSH versus lipid hydroperoxide.

Identification of a pharmacologically distinct prostaglandin H synthase in cultured epithelial cells.

Nonsteroidal anti-inflammatory drugs inhibit the action of prostaglandin H synthase (PGH synthase), and this effect may constitute the basis for therapeutic and idiosyncratic responses to these agents. We found that aspirin treatment of cultured ovine tracheal epithelial cells blocked PGH synthase-catalyzed formation of PG as expected but also caused a dose-dependent increase in 15-hydroxyeicosatetraenoic acid (15-HETE) production from arachidonic acid. In contrast, aspirin caused only inhibition of PG production without enhancing 15-HETE formation in ovine seminal vesicle and other tissues. The 15-HETE formed by aspirin-treated ovine tracheal epithelial cells was generated by a PGH synthase-dependent mechanism because: (i) the 15-HETE forming activity was just as sensitive as PG forming activity to selective inhibition by indomethacin; (ii) both 15-HETE and PG forming activities were quantitatively immunoprecipitated (depleted from supernatants and recovered in immune complex pellets) by a specific anti-PGH synthase antiserum. Additional immunoprecipitation experiments indicated that anti-PGH synthase monoclonal antibodies (cyo-1 and cyo-5) raised against the aspirin-inhibited form of the enzyme (contained in seminal vesicle) did not recognize the aspirin-stimulated 15-HETE-forming PGH synthase (contained in cultured epithelial cells). Thus, sequential immunoprecipitation of cultured epithelial cell material first with excess cyo-1 followed by anti-PGH synthase antiserum indicated that two isoforms of PGH synthase were expressed in these cells. SDS-polyacrylamide gel electrophoresis of immunoprecipitated PGH synthase from cultured epithelial cells revealed distinct protein bands for each form of the enzyme (M(r) = 70,000 and 72,000). The identification of a distinct PGH synthase which may be modified by aspirin so that selective oxygenation of fatty acid substrate is enhanced (while PG formation is inhibited) indicates that isozymes of PGH synthase exist which are pharmacologically distinct.