1. In this study, an attempt was made to distinguish between local and systemic effects of low doses of the topical glucocorticoid, budesonide. The effect of aerosolized budesonide administered to the lower airways versus intravenously administered budesonide on the acute and late response to nebulized Ascaris suum extract in the lung, was evaluated in the minipig after active sensitization with purified A. suum antigen. Budesonide was administered once, 1 h prior to A. suum challenge and airway reactions and mediator release were observed for 8 h after allergen challenge. 2. In the budesonide aerosol group (n = 6), 10.2 +/- 1.2 micrograms kg-1 budesonide was given locally and in the budesonide infusion group (n = 5), 5 micrograms kg-1 was given intravenously. The area under the plasma concentration curve for budesonide during the experiment was 11.4 +/- 1.2 and 10.3 +/- 1.2 nM h in the budesonide aerosol and budesonide infusion group, respectively (no significant difference). The lung tissue content of budesonide in the two groups was 45.2 +/- 4.9 and 18.4 +/- 3.5 nmol kg-1 dry tissue, respectively, 8 h after allergen challenge (P < 0.05). For comparison, 6 pigs were given budesonide vehicle as an infusion prior to A. suum challenge. 3. Total lung resistance (RL) increased acutely (maximal response within 15 min) in the budesonide aerosol, budesonide infusion and budesonide vehicle groups (by 91 +/- 40, 150 +/- 86 and 80 +/- 27%, respectively). The acute reaction partially resolved at about 1 h and was followed by a late increase in RL in the budesonide infusion and budesonide vehicle groups (by 251 +/- 148 and 281 +/- 136% at 8 h, respectively). However, no late change in RL was seen in the budesonide aerosol group (7 +/- 24%). 4. Aerosolized budesonide had a protective effect in that it attenuated the late changes in arterial blood gas and pH as well as the late elevation of plasma catecholamines. Budesonide given as an infusion did not protect against the late changes in these parameters. However, budesonide aerosol or infusion did not inhibit the late vasodilation in the bronchial circulation. 5. Histamine and cysteinyl-leukotrienes were released during the acute reaction as measured by urinary concentration of methylhistamine and leukotriene E4 respectively. There was no release of histamine during the late reaction. A late increase in leukotriene E4 was observed in 2 of the budesonide infusion and 3 of the budesonide vehicle pigs, whereas no such increase was seen in any of the budesonide aerosol pigs. 6. Budesonide concentration in lung tissue, but not in plasma at 8 h correlated negatively with the late increase in RL (P < 0.05, r = -0.53, n = 10), whereas budesonide concentration in plasma but not in lung tissue correlated negatively with the late decrease in dynamic compliance (P < 0.05, r = -0.67, n = 12). 7. This study has shown that a single low dose of locally administered budesonide can inhibit the late allergic reaction in the pig lower airways. If budesonide was given as an intravenous infusion in a dose yielding a plasma concentration similar to that seen after the aerosol treatment, the protective effect of budesonide was poor. It may be suggested that the tissue-bound portion of budesonide affects local mechanisms involved in the development of late changes in the airways (RL), although it does not affect the late increase in bronchial blood flow. We conclude that the inhibitory effect of budesonide on the allergen-induced late reaction in the pig airways relates to tissue-bound steroid, and that the systemic component is of less importance.
1. Pretreatment of guinea-pigs with endotoxin (1 mg kg-1 b.w., i.p., 4 days before the experiments) results in respiratory airway hyperreactivity in vitro. Dose-response curves with either arecoline or histamine on isolated tracheae from these animals display increased maximal contractions, and decreased EC50 values. 2. Tracheae denuded of epithelium respond with a similar hyperreactivity to histamine as observed in preparations from endotoxin pretreated animals. Removal of the epithelial layer of tracheae from endotoxin pretreated guinea-pigs did not additionally affect the histamine dose-response curve. 3. The cyclo-oxygenase inhibitor indomethacin (10 microM) induces histamine hyperreactivity which is equal in intact and epithelium-denuded tracheae from saline or endotoxin pretreated guinea-pigs. Similar results are obtained with the combined lipoxygenase/cyclo-oxygenase inhibitor nordihydroguaiaretic acid (10 microM). 4. Histamine (0.1 mM) induces an increase in prostaglandin E2 (PGE2) formation by the tracheal spiral in vitro, which is reduced by 34% by endotoxin pretreatment, and by about 60% following epithelium removal irrespective of endotoxin pretreatment. 5. Arachidonic acid (AA, 22 microM) stimulation of the guinea-pig trachea in vitro induces a relaxation, and an increase in PGE2 production. In preparations lacking the epithelium, AA induces a contraction which coincides with a 60% reduced increase in PGE2 formation. These effects are not altered by endotoxin pretreatment. 6. It is concluded that the endotoxin-induced respiratory airway hyperreactivity may be caused by a disturbed ability of epithelial cells to synthesize PGE2. The decreased formation of this prostaglandin is rather the consequence of a diminished liberation of AA from the phospholipid stores than a dysfunction of the cyclo-oxygenase enzyme.
Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID) and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa.
airway cell differentiation; airway cell proliferation; human airway epithelium; nude mouse; regeneration; SCID mouse; stem cell; xenograft
The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease.
nucleotide-binding and oligomerization domain-like receptors (NLRs); intestinal epithelial cells (IECs); wound healing; colitis; inflammatory bowel diseases (IBD); inflammasome; growth factors; innate immunity; toll-like receptors (TLRs)
1. Removal of the epithelium resulted in a threefold increase in guinea-pig tracheal sensitivity to histamine without increasing the maximal response. 2. Preincubation of epithelially-denuded guinea-pig tracheal smooth muscle with leukotriene E4 (LTE4) in vitro increased the subsequent maximal response of the tissues to histamine. The sensitivity of the tissues to histamine was unaffected by LTE4 pretreatment. 3. Pretreatment of the epithelially-denuded tissues with the LTE4-analogue, 20-COOH LTE4, did not affect the maximal response to histamine. 4. LTE4 pretreatment increased the maximal response of the epithelially-denuded tissues to substance P (SP) but did not affect the maximal response to carbachol, KCl nor to the beta-adrenoceptor agonist, isoprenaline. 5. LTE4-induced airway histamine hyperresponsiveness was blocked by indomethacin (5 microM), GR32191 (3 microM) and atropine (1 microM). 6. Both LTE4 and U46619 pretreatment increased the contractile response of tracheal smooth muscle to electrical field stimulation. 7. It is proposed that LTE4 induces an increased maximal response of epithelially-denuded guinea-pig airway smooth muscle to both histamine and substance P via a facilitation of cholinergic neurotransmission, which is dependent upon the secondary generation of prostanoid mediator(s) acting on TP-receptors situated on cholinergic nerve terminals. Further, it is suggested that the increased maximal response of the epithelially-intact tissues to both histamine and substance P, after LTE4 pretreatment, may be suppressed by an epithelially-derived factor.
1. Endothelin (1 nM-0.3 microM) produced a concentration-dependent contraction of guinea-pig epithelium-containing (intact) trachea (EC50 = 30.9 nM). Endothelin was a less potent agonist than leukotriene D4 (LTD4; EC50 = 0.77 nM), but was more potent than carbachol (EC50 = 0.15 microM) or substance P (EC50 = 1.4 microM). Endothelin was a more potent contractile agent in rat endothelium-denuded aorta (EC50 = 2.1 nM) than in guinea-pig trachea. 2. Endothelin-induced contraction in guinea-pig trachea was unaffected by mepyramine (10 microM), atropine (1 microM), SK&F 104353 (10 microM), a leukotriene receptor antagonist, or SQ 29,548 (1 microM), a thromboxane receptor antagonist. The contraction produced by 0.3 microM endothelin was potentiated by cyclo-oxygenase inhibition with 5 microM indomethacin. 3. Nicardipine (0.01 or 0.1 microM) or incubation in calcium-free medium +0.1 mM EGTA for 30 min had a relatively minor or no effect on endothelin concentration-response curves in guinea-pig intact trachea, but markedly inhibited responses produced by endothelin in endothelium-denuded aorta of the rat. Increasing the EGTA concentration in calcium-free medium to 1 mM abolished endothelin-induced contraction in guinea-pig trachea. 4. In guinea-pig trachea, ryanodine (10 microM) produced a 2.1 fold shift to the right of endothelin concentration-response curves and reduced the maximum response elicited by 0.3 microM endothelin. 5. Staurosporine (0.01 microM and 0.1 microM), a protein kinase C inhibitor, was without effect on endothelin- or carbachol-induced contraction in guinea-pig trachea, but markedly inhibited the response produced by endothelin in rat aorta. 6. Endothelin (3 nM-0.3 microM) produced a concentration-dependent stimulation of phosphatidylinositol (PI) turnover in guinea-pig intact trachea, with an EC50 value of 45.9 nM. 7. Removal of the epithlium markedly potentiated endothelin-induced contraction in guinea-pig trachea, producing a 4.7 fold leftward shift in endothelin concentration-response curves and an increase in the contractile response elicited by 0.3 microM endothelin. 8. These data indicate that endothelin is a potent agonist in guinea-pig trachea whose response is markedly enhanced by removal of the airway epithelium. Endothelin-induced contraction is not mediated to a marked extent by calcium influx via dihydropyridine-sensitive calcium channels and does not involve the release of histamine, acetylcholine, leukotrienes or thromboxane. Rather, endothelin appears to produce contraction of guinea-pig trachea via a direct action which involves stimulation of PI turnover and utilization of calcium from intracellular stores and, also, calcium influx via a pathway that is not sensitive to dihydropyridine calcium channel inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)
1. We have studied the effect of epithelium removal on relaxation of guinea-pig isolated tracheal smooth muscle induced by vasoactive intestinal peptide (VIP) or stimulation of non-adrenergic, non-cholinergic (NANC) inhibitory nerves. Also examined were the effects of inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE). 2. Epithelium removal produced a 3.6 +/- 0.4 fold leftward shift in the VIP concentration-response curve. The supersensitivity to VIP, following epithelium removal was abolished by phosphoramidon or thiorphan (NEP inhibitors), but unaffected by captopril (an ACE inhibitor). In intact trachea, the NEP inhibitors produced leftward shifts in the VIP curves similar to those produced by epithelium removal. 3. In contrast to responses to exogenous VIP, neurogenic NANC inhibitory responses to electrical field stimulation were affected neither by epithelial denudation nor by the peptidase inhibitors. 4. As in previous studies, epithelium removal increased tracheal sensitivity to isoprenaline. This was not altered by pretreatment with a cocktail of peptidase inhibitors. Thus, the effect of the NEP inhibitors on responses to VIP appears to be relatively specific. 5. These data indicate that exogenous VIP is a substrate for airway NEP, since inhibition of the enzyme potentiates the peptide. This is further evidence that the airway epithelium provides a source for the metabolism of mediators. 6. In guinea-pig trachea the NEP responsible for cleaving VIP may be located largely in the epithelial layer, since NEP inhibition was without effect on sensitivity to VIP in epithelium-denuded preparations. If VIP is a NANC inhibitory neurotransmitter in this tissue its degradation endogenously does not appear to involve epithelial NEP.
BACKGROUND AND PURPOSE
TNF-α is an inflammatory cytokine implicated in the pathogenesis of asthma and it causes airway inflammation, bronchoconstriction and airway hyperresponsiveness to a number of spasmogens following inhalation.
We compared contractions of guinea pig isolated trachea incubated with saline or TNF-α for 1, 2 or 4 days to electrical field stimulation (EFS), 5-HT or methacholine. In addition, we compared bronchoconstriction in anaesthetized guinea pigs 6 h after intratracheal instillation of saline or TNF-α to vagal nerve stimulation, i.v. 5-HT or methacholine. Differential counts were performed on the bronchoalvelolar lavage fluid (BALF).
Maximum contractions to methacholine, 5-HT and EFS were not different between freshly prepared and saline-incubated tissues. Exposure to TNF-α concentration-dependently potentiated contractions to 5-HT and EFS, but not methacholine. All contractions were atropine-sensitive, but not hexamethonium-sensitive. 5-HT-evoked contractions were inhibited by ketanserin or epithelial denudation. Only EFS-evoked contractions were tetrodotoxin-sensitive. Vagal stimulation, i.v. 5-HT or MCh caused a significant atropine-sensitive, frequency- and dose-dependent bronchoconstriction and decreased blood pressure similarly in both saline and TNF-α pre-treated animals. TNF-α potentiated the bronchoconstriction to vagal stimulation and 5-HT, but not MCh. The BALF from saline-treated animals contained predominantly macrophages, whereas that from TNF-α–treated animals contained neutrophils.
CONCLUSIONS AND IMPLICATIONS
TNF-α caused airway hyperresponsiveness to nerve stimulation in vivo and increased contractility in vitro. However, responsiveness to MCh was unchanged, suggesting a pre-synaptic action of TNF-α on parasympathetic nerves. TNF-α-induced airway hyperresponsiveness to 5-HT suggested an increased 5-HT2A receptor-mediated acetylcholine release from epithelial cells.
TNF-α; airway hyperresponsiveness; guinea pig; trachea; bronchoconstriction; inflammation; contraction; asthma; 5HT; acetylcholine; cholinergic
The efficacy of topical glucocorticosteroids in rhinitis and asthma is likely to depend on drug retention in the airway mucosa. With fluticasone propionate, retention may be achieved exclusively by lipophilicity, whereas for budesonide an additional possibility may be provided by its ability to form fatty acid esters in the airway mucosa that release the active drug. The aim of the present study was to determine the nasal mucosal retention of budesonide and fluticasone propionate, and the occurrence of budesonide-esters (budesonide-oleate, budesonide-palmitate) in the nasal mucosa.
In the present study, involving 24 healthy subjects, we have examined nasal mucosal drug retention of single doses of topical budesonide (256 µg) and fluticasone propionate (200 µg). Treatments were given consecutively and the administration sequence was randomised. Subjects were randomised into four parallel groups and two nasal biopsies were taken from each subject, i.e. before and at 2 h, at 2 and 6 h, at 6 and 24 h, or before and at 24 h after drug administration, resulting in 12 biopsies/time point. The measurement of unesterified budesonide, budesonide-oleate, budesonide-palmitate, and fluticasone propionate was based on microwave extraction procedures combined with liquid-chromatography/tandem mass-spectrometry.
Neither of the analytes was detected in samples taken before glucocorticosteroid administration. After administration, unesterified budesonide, budesonide-esters, and fluticasone propionate were detected in the tissue from 23, 20, and 19 subjects, respectively. The mean tissue levels of budesonide at 2 and 6 h were 1051 and 176 pmol g−1; the mean levels of fluticasone propionate at these time points were 237 and 10 pmol g−1. The dose-corrected budesonide/fluticasone propionate tissue concentration ratios were 3.5 (P = 0.07) and 13.7 (P < 0.0002), respectively. At 24 h, budesonide and fluticasone propionate were detected in 8/12 and 3/12 of the biopsies, respectively.
The present study demonstrates the formation of budesonide-esters in the human nasal mucosa in vivo, and that budesonide is retained in the nasal mucosa to a greater extent than fluticasone propionate. It is suggested that the formation of budesonide-esters and their subsequent release of budesonide contributes to an extended retention of budesonide in the airway mucosa.
airway; glucocorticosteroid; human; nasal; rhinitis; treatment
The intestine is not only the main target attacked by sepsis but also the vital organ which mediated sepsis. The recovery of the damaged intestinal barrier structure and function is related to the occurrence and outcome of multiple organ dysfunction syndrome (MODS). How to protect and reduce the damage of the intestinal mucosa and how to promote the reconstruction of the intestinal mucosa have been the important topics in sepsis for many years. This study aimed to investigate the influential factors of intestinal mucosal reconstruction after intestinal epithelial injury in vivo in a mouse model of sepsis.
Mice were subjected to cecal ligation and puncture (CLP) for induction of sepsis to assess intestinal mucosal damage, epithelial cell apoptosis, and transformed number of goblet cells, and to detect the concentration of TNF-α, IL-1 and TGF-β1 and TFF3 (trefoil factor 3) expression in the small intestinal mucosa. All above were performed by HE staining, western blot, ELISA and immunohistochemistry respectively. The experimental animals were divided into a sepsis group and a sham-operation group. The animals with sepsis were separately killed at 6 (7 animals), 24 (7 animals) and 48 hours (7 animals) after CLP.
Injured intestinal mucosa was observed in the 3 groups under a light microscope, in which damage scores in the 24-hour and 48-hour groups were higher than in the 6-hour group and no difference was found between the two groups. Moreover, less of goblet cells or other epithelial cells adjacent to the injured surface migrated into the wound to cover the denuded area. The number of goblet cells was substantially decreased in the three CLP groups compared with the sham-operation group. Protein levels of IL-1 and TNF-α were significantly increased by 3–4 fold at all time points when compared with the sham-operation group, and cleaved caspase-3 by 4 fold. Although TFF3 expression was modestly increased for 6 hours after the onset of CLP, it appeared to decline at 24 hours and 48 hours as shown by Western blot. A similar tendency was observed upon TGF-β1, i.e. the protein level was not elevated at 24 hours and 48 hours, but increased modestly at 6 hours.
Sepsis from CLP shows less restitution on the surface of injured intestinal mucosa. There is evidence that both constant inflammatory reaction and epithelial cell apoptosis may affect mucosal reestablishment of the intestine at the onset of sepsis. Mucosa after severe sepsis showed the state of high inflammation, and declined goblet cell function and mucosal reconstruction, which affected the repair of damaged intestinal barrier. Constant inflammatory reaction, and declined goblet cell function and mucosal reconstruction ability may affect the reestablishment of intestinal mucosa at the onset of sepsis.
Sepsis; Cecal ligation and puncture; Intestinal mucosa; Restitution; Goblet cells; Intestinal trefoil factor 3; Transforming growth factor β1; Cysteine-containing aspartate-specific proteases
The trefoil peptides, a recently recognized family of protease-resistant peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as growth factors, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil factor (RITF and HITF) to subconfluent nontransformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or nontransformed fibroblasts (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6-fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a 15-fold enhancement in restitution. No synergistic effect was observed by the addition of nonmucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGF beta bioactivity, trefoil peptide, and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGF-beta and was not affected by the presence of immunoneutralizing anti-TGF beta antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.
Defective epithelial repair in the setting of chronic lung disease has been suggested to contribute to uncontrolled extracellular matrix (ECM) deposition and development of fibrosis. We sought to directly test this hypothesis through gene expression profiling of total lung RNA isolated from mouse models of selective epithelial cell injury that are associated with either productive or abortive repair. Analysis of gene expression in repairing lungs of naphthalene-exposed mice revealed prominent clusters of up-regulated genes with putative roles in regulation of the extracellular matrix and cellular proliferation. Further analysis of tenascin C (Tnc), a representative matrix protein, in total lung RNA revealed a transient 4.5-fold increase in mRNA abundance 1 day after injury and a return to steady-state levels by Recovery Day 3. Tnc was deposited by the peribronchiolar mesenchyme immediately after injury and was remodeled to basement membrane subtending the bronchiolar epithelium during epithelial repair. Epithelial restitution was accompanied by a decrease in Tnc mRNA and protein expression to steady-state levels. In contrast, abortive repair using a transgenic model allowing ablation of all reparative cells led to a progressive increase in Tnc mRNA within lung tissue and accumulation of its gene product within the subepithelial mesenchyme of both conducting airways and alveoli. These data demonstrate that the ECM is dynamically remodeled in response to selective epithelial cell injury and that this process is activated without resolution in the setting of defective airway epithelial repair.
airway epithelium; repair; Clara cell; extracellular matrix; fibrosis
Early rapid mucosal restitution occurs as a consequence of epithelial cell migration to reseal superficial wounds, a process independent of cell proliferation. Our previous studies revealed that the canonical transient receptor potential‐1 (TRPC1) functions as a store‐operated Ca2+ channel (SOCs) in intestinal epithelial cells (IECs) and regulates epithelial restitution after wounding, but the exact mechanism underlying TRPC1 activation remains elusive. Caveolin‐1 (Cav1) is a major component protein that is associated with caveolar lipid rafts in the plasma membrane and was recently identified as a regulator of store‐operated Ca2+ entry (SOCE). Here, we showed that Cav1 plays an important role in the regulation of mucosal restitution by activating TRPC1‐mediated Ca2+ signaling. Target deletion of Cav1 delayed gastric mucosal repair after exposure to hypertonic NaCl in mice, although it did not affect total levels of TRPC1 protein. In cultured IECs, Cav1 directly interacted with TRPC1 and formed Cav1/TRPC1 complex as measured by immunoprecipitation assays. Cav1 silencing in stable TRPC1‐transfected cells by transfection with siCav1 reduced SOCE without effect on the level of resting [Ca2+]cyt. Inhibition of Cav1 expression by siCav1 and subsequent decrease in Ca2+ influx repressed epithelial restitution, as indicated by a decrease in cell migration over the wounded area, whereas stable ectopic overexpression of Cav1 increased Cav1/TRPC1 complex, induced SOCE, and enhanced cell migration after wounding. These results indicate that Cav1 physically interacts with and activates TRPC1, thus stimulating TRPC1‐mediated Ca2+ signaling and rapid mucosal restitution after injury.
Early rapid mucosal restitution occurs as a consequence of epithelial cell migration to reseal superficial wounds, a process independent of cell proliferation. Caveolin‐1 (Cav1) is a major component protein that is associated with caveolar lipid rafts in the plasma membrane and was recently identified as a regulator of store‐operated Ca2+ entry (SOCE). Our results indicate that Cav1 physically interacts with and activates TRPC1, thus stimulating TRPC1‐mediated Ca2+ signaling and rapid mucosal restitution after injury.
Ca2+ influx; Cav−/−mice; cell migration; cyclopiazonic acid; hypertonic NaCl injury; intracellular Ca2+; TRPC1
This study was designed to investigate the possibility that tracheal epithelium generates a relaxant factor analogous to the endothelial-derived relaxant factor (EDRF) of vascular tissue. The absence of such a factor following epithelial damage in diseases such as asthma might help to explain the airway hyperreactivity characteristic of such diseases. Removal of epithelium by rubbing enhanced the sensitivity of guinea-pig isolated tracheal chains to 5-hydroxytryptamine, histamine, acetylcholine, adenosine, isoprenaline and also minimally to KCl. Responses to LaCl3 and electrical field stimulation were not affected. Low concentrations of adenosine produced contractions only in tissues denuded of epithelium. In the presence of indomethacin 1.4 microM or dithiothreitol (DTT) 1 microM, dose-response curves to histamine were moved to the left in both control and rubbed tissues, and the maximum response was increased. The difference in sensitivity between tissues with and without epithelium was not affected by indomethacin, but was slightly reduced by DTT. Phenidone (0.1 mM) also increased the maximum responses, but increased the sensitivity only of the tissues with intact epithelium, to the same level as that seen in the tissues denuded of epithelium. Superfusion cascade studies provided no evidence for the generation of a relaxant factor from tracheal epithelium. It is suggested that the supersensitivity produced by removal of the epithelium is not due to the removal of a relaxant factor, but rather to the removal of a permeability barrier, allowing a greater concentration of agonist at the level of the underlying smooth muscle.
Combination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells.
To test this hypothesis, bronchial epithelial (BE) cells were obtained from airway brushings of 8 subjects with moderate-to-severe allergic asthma and 9 with neither asthma nor respiratory allergies. Cultured BE cells were incubated for 24 hours with budesonide (1.77 μM), formoterol (0.1 μM), both, or neither, and then inoculated with RV-16 (5×106 plaque forming units [PFU]/mL). After 24 hours, viral replication (RV RNA), cytokine secretion (CXCL8, CXCL10, TNFα, IFN-β, IL-28) and mRNA expression (CXCL8, CXCL10, TNF, IFNB1, IL28A&B) were analyzed.
RV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFα under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma.
Budesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.
Budesonide; Formoterol; Human rhinovirus; Bronchial epithelial cells; Asthma
BACKGROUND: Epithelial shedding processes in airway inflammation and defence may produce damaged areas where basal cells are the main remaining epithelial cell type. The present study examines the capacity of basal cells to form an epithelial barrier structure after loss of columnar epithelial cells. METHODS: A technique was developed which allows selective removal of columnar epithelial cells from isolated airways. A drop of tissue adhesive glue was applied on the mucosal surface shortly after excision of guinea pig trachea and human bronchus. Gentle removal of the glue, together with attached columnar cells, left a single layer of cobbled, solitary basal cells. The tissue was kept in culture media. Morphological changes of the basal cells were monitored by immuno-histochemistry and scanning and transmission electron microscopy at several time points. RESULTS: After 20 minutes the basal cells had undergone extensive flattening and established contact with each other. The basement membrane thus became covered by a poorly differentiated epithelium in both guinea pig and human airways. Abundant interdigitating cytoplasmic protrusions were observed at cell borders. CONCLUSIONS: Basal cells promptly flatten out to cover the basement membrane at loss of neighbouring columnar cells. These data may explain why the epithelial barrier function may be uncompromised in desquamative airway diseases. Furthermore, they suggest the possibility that sacrificial release of columnar epithelial cells and prompt creation of a barrier structure constitute important roles of basal cells in airway defence against severe insults.
The present study was undertaken to investigate the interaction of the renin–angiotensin system (RAS), bradykinin and the sympathetic nervous system with cholinergic transmission in the rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. Tracheal preparations were subjected to electrical field stimulation (trains of 1 ms pulses, 5 Hz, 15 V) and the stimulation-induced (S-I) efflux taken as an index of transmitter acetylcholine release.In both epithelium-intact and epithelium-denuded tracheal preparations, the α2-adrenoceptor agonist UK14304 (0.1 and 1 μM) inhibited the S-I efflux, in a concentration-dependent manner. The inhibition of S-I efflux produced by UK14304 (1 μM) was antagonized by the selective α2-adrenoceptor antagonist idazoxan (0.3 μM). Idazoxan (0.3 μM) alone had no effect on the S-I efflux.Angiotensin II (0.1 and 1 μM) was without effect on the S-I efflux in either epithelium-intact or epithelium-denuded tracheal preparations. When angiotensin-converting enzyme was inhibited by perindoprilat (10 μM), angiotensin II (1 μM) was also without effect on the S-I efflux. Similarly, in the presence of idazoxan (0.3 μM), to block prejunctional α2-adrenoceptors, angiotensin II (0.1 and 1 μM) did not alter the S-I efflux. When added alone, perindoprilat (10 μM) did not alter the S-I efflux.In epithelium-denuded preparations, bradykinin (0.01–1 μM) inhibited the S-I efflux. In epithelium-intact preparations, there was also a tendency for bradykinin (0.1 and 1 μM) to inhibit the S-I efflux but this was not statistically significant. However, when angiotensin-converting enzyme and neutral endopeptidase were inhibited by perindoprilat (10 μM) and phosphoramidon (1 μM), respectively, bradykinin (1 μM) significantly inhibited the S-I efflux in epithelium-intact preparations as well as in epithelium-denuded preparations. The inhibition of the S-I efflux produced by bradykinin, in the combined presence of perindoprilat (10 μM) and phosphoramidon (1 μM), was unaffected by the additional presence of the cyclo-oxygenase inhibitor indomethacin (10 μM) and/or the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 μM), in either epithelium-intact or epithelium-denuded preparations.In conclusion, the findings of the present study suggest that airway parasympathetic nerves are endowed with α2-adrenoceptors which subserve inhibition of transmitter acetylcholine release. Under the present conditions, however, transmitter acetylcholine release is not subject to transneuronal modulation by noradrenaline released from adjacent sympathetic nerves in the airways. Moreover, angiotensin II and perindoprilat do not appear to modulate acetylcholine release from parasympathetic nerves of the airways. In contrast, bradykinin inhibits acetylcholine release from airway parasympathetic nerves but this action of bradykinin is limited by the activity of epithelial angiotensin-converting enzyme and/or neutral endopeptidase. The inhibitory action of bradykinin on cholinergic transmission in the airways does not appear to involve the liberation of prostaglandins or nitric oxide.
Renin-angiotensin system; angiotensin II; angiotensin-converting enzyme; bradykinin; neutral endopeptidase; prejunctional α2-adrenoceptors; sympathetic nerves; cholinergic transmission; rat airways
Small bowel epithelium is at the frontline of intestinal barrier function. Restitution is considered to be the major determinant of epithelial repair as function recovers in parallel with restitution after acute injury. As such, studies of intact mucosa have largely been replaced by migration assays of cultured epithelia. These latter studies fail to account for the simultaneous roles played by villous contraction and paracellular permeability in recovery of barrier function. Non-steroidal anti-inflammatory drugs (NSAID) result in increased intestinal permeability and disease exacerbation in patients with IBD. Thus, we examined the reparative attributes of endogenous prostaglandins (PG) after injury of ileal mucosa by deoxycholate (6 mM) in Ussing chambers. Recovery of transepithelial resistance (TER) from 20–40 Ω.cm2 was abolished by indomethacin (INDO), whereas restitution of 40–100% of the villous surface was unaffected despite concurrent arrest of villous contraction. In the presence of PG, resident crypt and migrating epithelial cells were tightly apposed. In tissues treated with INDO, crypt epithelial cells had dilated intercellular spaces that were accentuated in the migrating epithelium. TER was fully rescued from the effects of INDO by osmotic-driven collapse of the paracellular space and PG-mediated recovery was significantly impaired by blockade of Cl− secretion. These studies are the first to clearly distinguish the relative contribution of paracellular resistance versus restitution to acute recovery of epithelial barrier function. Restitution was ineffective in the absence of PG-mediated paracellular space closure. Failure of PG-mediated repair mechanisms may underlie barrier failure resulting from NSAID use in patients with underlying enteropathy.
resistance; permeability; tight junction; deoxycholate
Communication between the airway epithelium and stroma is evident during embryogenesis, and both epithelial shedding and increased smooth muscle proliferation are features of airway remodeling. Hence, we hypothesized that after injury the airway epithelium could modulate airway smooth muscle proliferation. Fully differentiated primary normal human bronchial epithelial (NHBE) cells at an air–liquid interface were co-cultured with serum-deprived normal primary human airway smooth muscle cells (HASM) using commercially available Transwells. In some co-cultures, the NHBE were repeatedly (×4) scrape-injured. An in vivo model of tracheal injury consisted of gently denuding the tracheal epithelium (×3) of a rabbit over 5 days and then examining the trachea by histology 3 days after the last injury. Our results show that HASM cell number increases 2.5-fold in the presence of NHBE, and 4.3-fold in the presence of injured NHBE compared with HASM alone after 8 days of in vitro co-culture. In addition, IL-6, IL-8, monocyte chemotactic protein (MCP)-1 and, more markedly, matrix metalloproteinase (MMP)-9 concentration increased in co-culture correlating with enhanced HASM growth. Inhibiting MMP-9 release significantly attenuated the NHBE-dependent HASM proliferation in co-culture. In vivo, the injured rabbit trachea demonstrated proliferation in the smooth muscle (trachealis) region and significant MMP-9 staining, which was absent in the uninjured control. The airway epithelium modulates smooth muscle cell proliferation via a mechanism that involves secretion of soluble mediators including potential smooth muscle mitogens such as IL-6, IL-8, and MCP-1, but also through a novel MMP-9–dependent mechanism.
remodeling; NHBE; injury; MMP-9; HASM
Inflammatory bowel disease is characterized by a chronic inflammation of the intestinal mucosa. The mucosal epithelium of the alimentary tract constitutes a key element of the mucosal barrier to a broad spectrum of deleterious substances present within the intestinal lumen including bacterial microorganisms, various dietary factors, gastrointestinal secretory products and drugs. In addition, this mucosal barrier can be disturbed in the course of various intestinal disorders including inflammatory bowel diseases. Fortunately, the integrity of the gastrointestinal surface epithelium is rapidly reestablished even after extensive destruction. Rapid resealing of the epithelial barrier following injuries is accomplished by a process termed epithelial restitution, followed by more delayed mechanisms of epithelial wound healing including increased epithelial cell proliferation and epithelial cell differentiation. Restitution of the intestinal surface epithelium is modulated by a range of highly divergent factors among them a broad spectrum of structurally distinct regulatory peptides, variously described as growth factors or cytokines. Several regulatory peptide factors act from the basolateral site of the epithelial surface and enhance epithelial cell restitution through TGF-β-dependent pathways. In contrast, members of the trefoil factor family (TFF peptides) appear to stimulate epithelial restitution in conjunction with mucin glycoproteins through a TGF-β-independent mechanism from the apical site of the intestinal epithelium. In addition, a number of other peptide molecules like extracellular matrix factors and blood clotting factors and also non-peptide molecules including phospholipids, short-chain fatty acids (SCFA), adenine nucleotides, trace elements and pharmacological agents modulate intestinal epithelial repair mechanisms. Repeated damage and injury of the intestinal surface are key features of various intestinal disorders including inflammatory bowel diseases and require constant repair of the epithelium. Enhancement of intestinal repair mechanisms by regulatory peptides or other modulatory factors may provide future approaches for the treatment of diseases that are characterized by injuries of the epithelial surface.
Intestines; Wound healing; Inflammation; Restitution
The potency (pD2) and maximal contractile effect (Emax) of histamine, acetylcholine, carbachol and K+ were assessed from cumulative concentration-effect curves in guinea-pig isolated tracheal ring preparations with and without an intact epithelium. Estimates of Emax were not significantly different in epithelium-denuded preparations compared with those measured in intact preparations; pD2 values for acetylcholine, carbachol and K+ were not significantly altered. In contrast, the potency of histamine was significantly increased by about 4 fold in preparations devoid of epithelial cells. Estimates of potency and Emax were also determined for the smooth muscle relaxants isoprenaline, forskolin and theophylline (which increase intracellular cyclic AMP) and for nitroglycerin (which increases cyclic GMP) in both intact and epithelium-stripped tracheal rings. The pD2 values for these relaxants were not significantly altered by the removal of the epithelium. However, with the exception of nitroglycerin, Emax values for these relaxants were significantly lower in stripped than in intact tracheal rings that had been maximally precontracted with carbachol. The autoradiographic localisation of binding sites for the non-selective beta-adrenoceptor ligand [125I]-iodocyanopindolol (I-CYP) showed that the epithelium of the guinea-pig trachea had a 75 +/- 16% greater density of beta-adrenoceptors than the smooth muscle. Removing the epithelium did not significantly alter either the density of smooth muscle binding sites or the affinity of I-CYP binding. It was concluded that the reduced functional response of guinea-pig trachea to isoprenaline was probably not due to smooth muscle beta-adrenoceptor dysfunction. Results indicate that the epithelium plays an important role in the modulation of responsiveness of guinea-pig trachea to histamine and relaxants that mediate their effects by selectively increasing intracellular cyclic AMP levels.
1. The effect of the potassium channel openers, NIP-121 and cromakalim, on agonist-induced relaxation of the guinea-pig isolated trachea was investigated and the results were compared with those in the epithelium-denuded trachea. 2. Tracheal strips were incubated with a potassium channel opener or vehicle for 30 min in the presence of 5 microM indomethacin and then contracted with 30 nM leukotriene D4 (LTD4). Relaxant agents were added to the organ bath after the LTD4-elicited contraction had reached a plateau. 3. In epithelium-intact trachea, NIP-121 0.1 microM and cromakalim 1 microM, which did not modify the LTD4 (30 nM)-induced contraction, significantly enhanced the sodium nitroprusside (SNP)-induced relaxation. This enhancement of relaxation was not seen in the case of relaxation induced by the cyclic AMP-dependent bronchodilators isoprenaline, vasoactive intestinal peptide or prostaglandin E2. The enhancement of SNP-induced relaxation by NIP-121 and cromakalim was abolished in the presence of the ATP-sensitive potassium channel blocker, glibenclamide (1 microM). NIP-121 and cromakalim did not produce any significant changes in the relaxation induced by 8-bromoguanosine-cyclic monophosphate (8-Br-cyclic GMP), a cyclic GMP analogue. 4. In epithelium-denuded trachea, SNP-induced relaxation alone was significantly enhanced but that induced by 8-Br-cyclic GMP was not changed. Neither NIP-121 nor cromakalim enhanced SNP-induced relaxation in denuded trachea.(ABSTRACT TRUNCATED AT 250 WORDS)
1. An experimental set up is used whereby the serosal (out)side or mucosal (in)side of the guinea-pig isolated tracheal tube can be stimulated selectively with drugs and reactivity measured. 2. Potassium induces a concentration-dependent (5-70 mM) monophasic contraction of tracheal tubes when added on the outside. In contrast, on the inside, potassium induces a concentration-dependent relaxation at low concentrations (5-40 mM) which was reversed into a contraction up to approximately basal tone at higher concentrations (50-70 mM). 3. Epithelium denudation reversed the potassium-induced relaxation into a contraction. Interestingly, in the 'half' epithelium-denuded trachea the contractions were significantly (P < 0.01) reduced by 46% compared to complete epithelium-denuded tissues. 4. Incubation with the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 120 microM) for 30 min on the inside of the tracheal tube completely prevented the relaxation. However, L-NAME did not reverse the potassium-induced relaxation into a contraction. This indicates that potassium does not penetrate through the epithelial layer. 5. It is concluded that depolarization of smooth muscle cells leads to a monophasic contraction and that depolarization of the epithelium leads to a relaxation of tracheal smooth muscle. The epithelial layer has an important barrier function and can release relaxing factors like NO.
1. The effects of the inhaled corticosteroid budesonide and a novel PDE 4 inhibitor CDP840 given systematically, were evaluated in a model of antigen-induced airway inflammation in the rabbit. 2. Adult litter-matched NZW rabbits (2.4-3.5 kg) immunised within 24 h of birth with Alternaria tenuis antigen were pretreated with budesonide (total dose 100 micrograms, inhaled over 2 days) or CDP840 (total dose 7 mg kg-1, i.p. over 3 days), before antigen challenge. For each drug-treated group a parallel group of rabbits was pretreated with the appropriate vehicle. In all groups airway responsiveness to inhaled histamine was assessed and bronchoalveolar lavage (BAL) performed 24 h before and after antigen challenge. 3. Basal lung function in terms of total lung resistance (RL; cmH2O l 1s-1) and dynamic compliance (Cdyn; ml cmH2O-1) were unaltered by pretreatment with budesonide or CDP840 compared to their respective vehicles 24 h before or after antigen challenge. 4. The RL component of the acute bronchoconstriction induced by inhaled Alternaria tenuis aerosol was unaffected by pretreatment with budesonide. However, budesonide prevented the fall in Cdyn due to antigen. Treatment with CDP840 significantly reduced antigen-induced acute bronchoconstriction in terms of both RL and Cdyn. 5. Airway hyperresponsiveness (AHR) to inhaled histamine was indicated by reduced RL PC50 (2.4-4.5 fold) and Cdyn PC35 (2.1-3.9 fold) values 24 h after antigen challenge. Treatment with either budesonide or CDP840 abolished the antigen-induced increase in responsiveness to inhaled histamine. 6. Total cells recovered per ml of BAL fluid increased 24 h after antigen challenge. Antigen-induced pulmonary eosinophilia was reduced (93%) in budesonide and (85%) in CDP840 treated rabbits. Antigen-induced increases in neutrophil numbers were reduced (76%) with budesonide but not CDP840 pretreatment. 7. Inhalation of Alternaria tenuis aerosol elicited an acute bronchoconstriction, followed 24 hours later by an increased responsiveness to inhaled histamine and pulmonary neutrophil and eosinophil recruitment. CDP840 was more effective than budesonide in preventing the antigen-induced increase in total lung resistance (RL); however, both drugs prevented the antigen-induced reduction in dynamic compliance (Cdyn). CDP840 and budesonide also prevented antigen-induced AHR and eosinophilia in the immunised rabbit.
Influenza virus infections increase susceptibility to secondary bacterial infections, such as pneumococcal pneumonia, resulting in increased morbidity and mortality. Influenza-induced tissue damage is hypothesized to increase susceptibility to Streptococcus pneumoniae infection by increasing adherence to the respiratory epithelium. Using a mouse model of influenza infection followed by S. pneumoniae infection, we found that an influenza infection does not increase the number of pneumococci initially present within the trachea, but does inhibit pneumococcal clearance by 2 hours after infection. To determine whether influenza damage increases pneumococcal adherence, we developed a novel murine tracheal explant system to determine influenza-induced tissue damage and subsequent pneumococcal adherence. Murine tracheas were kept viable ex vivo as shown by microscopic examination of ciliary beating and cellular morphology using continuous media flow for up to 8 days. Tracheas were infected with influenza virus for 0.5–5 days ex vivo, and influenza-induced tissue damage and the early stages of repair to the epithelium were assessed histologically. A prior influenza infection did not increase pneumococcal adherence, even when the basement membrane was maximally denuded or during the repopulation of the basement membrane with undifferentiated epithelial cells. We measured mucociliary clearance in vivo and found it was decreased in influenza-infected mice. Together, our results indicate that exposure of the tracheal basement membrane contributes minimally to pneumococcal adherence. Instead, an influenza infection results in decreased tracheal mucociliary velocity and initial clearance of pneumococci, leading to an increased pneumococcal burden as early as 2 hours after pneumococcal infection.
influenza virus; Streptococcus pneumoniae; mucociliary velocity; bacterial clearance and adherence; tracheal explants