Non-genomic action of beclomethasone dipropionate on bronchoconstriction caused by leukotriene C4in precision cut lung slices in the horse
© Fugazzola et al.; licensee BioMed Central Ltd. 2012
Received: 29 November 2011
Accepted: 13 August 2012
Published: 10 September 2012
Glucocorticoids have been proven to be effective in the therapy of recurrent airway obstruction (RAO) in horses via systemic as well as local (inhalative) administration. Elective analysis of the effects of this drug on bronchoconstriction in viable lung tissue offers an insight into the mechanism of action of the inflammatory cascade occurring during RAO which is still unclear. The mechanism of action of steroids in treatment of RAO is thought to be induced through classical genomic pathways. We aimed at electively studying the effects of the glucocorticoid beclomethasone dipropionate on equine precision-cut lung slices (PCLS).
PCLS were used to analyze ex-vivo effects of beclomethasone on inhibiting bronchoconstriction in the horse. The inhibiting effect was measured through instillation of a known mediator of inflammation and bronchoconstriction, leukotriene C4. For this, the accessory lobes of 13 horses subjected to euthanasia for reasons unrelated to the respiratory apparatus were used to obtain viable lung slices.
After 30 minutes of PCLS incubation, beclomethasone showed to significantly inhibit the contraction of the bronchioles after instillation with leukotriene C4. The EC50 values of the two contraction curves (LTC4 with and without BDP) differed significantly from each other (p = 0.002). The possibility of a non-genomic rapid mechanism of action seems likely since transcriptional activities require a longer lag period.
In human neuroendocrinology, high levels of glucocorticoids have been proven to function via a non-genomic mechanism of membrane receptors. The concentration of beclomethasone used on the lung slices in our study can be considered as high. This allows speculation about similar rapid non-genomic mechanisms of high-dosage inhaled glucocorticoids in the lower airways of horses. However, further assessment on a molecular basis is needed to confirm this.
KeywordsBeclomethasone Horse Leukotriene Non-genomic membrane receptors Recurrent airway obstruction RAO Precision-cut lung slices PCLS
The exact interactions of the single inflammatory mediators within the inflammatory cascade taking place during recurrent airway obstruction (RAO) in the horse are still unclear. Ex-vivo precision-cut lung slices (PCLS) from terminal airways of the horse offer a unique condition where the interaction of single inflammatory agents (i.e. bronchoconstrictors) and anti-inflammatory agents (i.e. bronchodilators) can be observed without multifactorial influence as occurs in vivo. This may be useful to further understand the pathogenesis of RAO. The aim of the study was to take a first step to analyse the interaction of two substances involved in bronchoconstriction and bronchodilation in healthy pulmonary tissue of 13 horses.
Synthetic glucocorticoids are recognized to be effective in the treatment of RAO in horses and have been widely used via various routes of administration in the past 15 years. Their beneficial effects are thought to be mainly achieved through their potent anti-inflammatory properties. They inactivate NF-kB, blocking transcription of genes coding for pro-inflammatory cytokines, up-regulate and increase the sensitivity of β2-adrenoceptors and reduce production of pro-inflammatory eicosanoids . Their mechanism of action is mainly genomic with lag periods ranging from at least four to several hours or even days the manifest action of drug-induced response takes place [2–4].
Inhalation of glucocorticoids has become a good alternative to systemic administration in chronic obstructive airway diseases where high local concentration of the drug, lower dosage and the low risk of unwanted systemic side effects represent the major advantages of way of administration [5, 6]. Beclomethasone dipropionate (BDP) is a widely administered inhaled corticosteroid in equine practice [7, 8], although in vivo studies have shown that the marked improvement of clinical signs of airway obstruction after administration of low-dose inhaled beclomethasone was not accompanied by a decrease in airway inflammatory cells or a suppression of transcription factors NF-kB and AP-1 DNA-binding activity .
In the past, leukotrienes have been shown to mimic many of the pathophysiological processes in allergic airway disease [12, 13] and since leukotriene C4 is renowned for its potent bronchoconstricting action, it was chosen as bronchoconstrictor in our study.
Scientific investigations on the possible role of the cysteinyl leukotrienes in RAO have been reported in recent years. Lavoie et al. could not demonstrate improved lung function in horses with RAO after administration of a leukotriene D4 antagonist . A more recent study suggested that there is increased leukotriene B4 and C4 production in airways of horses affected with RAO which may contribute to infiltration of neutrophils into the lungs and the sustained inflammation associated with RAO . In an earlier study, the inhalative administration of LTD4 and LTB4 to healthy horses caused bronchoconstriction and neutrophil accumulation respectively . Specifically, ex vivo LTC4 caused marked bronchoconstriction in equine PCLS which could be antagonized by the receptor antagonist MK-571 . A hyper-reactivity of the bronchioles of RAO-affected horses in comparison to healthy horses was also demonstrated . To date, no in vivo studies on effects of LTC4 have been reported.
We aimed at examining the effects of directly instilled beclomethasone on viable equine terminal airways to determine its capacity of inhibiting bronchoconstriction.
13 horses (8 geldings, 4 mares, 1 stallion; 16 ± 6 years of age [mean ± SD]; 500 ± 90 kg of body weight) which were to be euthanized due to conditions unrelated to this study or pulmonary disease were used for this study. After a general clinical examination, a specific examination of the respiratory apparatus consisting of auscultation, percussion, bronchoscopy, TBS-analysis and blood gas analysis was performed to exclude respiratory disease. Seven of the horses had received non-steroidal analgesics and antibiotic therapy related to the primary problem in the period preceding our study. After sedation (Detomidine, 0,02 mg/kg iv), euthanasia was performed using an overdose of pentobarbital.
Immediately post mortem, the accessory lobe was removed with a cutting wire loop which was placed around the base of the lobe after opening the thorax and subsequently chilled to 4°C on ice, decreasing metabolic activity in the tissue without losing viability. In order to harden the lung tissue, the lobe was filled with a 1,5% low melting point agarose solution (type VII, Sigma-Aldrich Chemie, Germany) . The agarose was enriched with cell culture medium (RPMI-1640 Medium, Biochrom AG Berlin, Germany) to provide nutrition to the tissue throughout the cutting procedures. Subsequently, precision cut lung slices with a diameter of 8 mm and a thickness of 400 μm were obtained from the lung tissue with a microtome (Krumdieck Tissue Slicer Model MD 4000, Alabama Research and Development). At the centre of each slice, the integrity, i.e. the presence of a terminal bronchiolus which was orientated perpendicular to the cut surface was condition of selection. After bedding the obtained PCLS in RPMI culture medium, viability was checked for each slice by contraction with metacholine (10-5 mol/l). A minimum of 50% lumen reduction was chosen as selection criterion. Viable slices were then washed with RPMI medium three times and incubated for 30 minutes to allow dilatation. Absence of pre-contraction was assessed by comparing initial values of bronchiolar lumen diameter with those after the reopening phase before the first contraction with leukotriene C4 was performed.
The concentration of LTC4 used for bronchoconstriction was based on an earlier study on PCLS where CysLT1 antagonists had been examined .
Additional file 1: Contraction of bronchiolus after instillation of 10 -13 mol/l LTC 4 .(WMV 5 MB)
The effects of the bronchoconstrictor LTC4 with and without beclomethasone dipropionate were recorded through digital photography after every increase of concentration.
As a final step, the PCLS were again rinsed and incubated for 30 to 40 minutes in order to dilate. A further contraction with 10-8 mol/l LTC4 was performed to assure viability of the slices throughout the procedures. If a 50% lumen reduction was not achieved at this point, slices where excluded from statistical analysis.
The photographed lumen area of the bronchioles after each concentration increase was measured three times manually with a digitalising tablet (Trust®) and the average value was used. This value was quantified with percentage digital measurement (“Scion Image Version Beta 4.0.2”) and statistically analysed afterwards.
In order to compare reactivity of PCLS to LTC4 in our study to results of earlier studies, an EC50 was calculated. EC50 was defined as the concentration at which the PCLS contracted their lumen to 50% of the original surface area. Where a minimum of 50% lumen reduction was not reached, the EC50 concentration was defined as > −8 mmol/l. The effect of beclomethasone was compared with untreated control slices by comparing the EC50 values of the two groups using the Wilcoxon test. In order to obtain one data point per increasing LTC4 concentration, the average of 3 PCLS measurements per horse was used.
A p-value < 0.05 was regarded significant.
The software used for statistic evaluation was Graph Pad Prism 5.
Results and discussion
The calculated EC50 for LTC4 during the first contraction series of all included PCLS was 2 x 10-10 mol/l whilst the calculated EC50 for LTC4 after 30 minutes incubation with BDP (10-5 mol/l) was 3 x 10-9 mol/l.
The well-investigated genomic anti-inflammatory effect of glucocorticoids is mediated by their binding to a cytoplasmic receptor which, when activated, migrates to the nucleus. The activated receptor can interact with transcription factors (i.e. NF-kappaB, AP-1) inhibiting the synthesis of pro-inflammatory proteins (i.e. cytokines, COX-2). This trans-repressive activity explains most of the anti-inflammatory effects of glucocorticoids . Since this mechanism is dependent on protein synthesis, the manifest action of the glucocorticoid takes place only after the characteristic lag period which ranges from at least four to several hours or even days [2–4, 23].
In our study, incubation periods of 30 minutes with BDP seem too short for transcriptional procedures so it is likely that the inhibiting effect was mediated by a non-genomic mechanism of action which occurs instantaneously after exposure or within a very short latency period [2–4, 24, 25]. The non-genomic receptors directly activate various second messengers (e.g. Ca++-dependant protein kinases, cAMP) [26, 27] which regulate specific biological actions within the cells such as bronchodilation, secretion, absorption etc. A controversy regarding the identity of receptors that mediate non-genomic, transcription-independent cellular responses to steroids is presently attracting considerable scientific interest [2, 4, 28, 29]. There is strong evidence that classic receptors belonging to the nuclear receptor superfamily mediate non-genomic steroid effects in some cases . In the human brain, the rapid non-genomic effect of corticoids seems to depend on classical mineralocorticoid receptors which are accessible from the outside of the plasma membrane and display a 10-fold lower affinity for corticosterone than the nuclear version involved in neuroprotection. Consequently, this type of receptor seems to play an important role while corticosteroid levels are high, i.e. during the initial phase of the stress response .
In our case, the concentration of BDP (10-5 mol/l) used for our study was based on Chanoine´s studies on rat lungs where intratracheal administration of the same concentration of BDP was used to assess pharmacokinetics . Considering the short distance of diffusion and the small amount of lung tissue in PCLS treated with each application of BDP, 10-5 mol/l may be considered a high dosage. Since this dosage might be difficult to achieve in vivo, lower dosages should be assessed in following studies in order to confirm clinical relevance of these findings.
Another consideration is that a classic genomic mechanism would prevent bronchoconstriction by inhibiting at a higher level of the inflammatory signal cascade. Thus, final products such as leukotriene would not be produced. Since, in our study, the inflammatory mediator was directly instilled on the PCLS, a genomic mechanism of action could not occur; instead, a direct action of the glucocorticoid is most likely.
The pharmacological action of beclomethasone on LTC4 catabolism in transformed human bronchial epithelial cell lines accelerates the degradation of LTC4 into less active (less contractile) LTE4 and LTD4. This mechanism is unlikely the same as in our study since increase of LTC4 catabolism was observed only after 2-day BDP incubation period of the cells, suggesting new protein synthesis.
In vivo BDP has proven to be effective in the therapy of RAO in horses [34, 35]. The first signs of clinical improvement could only be seen after four days of treatment. Although this would indicate a complex trans-repressive mechanism of action, Couteil et al. could not show a decrease in pro-inflammatory transcription factors (NF-kappaB, AP-1) in the BALF despite clinical signs improving significantly . These findings are in line with our results and suggest that clinical improvement (in our study inhibition of bronchoconstriction) was achieved through a fast-acting non-genomic mechanism.
In this study, the mechanisms of action through which inhibition of bronchoconstriction was achieved were not investigated. There is evidence for a rapid and non-genomic reduction of intracellular [Ca2+] induced by aldosterone in human bronchial epithelium . This could give reason to speculate about an inhibiting action of BDP on bronchoconstriction in PCLS through rapid calcium reduction in smooth muscle cells. In conclusion, we may say that further investigation is needed to understand the exact mechanism of action with which BDP at a high dosage inhibits bronchoconstriction in terminal airways through a non-genomic pathway.
Broncho-alveolar lavage fluid
Precision cut lung slices
Recurrent airway obstruction
We thank the Department of Pharmacology for technical support during pharmacologic tests and the Department of Toxicology for support in the tissue slicing procedure.
The study was founded with the didactical funds of the Institute of Pharmacology and the Clinic for Horses of Hanover Veterinary School.
- Barnes PJ: Molecular mechanisms of glucocorticoid action in asthma. Pulmonary Pharmacology & Therapeutics. 1997, 10: 1-65. 10.1006/pupt.1997.0079. 3–19View Article
- Alangari A: Genomic and non-genomic actions of glucocorticoids in asthma. Annals of Thoracic Medicine. 2010, 5: 133-139. 10.4103/1817-1737.65040.PubMed CentralView ArticlePubMed
- Reichardt HM, Tuckermann JP, Bauer A, Schutz G: Molecular genetic dissection of glucocorticoid receptor function in vivo. Zeitschrift Fuer Rheumatologie. 2000, 59: 1-5. 10.1007/s003930050001.View Article
- Gibson PG, Saltos N, Fakes K: Acute anti-inflammatory effects of inhaled budesonide in asthma: a randomized controlled trial. Am J Respir Crit Care Med. 2001, 163: 32-36.View ArticlePubMed
- Lavoie JP: Heaves (recurrent airway obstruction): practical management of acute episodes and prevention of exacerbations Current Therapy in Equine Medicine2003. Edited by: Robinson ME, Sprayberry KA. St. Louis, Saunders
- Hanania NA: The impact of inhaled corticosteroid and long-acting beta-agonist combination therapy on outcomes in COPD. Pulm Pharmacol Ther. 2008, 21: 540-550. 10.1016/j.pupt.2007.12.004.View ArticlePubMed
- Corcoran BM, Mair TS, Taylor DJ: Drugs used in the treatment of disorders of the respiratory system. The Veterinary Formulary, sixth edition 2005. 2005, The Pharmaceutical Press, London
- Leguilette R: Recurrent airway obstruction—heaves. The Veterinary Clinics Equine Practice. 2003, 19: 63-86. 10.1016/S0749-0739(02)00067-6.View Article
- Couetil LL, Art T, Demoffarts B, Becker M, Melotte D, Jaspar F, Bureau F, Lekeux P: Effect of beclomethasone dipropionate and dexamethasone isonicotinate on lung function, bronchoalveolar lavage fluid cytology, and transcription factor expression in airways of horses with recurrent airway obstruction. J Vet Intern Med. 2006, 20: 399-406. 10.1111/j.1939-1676.2006.tb02875.x.View ArticlePubMed
- Davis E, Rush BR, Equine RAO: Pathogenesis and patient management. Veterinary Clinics of North America: Equine Practice. 2002, 18: 453-467. 10.1016/S0749-0739(02)00026-3.PubMed
- Ainsworth DM, Cheetam J: Disorders of the respiratory system. Equine Internal Medicine. Edited by: Reed , Warwick , Sellon . 2010, Saunders, St. Lois, 340-344. third
- Nagase T, Fukuchi Y, Dallaire MJ, Martin JG, Ludwig MS: In vitro airway and tissue response to antigen in sensitized rats - role of serotonin and leukotriene D-4. Am J Respir Crit Care Med. 1995, 152: 81-86.View ArticlePubMed
- Drazen JM: Leukotrienes as mediators of airway obstruction. Am J Respir Crit Care Med. 1998, 158: 193-200.View Article
- Lavoie JP, Leguillette R, Pasloske K, Charette L, Sawyer N, Guay D, Murphy T, Hickey GJ: Comparison of effects of dexamethasone and the leukotriene D4 receptor antagonist L-708,738 on lung function and airway cytologic findings in horses with recurrent airway obstruction. Am J Vet Res. 2002, 63: 579-585. 10.2460/ajvr.2002.63.579.View ArticlePubMed
- Lindberg A, Robinson NE, Naesman-glaser B, Jensen-waern M, Lindrgren JA: Assessment of leukotriene B4 production in leukocytes from horses with recurrent airway obstruction. American Journal of Veterinary Reasearch. 2004, 65: 289-295. 10.2460/ajvr.2004.65.289.View Article
- Marr K, Lees P, Page CP, Cunningham FM: Inhaled leukotrienes cause bronchoconstriction and neutrophil accumulation in horses. Res Vet Sci. 1998, 64: 219-224. 10.1016/S0034-5288(98)90129-1.View ArticlePubMed
- Schwalfenberg B: Untersuchung der Leukotrienwirkung am Pferdebronchus mittels. 2007, Tierärztliche Hochschule, Dissertation, Hannover
- Barton AK, Niedorf F, Gruber AD, Kietzmann M, Ohnesorge B: Pharmacological studies of bronchial constriction inhibited by parasympatholytics and cilomilast using equine precision-cut lung slices. Berliner und Münchner Tierarztliche Wochenschrift. 2010, 123: 229-235.
- Vietmeier J, Niedorf F, Baumer W, Martin C, Deegen E, Ohnesorge B, Kietzmann M: Reactivity of equine airways - a study on precision-cut lung slices. Vet Res Commun. 2007, 31: 611-619. 10.1007/s11259-007-3501-y.View ArticlePubMed
- Barton AK, Niedorf F, Rohwer J, Kietzmann M, Ohnesorge B: Pilot study on passive sensitization against ovalbumin using equine precision-cut lung slices (PCLS). Pferdeheilkunde. 2010, 26: 789-796.
- Martin C, Uhilg S: Ullrich: Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices. Eur Respir J. 1996, 9: 2479-2487. 10.1183/09031936.96.09122479.View ArticlePubMed
- Devillier P: Pharmacology of corticosteroids and ENT diseases: 10 key points. Presse Medicale. 2001, 30: 59-69.
- Van Dv: Glucocorticoids: mechanisms of action and anti-inflammatory potential in asthma. Mediat Inflamm. 1998, 7: 229-237. 10.1080/09629359890910.View Article
- Ferguson DC, Hoenig M: Glucocorticoids, mineralocorticoids, and steroid synthesis inhibitors Veterinary Pharmacology and Therapeutics Editor: Adams. 2001, Iowa State University Press, Ames (USA), 649-668.
- Croxtall JD, Choudhary Q, RJ FLOWER: Br J Pharmacol. 2000, 130: 289-298. 10.1038/sj.bjp.0703272.PubMed CentralView ArticlePubMed
- Muto S, Ebata S, Okada K, Saito T, Asano Y: Glucocorticoid modulates Na+/H + exchange activity in vascular smooth muscle cells by non-genomic and genomic mechanisms. Kidney Int. 2000, 57: 2319-2333. 10.1046/j.1523-1755.2000.00092.x.View ArticlePubMed
- Harvey BJ, Alzamora R, Healy V, Renard C, Doolan CM: Rapid responses to steroid hormones: from frog skin to human colon. An homage to Hans Ussing. Biochimica Et Biophysica Acta-Biomembrane. 2002, 1566: 116-128. 10.1016/S0005-2736(02)00589-8.View Article
- Stahn C, Buttgereit F: Genomic and non-genomic effects of glucocorticoids. Nat Clin Pract Rheumatol. 2008, 4: 525-533. 10.1038/ncprheum0898.View ArticlePubMed
- Lecoq L, Vincent P, Lavoie-Lamoureux A, Lavoie JP: Genomic and non-genomic effects of dexamethasone on equine peripheral blood neutrophils. Vet Immunol Immunopathol. 2009, 128: 126-131. 10.1016/j.vetimm.2008.10.303.View ArticlePubMed
- Wehling M, Losel R: Non-genomic steroid hormone effects: Membrane or intracellular receptors?. Journal of Steroid Biochemistry and Molecular Biology. 2006, 102: 180-183. 10.1016/j.jsbmb.2006.09.016.View ArticlePubMed
- DE Kloet ER, Karst H, Joels M: Corticosteroid hormones in the central stress response: Quick-and-slow. Front Neuroendocrinol. 2008, 29: 268-272. 10.1016/j.yfrne.2007.10.002.View ArticlePubMed
- Chanoine F, Grenot C, Heidmann P, Junien JL: Pharmacokinetics of butixocort 21-propionate, budesonide, and beclomethasone dipropionate in the rat after intratracheal, intravenous, and oral treatments. Drug Metabolism and Disposition. 1991, 19: 546-553.PubMed
- Zaitsu M, Hamasaki Y, Aoki Y, Miyazaki S: A novel pharmacologic action of glucocorticosteroids on leukotriene C4 catabolism. Journal of Allergy and Clinical Immunology. 2001, 108: 122-124. 10.1067/mai.2001.116000.View ArticlePubMed
- Ammann VJ, Vrins AA, Lavoie JP: Effects of inhaled beclomethasone dipropionate on respiratory function in horses with chronic obstructiove pulmonary disease (COPD). Equine Veterinary Journal. 1998, 30: 152-15734. 10.1111/j.2042-3306.1998.tb04475.x.View ArticlePubMed
- Rush BR, Flaminio MJ, Matson CJ, Hakala JE, Shuman W: Cytologic evaluation of broncho-alveolar lavage fluid from horses with recurrent airway obstruction after aerosol and parenteral administration of beclomethasone dipropionate and dexamethasone, respectively. Am J Vet Res. 1998, 59: 1033-1038.PubMed
- Urbach V, Harvey BJ: Rapid and non-genomic reduction of intracellular [Ca2+] induced by aldosterone in human bronchial epithelium. J Physiol. 2001, 537 (1): 267-27. 10.1111/j.1469-7793.2001.0267k.x.PubMed CentralView ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.