Δ9-Tetrahydrocannabinol reverses TNFα-induced increase in airway epithelial cell permeability through CB2 receptors
Graphical abstract
Introduction
The airway epithelium provides a physical barrier, which prevents harmful agents from penetrating into the smooth muscle compartment and activating inflammatory responses [1]. This barrier function is regulated by tight junctions between cells, comprising of a complex of proteins, including occludin, claudin, junctional adhesion molecules, and zonula occludens (ZO-1, ZO-2, and ZO-3) [2]. Reduced expression of these proteins results in altered tight junction function, increased epithelial permeability and, consequently, increased transit of pro-inflammatory mediators and cytokines, leading to stimulation of the afferent sensory nerves and airway hyperreactivity [3]. Cytokines, such as TNFα have been shown to lead to loss of occludin staining, which is associated with increased epithelial permeability [4]. Therefore, regulation of tight junction protein expression and hence airway epithelial permeability is a target for preventing aggravation or progression of inflammatory airway diseases such as asthma [5]. Interestingly, tight junction disruption, with increased permeability and reduced expression of ZO-1 and occludin, is present in biopsies from patients with asthma irrespective of treatment suggesting that current treatments for asthma may not prevent epithelial dysfunction [6]. Δ9-tetrahydrocannabinol (THC), the main phytocannabinoid derived from the Cannabis plant, binds readily to both CB1 and CB2 receptors as a partial agonist [7]. Cannabinoid receptors have been shown to have anti-inflammatory effects in the airways. For example, THC prevents the enhanced nerve-evoked airway contractions in guinea pig trachea exposed to TNFα [8] through stimulation of both CB1 and CB2 cannabinoid receptors. The cannabinoid receptor agonist CP55,940 prevents inflammation-induced bronchoconstriction and mast cell degranulation in ovalbumin-sensitised guinea-pigs [9] and the endocannabinoid anandamide reverses leukotriene D4-induced airway constriction [10]. Although the effects of THC on inflammation-induced changes in airway epithelial permeability are unknown, it has recently been shown that THC reverses the increase in colonic epithelial permeability caused by cytokines through activation of CB1 receptors [11]. It is not known whether the effects of THC on colonic epithelial cells can be replicated in airway epithelial cells and, hence, whether cannabinoid receptor agonists might be exploited therapeutically to reverse the increase in airway epithelial permeability as seen in airway inflammation. Therefore, this present study determined the effect of THC on TNFα-induced increase in permeability and reduced tight junction protein expression in airway epithelial cells.
Section snippets
Cell culture
Calu-3 cells obtained from ATCC (Rockville, MD, USA) were cultured (from passages 5–20) on polyester membrane of Transwells® (pore size 0.4 μm, inserts surface area 1.12 cm2) (Corning CoStar, Arlington, UK). Cell culture medium, Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12 Ham contained 10% of foetal bovine serum, 1% l-glutamine 1% non-essential amino acids, and 1% penicillin/streptomycin. Cells were seeded at a density of 1 × 105 cells per well until day 5 until confluent. Medium from
Effect of THC on cytokine-induced reductions in TEER
Basolateral application of THC alone at 3 and 10 μM had no effect on the Transepithelial Electrical Resistance (TEER) reading in Calu-3 airway epithelial cells (Fig. 1). At 30 μM THC, there was a small increase in TEER (Fig. 1). Resistance readings for vehicle control 0.3% (v/v) EtOH were maintained throughout the whole duration of experiment. In other experiments, THC was added 24 h after addition of TNFα (10 ng/ml; Fig. 2). In these experiments, TNFα caused a reduction in TEER over the first 24 h,
Discussion
The phytocannabinoid THC has been previously shown to improve airway function in asthmatic patients [15] and activation of cannabinoid receptors prevents inflammation-induced changes in the airways [9]. Airway inflammation leads to increased permeability of the epithelial layer, resulting in a loss of barrier function, which is thought to be involved in development of airway hyperreactivity. This present study has demonstrated that pre-treatment with THC prevents the increase in permeability
Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Specific contributions
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VCMS carried out the experiments.
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VCMS, DAK, & RER designed the experiments.
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VCMS, DAK, & RER analysed and interpreted the data.
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VCMS, DAK, & RER wrote the paper.
Acknowledgements
We thank Drs Saoirse O’Sullivan and William Hind, Graduate Entry Medical School, University of Nottingham for the technical support on TEER experiments during the initial part of our study.
We thank Dr. Cynthia Bosquillon, School of Pharmacy, University of Nottingham for the help and advice with the permeability assays.
Funded by the University of Nottingham.
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