Cell culture

Caco-2 cells (ECACC, Wiltshire, UK, passages 56–72) were cultured in Minimum Essential Medium Eagle supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% penicillin/streptomycin. Cells were kept at 37°C in 5% CO2 and 95% humidity. Cells were grown in 12-well plates and seeded at 50 000 cells per insert on 12 mm diameter, 0.4 µm pore polycarbonate membrane inserts. Cells were grown for a minimum of 14 days and used for experimentation between days 14 and 21, when each insert had a transepithelial electrical resistance (TEER) value greater than 1000 Ω cm2.

TEER measurement

The TEER measurement was used to evaluate the paracellular permeability of cell monolayers (Madaraet al., 1988). The TEER of the monolayer was determined using an EVOM™ voltohmmeter (World Precision Instruments, Sarasota, FL, USA) according to the methods of Wells and colleagues (Wells et al., 1998).

Inflammatory protocol

Initial TEER readings were made before the addition of 10 ng·mL−1 IFNγ (basolateral compartment). After 8 h, TEER was measured again, and 10 ng·mL−1 TNFα was added for another 16 h. TEER was measured again after a total of 24 h incubation with the cytokines, which caused an average fall in TEER of 20–25%, representing increased epithelial permeability.

Permeability studies

Intestinal permeability to fluorescein isothiocyanate (FITC)-dextran molecular mass 4 kDa (FD4), a tracer for the paracellular pathway, was evaluated by measuring the flux of FD4 across cell monolayers. Cannabinoids [cannabidiol (CBD, 1 µM), AM251 (100 nM) and methandamide, mAEA 100 Nm] were applied apically either concomitant with the cytokines (0 h) or following the inflammatory protocol (24 h), for a further 6 h. Cell layers (30 h) were then washed with HBSS/20 mM HEPES (pH 7.4) and left for 30 min at 37°C to equilibrate. FD4 (3 mg·mL−1) was applied apically and 100 µL aliquots were collected from the basolateral side of each insert after 30 min and 1 h. FD4 levels in the medium were measured using a fluorescence microplate reader at an excitation wavelength of 490 nm and emission wavelength of 520 nm (VICTOR, Perkin Elmer, USA). FD4 flux was calculated as the average fluorescence value of two samples taken from the same well, and expressed as a percentage of the FD4 permeability of vehicle control monolayers in the same experiment.

Cell viability (MTS) and membane integrity (lactate dehydrogenase release) assays

To show that the effect of cytokine application was not due to cellular damage and changes in transcellular permeability, we performed MTS (Promega, Madison, WI, USA) and lactate dehydrogenase (LDH) assays (Bio Vision, CA, USA), according to the manufacturer’s instructions, on Caco-2 treated with 10 ng·mL−1 IFNγ and 10 ng·mL−1 TNFα for up to 72 h.

Effects of cannabinoids on Caco-2 cell monolayer integrity (apical application)

Fresh medium, with or without cannabinoids [Δ9-tetrahydrocannabinol (THC), CBD, AEA or 2-AG (all 10 µM)], was applied apically to plates where inflammation had been established (i.e. after 24 h). Vehicle (0.1% ethanol) was applied to control wells. TEER values were measured every 1 h for the next 8 h, and then again 48 and 72 h after cannabinoid administration. Our initial experiments showed that a single dose of THC or CBD (10 µM) ameliorated the fall in TEER caused by cytokines, while a single dose of AEA or 2-AG (10 µM) worsened this (see Figure 1). Therefore, we proceeded to perform concentration–response curves to THC, CBD, AEA and 2-AG by adding increasing concentrations of each drug to inserts. TEER values were monitored at all time points as described above.

Figure 1

The effects of phytocannabinoids (THC and CBD, 10 µM, A) and endocannabinoids (AEA and 2-AG, 10 µM, C) applied apically on the fall in TEER values caused by the inflammatory cytokines (IFNγ and TNFα, 10 ng·mL−1 …

In some experiments, 10 µM of either THC or CBD was applied at the apical compartment at 0 h (i.e. at the same time as the cytokines) or 48 h after cytokine application. TEER values were measured as above.

Target sites of action of cannabinoids

The following antagonists were co-applied with cannabinoids (24 h after inflammation was established); AM251 (CB1 receptor antagonist), AM630 (CB2 receptor antagonist), capsazepine (TRPV1 antagonist), GW9662 (PPARγ antagonist), GW6471 (PPARα antagonist) and O-1918 (proposed cannabinoid receptor antagonist). All antagonists were used at 1 µM except AM251, which was used at 100 nM (seeAlhamoruni et al., 2010) and appropriate vehicles were applied to control inserts. TEER values were measured as above.

In some experiments, 100 nM of either AM251 or AM630 was applied at the apical compartment at 0 h (i.e. at the same time as the cytokines) or 24 h after cytokine application (when increased permeability was induced). TEER values for each group were monitored over time.

Effects of cannabinoids on Caco-2 cell monolayer permeability (basolateral application)

Fresh medium, with or without cannabinoids (THC, CBD, AEA or 2-AG, all 10 µM), was applied basolaterally to plates where inflammation had been established.

Effects of enzyme inhibitors on increased permeability induced by cytokines

To establish the role of the FAAH enzyme on the AEA effect on intestinal permeability, AEA (10 µM) was applied to the apical side of inserts in the absence or presence of an FAAH inhibitor 24 h after inflammation was established (URB597, 1 µM). Similarly, 2-AG (10 µM) was applied to the apical side of inserts either alone or together with a monoacylglycerol lipase (MGL) inhibitor (JZL 184, 1 µM). In both experiments, the vehicle [ethanol and dimethyl sulfoxide (DMSO)] was applied to control wells. TEER values were measured hourly for the next 8 h, and then again at 48, and 72 h after cannabinoid administration.

In some experiments, 1 µM of URB597 or JZL 184, alone or together with CB1 antagonist AM251 (100 nM), were applied at the apical compartment at the same time as the cytokines.

Orlistat (1 µM), a 2-AG synthesis inhibitor, alone or together with CB1 antagonist (AM251, 100 nM) was applied at the apical compartment at the same time as cytokine application.

Chemicals and reagents

All chemicals were purchased from Sigma-Aldrich (Poole, UK) unless otherwise stated. IFNγ and TNFα were purchased from Invitrogen (Paisley, UK), and further dilutions in BSA stored at −80°C for IFNγ and −20°C for TNFα. All cannabinoids and antagonists were purchased from Tocris Bioscience (Bristol, UK) except THC and capsazepine, which were obtained from Sigma UK. CBD, THC, capsazepine, AEA and 2-AG were dissolved in ethanol to a stock concentration of 10 mM with further dilutions made in distilled water. GW9662, AM251 and AM630 were dissolved in DMSO to 10 mM, with further dilutions made in distilled water. URB597, JZL 184 and Orlistat were dissolved in DMSO to 10 mM, with further dilutions made in fresh media.

Statistical analysis

In each protocol, values are expressed as mean ± SEM. Area under the curve (AUC) values were calculated using GraphPad Prism 5 software using the trapezoidal method. Data were compared, as appropriate, by Student’s t-test or by anova with statistical significance between manipulations and controls determined by Dunnett’s post hoc test.

Cytokines increased permeability without affecting cell viability or membrane integrity

Combined application of IFNγ and TNFα (10 ng·mL−1) in Caco-2 cells caused a reversible decrease in TEER (i.e. increased permeability) over the 72 h measurement period. Application of IFNγ and TNFα to Caco-2 cells did not affect the Caco-2 cell mitochondrial activity at any point over the 72 h experimental period compared with the vehicle group, as indicated by the MTS assay (OD at 72 h; vehicle 0.54 ± 0.03, cytokine application, 0.52 ± 0.01, n= 4). The total LDH release from Caco-2 cells treated with cytokines was also not significantly different to vehicle at any point over the 72 h experimental period (OD at 72 h; vehicle 0.22 ± 0.01, cytokine application, 0.11 ± 0.01, n= 4).

Apical application of phytocannabinoids recovers cytokine-induced increased permeability

Twenty-four hours after exposure to IFNγ and TNFα, apical application of either THC or CBD (10 µM) accelerated the recovery of TEER values (see Figure 1A), and the total response over time (AUC) was significantly different to vehicle controls for both THC and CBD (P < 0.01, Figure 1B). Further experiments showed that the ability of THC and CBD to speed the recovery of TEER values after 24 h cytokine application was concentration-dependent (see Figure 2 and Table 1). When a sigmoidal concentration–response curve was plotted with the AUC data presented in Table 1, the logEC50 of THC and CBD were −6.03 and −5.68, respectively.

Figure 2

Concentration–response curves to THC (A), CBD (B), AEA (C) and 2-AG (D) applied apically on the fall in TEER caused by cytokine application. Data are given as means with error bars representing SEM. (n= 3, *P < 0.05, **P < 0.01, …

Table 1

Area under the curve values (%·min−1) for the concentration–responses to cannabinoids on TEER

Apical application of endocannabinoids further increases permeability after cytokine application

Twenty-four hours after exposure to IFNγ and TNFα, apical application of endocannabinoids (10 µM of either AEA or 2-AG) caused a further and sustained drop in TEER in addition to the effects of cytokines (P < 0.05, Figure 1C and D). Further experiments showed that this effect was concentration-dependent (see Figure 2 and Table 1). When a sigmoidal concentration–response curve was plotted with the AUC data presented in Table 1, the logEC50 of AEA and 2-AG were −3.95 and −3.78, respectively.

The effects of both phytocannabinoids and endocannabinoids are CB1 mediated

The effects of THC and CBD were only significantly inhibited by the cannabinoid CB1 receptor antagonist, AM251. Similarly, the effects of the endocannabinoids AEA and 2-AG were also only sensitive to AM251 (Figure 3 and Table 2).

Figure 3

The effects of various receptor antagonists on the effects of THC (10 µM, A), CBD (10 µM, B), AEA (10 µM, C) and 2-AG (10 µM, D) applied apically on the fall in TEER caused by cytokine application. Data are given as means …

Table 2

Area under the curve values (%·min−1) for the effects of cannabinoids on TEER in the presence of various receptor antagonists

Basolateral application of cannabinoids and permeability after cytokine application

When applied to the basolateral membrane after cytokine application, neither THC, CBD, AEA or 2-AG had any significant effect on TEER (data not shown).

Phytocannabinoids prevented increased permeability associated with cytokine application

When inserts were treated with cytokines (basolateral) and THC or CBD (apical) at the same time (0 h), THC and CBD (10 µM) completely inhibited the fall in TEER caused by the cytokines (see Figure 4A). However, when THC or CBD were applied 48 h after cytokine application, they had no effect on the response to these cytokines (Figure 4B).

Figure 4

The effect of phytocannabinoids (THC and CBD, 10 µM) applied apically at time 0 h (A), or after 48 h (B) on the fall in TEER caused by cytokine application. Data are given as means with error bars representing SEM. (n= 3, *P < 0.01, *** …

CB1 antagonism reduces the increased permeability associated with cytokines

To determine whether the effect of cytokines can be prevented by cannabinoid receptor antagonism, AM251 or AM630 (both 100 nM, apical application) were added at the same time as cytokine application (0 h) or after cytokine-induced increases in TEER were induced (24 h). When applied at time 0, AM251 significantly reduced the fall in TEER caused by cytokines. However, when AM251 was applied after 24 h, there was no effect of this compound (Figure 5A). AM630 did not affect TEER values when co-applied with cytokines, or when applied after inflammation was induced (Figure 5B), indicating no role for CB2receptor activation.

Figure 5

The effects of the CB1 receptor antagonist, AM251 (100 nM, A) on TEER applied apically at the same time as (0 h), or 24 h after cytokine application. The effects of the CB2 receptor antagonist, AM630 (100 nM, B) on TEER applied at the same time as (0 …

FAAH and MGL inhibition worsened endocannabinoids effects on increased permeability after cytokine application

URB597 alone caused no significant change in the recovery of TEER compared with the vehicle (see Figure 6A and B). As previously shown, AEA alone caused a significant drop in TEER in addition to the effects of cytokines compared with vehicle. However, application of URB597 together with AEA caused a significantly greater drop in TEER than AEA alone (Bonferroni’s multiple comparison test, Figure 6A and B). JZL 184 alone also caused no significant change in the recovery of TEER compared with vehicle. 2-AG alone caused a significant decrease in TEER as compared with vehicle group, and application of JZL 184 with 2-AG caused a significantly greater drop in TEER than 2-AG alone (Figure 6C and D).

Figure 6

The effect of the FAAH inhibitor URB597 (1 µM, A) and MGL inhibitor JZL 184 (1 µM, C) applied apically alone, or in combination with AEA or 2-AG on the fall in TEER values caused by inflammatory cytokines. (B, D) Integrated response over …

To test the hypothesis that locally produced AEA and 2-AG partly mediates the increase in permeability caused by cytokines, and whether these effects, if any, are mediated by CB1 receptors, URB597 or JZL 184 (1 µM each) were applied apically at the same time as cytokine application either alone or with AM251 (100 nM). When applied at the same time as cytokines, URB597 alone caused a further drop in TEER (i.e. increased permeability) than cytokine application alone, and this effect was inhibited by AM251 (Figure 7A and B). Similarly, JZL 184 application led to a decrease in TEER, and this effect was also inhibited by AM251 (Figure 7C and D).

Figure 7

The effect of endocannabinoid enzyme inhibitors (URB597, 1 µM, A; JZL 184, 1 µM, C; Orlistat, 1 µM, E) applied apically at the same time as cytokines, either alone or together with the CB1 antagonist AM251 (100 nM) on the fall …

To further investigate the possible role of locally produced 2-AG on the TEER reduction caused by cytokines, Orlistat (1 µM), a 2-AG synthesis inhibitor was applied either alone or together with AM251 (100 nM). It was observed that Orlistat inhibited the drop in TEER caused by cytokines as compared with vehicle group (Figure 7E and F). This was not further affected by AM251.

FD4 flux was increased by cytokines and modulated by cannabinoids

To support our TEER data, we performed key experiments to measure the functional outcome of junctional change, that is, paracellular permeability, by using FITC-conjugated dextran (FD4) as a tracer. Cytokine application [interferon gamma and TNF alpha (IT)] induced an increase of 22 ± 4% in permeability to FD4 when compared with basal flux (Figure 8A). CBD both reversed (IT + CBD) and prevented (CBD + IT) this increase, as previously observed in the TEER experiments. As before, the CB1antagonist AM251 (100 nM) blocked the CBD effect on cytokine-induced FD4 flux.

Figure 8

The effect of cannabinoids on cytokine-induced FD4 flux. (A) CBD (1 µM) or mAEA (100 nM) was applied apically at time zero together with the cytokines (IFNγ and TNFα, 100 ng·mL−1) or after 24 h of cytokine application …

In addition, reflecting the AEA effect on cytokine-induced TEER changes, mAEA (100 nM) further enhanced the increased permeability to FD4 to 35.2 ± 3.1% (an enhancement of approximately 12%), which was also blocked by AM251.

AM251 (100 nM) was able to both partially inhibit and prevent the cytokine-induced increase in FD4 flux, whereas the CB2 receptor antagonist/inverse agonist, AM630, had no effect (see Figure 8B), again in support of the previous TEER data.

We would like to thank the technical support of Mrs Averil Warren and Mr Andrew Lee.

AEA

arachidonyl-ethanolamide, anandamide

AM251

N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide

AM630

6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl) methanone

Caco-2

carcinoma colon cell line

CB1

cannabinoid receptor 1

CB2

cannabinoid receptor 2

CBD

cannabidiol

EVOM

epithelial tissue volt-ohm-meter

FAAH

fatty acid amide hydrolase

GW6471

[(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester

GW9662

2-chloro-5-nitro-N-phenylbenzamide

IBD

inflammatory bowel disease

JZL 184

4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate

MGL

monoacylglycerol lipase

O-1918

1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]benzene

TEER

transepithelial electrical resistance

THC

Δ9-tetrahydrocannabinol

URB597

3′-(aminocarbonyl)[1,1′-biphenyl]-3-yl)-cyclohexylcarbamate

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