Animal Treatment

All animal studies were carried out in accordance with the National Institutes of Health Guide for the Use and Care of Laboratory Animals. Male Sprague Dawley rats (200 − 225g) were pretreated for 1 h with CBD or vehicle, dimethylsulfoxide (DMSO), i.p. (CBD or DMSO : Cremophor : saline in a ratio of 1 : 1 : 18). After 30 min, rats were given 8-cyclopentyl-1,3-dipropylxanthine (CPX; Sigma, MO), 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol (ZM 241385; Tocris Bioscience, Ellisville, MO), or vehicle i.p. (CPX, ZM or DMSO : Cremophor : saline in a ratio of 1 : 1 : 8). After drug pretreatment, rats were anesthetized with isoflurane and given a single LPS (Salmonella enterica; Sigma, MO) injection at 0.35 mg/kg. After 24 hours, rats were killed by decapitation after anesthesia with isoflurane. Retinas and vitreous were collected for assay of total protein by the Bio-Rad DC protein assay, and for TNF-α levels by enzyme-linked immunosorbent assay (ELISA).

Primary Rat Retinal Microglia Cultures

Microglial cells were isolated from retinas of newborn (within 24 h) rats as described previously with minor modifications.9, 18 Briefly, retinas were collected and washed twice with ice-cold PBS and digested with 0.125% trypsin at 37° C for 3 min. Trypsin was inactivated by adding culture medium (DMEM/F12 (1:1) with 10% fetal bovine serum (FBS),100 units/ml penicillin and 100 μg/ml streptomycin). Tissues were triturated with a plastic pipette and washed twice. Cells were filtered through a Nitex nylon mesh (mesh opening 100 microns; Sefar America Inc.), collected by centrifugation, resuspended in culture medium and plated onto 100 cm2 cell culture flasks at a density of 2 × 105 cells/cm2. All cultures were maintained in a humidified incubator at 37° C and 5% CO2 and fed on the third day, then once every 4 days. After 2 weeks, microglial cells were harvested in culture medium by shaking the flasks at 100 rpm for 1.5 h. The cell suspension was centrifuged and the detached cells re-plated in culture medium at designated densities for various experiments. The purity of the microglial cultures was ~98% as determined by immunocytochemical staining analysis for OX42, a macrophage/microglial marker. The morphology of microglia in culture was carefully examined by phase contrast and fluorescent microscopy.18

Quantitative Real-Time RT-PCR

Total RNA was isolated from rat retinal microglial cells using RNAqueous – 4PCR (Ambion). Subsequently, 1μg of total RNA was reverse transcribed using a mixture of random and poly-T primers according to the manufacturer’s protocol (Invitrogen). Primers were designed for the rat A1 (FWD, 5′- TCATTGCCTTGGTCTCTG-3′; REV, 5′- GTGGAAGTAGGTCTGTGG-3′), A2A (FWD 5′- TGCTTCCATCATGTCCTTGC-3′; REV, 5′- GCCCTTCGCCTTCATACC-3′), A2B (FWD, 5′-TTGGCATTGGATTGACTC-3′; REV, 5′- TATGAGCAGTGGAGGAAG-3′), and A3AR (FWD, 5′-TGCTTCCATCATGTCCTTGC-3′; REV, 5′- TGCTTCCATCATGTCCTTGC-3′) using Beacon Design software (Bio-Rad; Kreckler et al 2006). PCR amplification (in SYBR Green Supermix) was performed using an iCycler iQ thermocycler (Bio-Rad) for 40 cycles of 25 s at 95°C followed by 45 s at an optimized annealing temperature for each AR. The cycle threshold, determined as the initial increase in fluorescence above background, was ascertained for each sample. Melt curves was performed upon completion of the cycles to ensure that nonspecific products were absent. For quantification of AR transcripts, a standard curve plotting cycle threshold versus copy number was constructed for each receptor subtype by analyzing 10-fold serial dilutions of plasmids containing the full-length rat AR clones.19 AR transcript levels were expressed as copies/50 ng of total RNA.

Cell Treatment

Microglial cells collected from culture flasks were seeded at a density of 1 × 105 cells/well in 96-well plates. One day after seeding, the culture wells were washed with Cellgro Complete (Mediatech, Manassas, VA) and incubated in the same media with various treatments. Cells were pretreated with 1 μM CBD (Cayman Chemical, Ann Arbor, MI) or vehicle dimethylsulfoxide (DMSO) for 30 min at 37°C, followed by adenosine (Sigma, MO), AR agonists (all from Sigma) or AR antagonists (all from Sigma except ZM 241385) at the indicated concentrations for 30 min at 37°C, finally treated with 30 ng/ml LPS (Escherichia coli OB4:1111; Sigma). After the indicated time course, culture media were collected and assayed for TNF-α by ELISA. Subsequently, the cells were lysed with 0.4 N NaOH and assayed for total protein by the Bio-Rad DC protein assay.

Adenosine Uptake Assays

Assays were carried out at 37°C in Earle’s normal balanced salt solution (EBSS). Cells seeded at a density of 5 × 105 cells/well in 24-well plates were washed once in EBSS and pre-incubated 30 min at 37°C with drug or DMSO vehicle. Uptake began after addition of 0.5 μCi [2-3H]adenosine (37 MBq; Amersham, UK). Nonspecific uptake was defined as uptake in the presence of 1 mM adenosine, which was added 1 min before [3H]adenosine. After 1 min, buffer was rapidly aspirated, and cells were washed once with ice-cold PBS. Cells were solubilized in 0.2 M NaOH/1% SDS, and radioactivity was determined.

Enzyme-Linked Immunosorbent Assay (ELISA) for TNF-α

TNF-α levels in the supernatants of culture media were estimated with ELISA kits (R & D Systems, Minneapolis, MN) per the manufacturer’s instructions. Briefly, diluted samples were added to a microplate pre-coated with a monoclonal antibody specific for rat TNF-α. After washing to remove unbound substances, an enzyme-linked polyclonal antibody specific for TNF-α was added. Following a wash to remove unbound antibody-enzyme reagent, a substrate solution was added. The colored product yielded by the enzyme reaction was measured at 450 nm. The minimum detectable levels for TNF-α with this assay is 5 pg/ml. The sample levels were calculated from a standard curve. Subsequently, the cells were lysed with 0.4 N NaOH and assayed for total protein by the Bio-Rad DC protein assay. TNF-α released was expressed, after adjusted for protein, as a percentage of maximal TNF-α released from vehicle-treated cells. Retinal microglial cells were treated with LPS and culture media were collected at 5, 15, 20 and 30 min and 1, 2, 4, 6, 12 and 24 h after treatment and assayed for TNF-α. Levels of TNF-α began to increase significantly after 4 h of LPS treatment, peaking at the 6th h, then slowly leveled off within 24 h period. The levels of TNF-α in control culture media without LPS treatment did not change throughout the experimental period.18

Retinal and vitreous lysates from rat eyes used for TNF-α assay by ELISA was according to the manufacturer’s instructions (R&D) with modifications. Pooled retinas and vitreous from each animal were placed in 150 μl of lysis buffer [20 mM imidazole HCl, 100 mM KCl, 1 mM MgCl2, 1% Triton X-100, 10 mM NaF, 1 mM Na3VO4, 1 mM EGTA,1 mM EDTA, pH 6.8]20 supplemented with a protease inhibitor cocktail (Sigma) followed by homogenization in a Mini-Bead beater with treated Ottawa sand.5 The lysate was cleared of debris by centrifugation at 10,000 × g for 15 minutes (4°C), and 50 μl of the supernatant was used for ELISA directly without further dilution. The sample TNF-α levels were calculated from a standard curve, corrected for protein concentration and expressed as a percentage of maximal TNF-α released from vehicle and LPS-treated animals.

Data Analysis

The results are expressed as mean ± SEM. Differences among experimental groups were evaluated by analysis of variance, and the significance of differences between groups was assessed by Fisher’s PLSD post hoc test when indicated. Significance was defined as P < 0.05.

AR Expression in Retinal Microglial Cells

We first quantified mRNA expression of the four AR subtypes by real-time RT-PCR. The absolute copy numbers of the AR transcripts were calculated based on standard curves generated with AR cDNA clones.19 As illustrated in Fig. 1, we detected abundant mRNA expression of A2AAR (940±95 copies/50 ng of RNA) mRNA in rat retinal microglial cells. mRNA expression of A1AR, A2BAR or A3AR mRNA were barely detectable above background levels.

Figure 1

Expression of AR subtypes in retinal microglial cells. Expression of the four AR subtypes in rat retinal microglia in the form of mRNA levels as quantified by real-time RT-PCR (n=4 − 5).

A2AAR Regulation of TNF-α in Retinal Microglial Cells

To identify the AR subtype(s) involved in inhibiting TNF-α release in the retinal microglia in response to LPS stimulation, we examined the effect of the nonselective AR agonist adenosine-5’-N-ethylcarboxamide (NECA) in the presence of AR subtype-selective antagonists. The concentrations of each antagonist chosen for this study were based on the affinity and selectivity for the recombinant mouse AR subtypes determined by radioligand binding studies.19 As shown inFig 2A, cells pretreated with vehicle showed a 10-fold increase in LPS-induced TNF-α release compared to vehicle-treated control cells. Treatment with NECA at a concentration of 1 μM potently inhibited LPS-stimulated TNF-α release (40%). When the cells were pretreated with the A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (CPX) (100 nM), the A2BAR antagonist 8-[4-[((4-Cyanophenyl) arbamoylmethyl) oxy]phenyl]-1,3-di(n-propyl) xanthine hydrate (MRS 1754) (1 μM), or the A3AR antagonist 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate (MRS 1523) (10 μM), the inhibitory effect of NECA on TNF-α release was not affected. However, this effect was successfully blocked by 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl} phenol (ZM241385) at concentrations (100 and 500nM) capable of blocking A2AARs. These results suggest that NECA inhibited LPS-induced TNF-α release from rat retinal microglia via the A2AAR.

Figure 2

Functional identification of AR subtypes in retinal microglial cells. TNF-α production was determined in LPS-treated (30 ng/ml, 12 hours) retinal microglial cells. A. In the presence of nonselective AR agonist NECA (1 μM) and subtype-selective …

To confirm the role of the A2AAR in regulating TNF-α release, we compared concentration-response curves generated with the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2AAR agonist 2-p-[2-Carboxyethyl]phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680), and the A3AR agonist N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA). As revealed in Fig. 2B, treatment with CGS 21680 concentration-dependently inhibited LPS-stimulated TNF-α release. In contrast, IB-MECA and CCPA inhibited TNF-α release, but their potencies were low reflecting their low affinity for the A2AAR. Although, it is noticed that the potencies of IB-MECA and CGS 21680 were very similar. This is probably because the A3AR-selective IB-MECA can inhibit TNF-α release via the A2A and/or A2BAR in A2AAR-dominant systems.19 These results suggest a TNF-α release regulatory effect mediated by the A2AAR.

CBD Effects on Adenosine Uptake in Retinal Microglial Cells

CBD has the ability to enhance adenosine signaling through competitive inhibition of adenosine uptake by equilibrative nucleoside transporter 1 (ENT1) in murine microglia and macrophages.16To determine whether ENT1, which is S-(4-nitrobenzyl)-6-thioinosine (NBMPR) sensitive, is expressed in retinal microglial cells, we demonstrated NBMPR sensitivity in these cells. [3H]adenosine uptake assays were carried out in cells in the presence of 0.1 μM NBMPR. To specifically examine inward [3H]adenosine transport, we examined uptake during the first minute after [3H]adenosine addition. As shown in Fig. 3, [3H]adenosine uptake was partially inhibited by NBMPR, suggesting the presence of ENT1 in these cells. To determine whether CBD inhibits adenosine uptake in retinal microglial cells via ENT1, we determined the effect of CBD on [3H] adenosine transport in the presence of 0, 0.02, 0.1, and 0.5 μM NBMPR. NBMPR alone dose-dependently inhibited [3H] adenosine transport. CBD at 0.5 μM also inhibited [3H] adenosine uptake. NBMPR at all levels did not enhance this inhibition, suggesting CBD is competing with NBMPR for ENT1 (Fig. 3). To determine if this inhibitory effect is dependent of known cannabinoid receptors, HU-210, a synthetic cannabinoid agonist which has an affinity for CB1 and CB2 receptors in the picomolar range21 was tested for its ability to decrease [3H]adenosine uptake. HU-210 at 1 μM did not inhibit [3H]adenosine uptake (Fig. 3). These data suggest an adenosine uptake inhibitory effect of CBD independent of known cannabinoid receptors.

Figure 3

CBD or NBMPR, but not HU210, inhibits [3H]adenosine uptake in retinal microglial cells. Cells were pretreated with NBMPR (0, 0.02, 0.1, and 0.5 μM), CBD (0.5 μM), NBMPR combined with CBD, or HU210 (1 μM) for 30 min at 37°C, …

Adenosine Mediation of CBD Effects on TNF-α in Retinal Microglial Cells

We tested the hypothesis that the TNF-α-inhibiting effect of CBD is mediated through enhancement of adenosine. This was done by comparing concentration-response curves generated with CBD or adenosine alone, then with the two reagents combined. Cells that were pretreated with adenosine alone from 1.5 μM to 100 μM followed by LPS showed a gradual decrease in TNF-α from 100% to 50% (Fig. 4). Cells that were pretreated with CBD alone from 0.03 μM to 1 μM followed by LPS showed a more drastic decrease in TNF-α from 100% to 20%. The drastic TNF-α-reducing effect by CBD at or near 1 μM may be due to CBD’s capability of being an anti-oxidant to scavenge oxygen radicals.22 Interestingly, cells that were co-treated with adenosine from 6 μM to 100 μM and CBD from 0.06 μM to1 μM showed a decrease in TNF-α that was much more drastic than the effect of either agent alone. This effect is particularly obvious at low concentrations of adenosine and CBD where TNF-α-inhibiting effect was not observed for either reagent alone (Fig. 4). Taken together, these results suggest that by inhibiting adenosine uptake, CBD synergistically enhances adenosine’s TNF-α suppression upon LPS treatment.

Figure 4

CBD enhances adenosine’s ability for TNF-α inhibition. Retinal microglial cells were pretreated with CBD (0.03 − 1 μM) or vehicle for 0.5 hr. Cells were then treated with vehicle or adenosine (1.5 − 100 μM) for …

Adenosine Mediation of CBD Effects on TNF-α in the Retina

Adenosine uptake inhibitors decrease TNF-α in LPS-treated mice by increasing the amount of endogenous adenosine available to bind the A2AAR.15 Because CBD inhibits adenosine uptake, we hypothesized that the A2AAR mediates TNF-α inhibition seen with CBD treatment.5, 18 Rats were pretreated with CBD (1 mg/kg, i.p.) and with the A1AR antagonist CPX (3 mg/kg, i.p.) or the A2AAR antagonist ZM 241285 (10 mg/kg, i.p.) before LPS treatment (0.35 mg/kg, footpad). CBD-pretreated rats showed significantly decreased retinal TNF-α levels, compared with vehicle-pretreated rats (Fig. 5). Although this decrease remained unchanged upon co-treatment with CPX, pretreatment with ZM 241385 reversed the effects of CBD on TNF-α (Fig. 5). Neither antagonist alone significantly altered the effect of LPS to increase TNF-α levels (data not shown). These results correlate with the in vitro data obtained using microglial cells showing that CBD enhances the ability of adenosine to inhibit LPS-induced TNF-α release via the A2AAR.

Figure 5

CBD decreases TNF-α via activation of A2AAR in the retina. Male SD rats were pretreated before LPS injection with a single dose of CBD (1 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were given vehicle, CPX (3 mg/kg, i.p), or ZM 241385 …

We thank Dr. Wenbo Zhang for helpful comments and suggestions in this manuscript. This research was supported in part by grants from National Institutes of Health: HL 077707 (JAA), DA09155 and NS041314 (CJH), and from American Diabetes Association 1-06-RA-82 and Knights Templar Educational Foundation (GIL).

AR

adenosine receptor

ENT

equilibrative nucleoside transporter

CBD

cannabidiol

LPS

lipopolysaccharide

TNF-α

tumor necrosis factor-α

RT-PCR

reverse transcription-polymerase chain reaction

FWD

forward

REV

reverse

NECA

adenosine-5’-N-ethylcarboxamide

CPX

8-cyclopentyl-1,3-dipropylxanthine

ZM 241385

4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol

MRS 1754

8-[4-[((4-cyanophenyl) arbamoylmethyl) oxy]phenyl]-1,3-di(n-propyl) xanthine hydrate

MRS

15233-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate

CCPA

2-chloro-N6-cyclopentyladenosine

CGS 21680

2-p-[2-Carboxyethyl]phenethylamino-5′-N-ethylcarboxamidoadenosine

IB-MECA

N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide

NBMPR

S-(4-nitrobenzyl)-6-thioinosine