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Originally published online as doi:10.1189/jlb.0307159 on May 30, 2007

Published online before print May 30, 2007
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(Journal of Leukocyte Biology. 2007;82:532-541.)
© 2007 by Society for Leukocyte Biology

Anti-inflammatory property of the cannabinoid receptor-2-selective agonist JWH-133 in a rodent model of autoimmune uveoretinitis

Heping Xu*,1,2, Ching L. Cheng*,{dagger},1, Mei Chen*, Ayyakkannu Manivannan{ddagger}, Laurence Cabay*, Roger G. Pertwee§, Angela Coutts§ and John V. Forrester*

Departments of
* Ophthalmology and
{ddagger} Radiology, School of Medicine, and
§ School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom; and
{dagger} Singapore National Eye Centre, Singapore Eye Research Institute, Singapore

2 Correspondence: Department of Ophthalmology, School of Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK. E-mail: h.xu{at}abdn.ac.uk

ABSTRACT

Previous studies have shown that cannabinoids have anti-inflammatory and immune-modulating effects, but the precise mechanisms of action remain to be elucidated. In this study, we investigated the effect of JWH 133, a selective agonist for cannabinoid receptor 2, the main receptor expressed on immune cells, in a model of autoimmune disease, experimental autoimmune uveoretinitis (EAU). JWH 133 suppressed EAU in a dose-dependent manner (0.015–15 mg/kg), and the suppressive effect could be achieved in the disease-induction stage and the effector stage. Leukocytes from mice, which had been treated with JWH 133, had diminished responses to retinal peptide and mitogen Con A stimulation in vitro. In vivo JWH 133 treatment also abrogated leukocyte cytokine/chemokine production. Further in vitro studies indicated that JWH 133 down-regulated the TLR4 via Myd88 signal transduction, which may be responsible for its moderate, suppressive effect on antigen presentation. In vivo JWH 133 treatment (1 mg/kg) also suppressed leukocyte trafficking (rolling and infiltration) in inflamed retina as a result of an effect on reducing adhesion molecules CD162 (P-selectin glycoprotein ligand 1) and CD11a (LFA-1) expression on T cells. In conclusion, the cannabinoid agonist JWH 133 has a high in vivo, anti-inflammatory property and may exert its effect via inhibiting the activation and function of autoreactive T cells and preventing leukocyte trafficking into the inflamed tissue.

Key Words: antigen presentation • autoimmunity • leukocyte trafficking

INTRODUCTION

Cannabinoids are hydrophobic compounds, which are derived from the plant Cannabis sativa. The medicinal uses of cannabinoids began centuries ago for diseases such as asthma, migraine, pain, convulsions, and anxiety [1 ]. More recently, pharmacological interests have been focused on the putative, beneficial effect of natural and synthetic cannabinoids on appetite [2 ], glaucoma [3 ], pain [4 ], and inflammation [5 6 ]. The discovery of cannabinoid receptors (CBs) has greatly enriched our knowledge of the molecular basis of cannabinoid action on the CNS and immune systems [7 ].

To date, two CBs have been cloned and characterized. CB1 is expressed primarily by neurons [8 ] and CB2, by immune cells [9 ]. Besides, there is evidence supporting the presence of as-yet-unidentified CBs mainly on the basis of pharmacological activity of CBs in CB1- and CB2-deficient mice [10 ]. However, their physiological roles remain unknown. The CB2 is the main peripheral molecular target responsible for the inhibitory properties of the cannabinoids on the immune system [5 6 ]. Previous studies have shown that synthetic cannabinoids, through activation of the CB2, suppressed autoimmunity in various animal models including collagen-induced arthritis [11 ], experimental autoimmune encephalomyelitis [12 ], and virus-mediated, demyelinating disease [13 ]. However, a clear molecular mechanism underlying the immune-suppressive effect of CB2 activation has not been fully identified.

The aim of the present study is to investigate the mechanism underpinning the immune-suppressive actions of CB activation during the development of autoimmune diseases. To address this, we have used a CB2-selective agonist, 3-(1'1'-dimethylbutyl)-1-deoxy-{Delta}8-THC (JWH 133) [14 ], to activate the CB2 in vivo and in vitro. The consequences of CB2 activation on the immune system in vivo have been evaluated in mice in a model of autoimmune uveitis, namely interphotoreceptor retinoid-binding protein peptide (IRBPp)-mediated experimental autoimmune uveoretinitis (EAU), which is a model for the sight-threatening, human autoimmune disease, panuveitis [15 16 ]. EAU is a CD4 T cell-mediated autoimmune disease, which can be induced in rodents by challenge with retinal antigens or their peptides [17 18 ] or by systemic administration of in vitro-activated, retinal antigen-specific T cells [19 20 ]. Our data show that EAU is strongly inhibited when the CB2 is engaged and that the effects of CB2 engagement appear to be mediated predominantly through down-regulation of T cell function with a less-marked effect on antigen presentation.

MATERIALS AND METHODS

Animals and induction of EAU
Inbred B10.RIII mice and BALB/c mice, 8–10 weeks of age, were supplied by the Medical Research Facility at the Medical School, University of Aberdeen (UK). The procedures adopted conformed to the regulations of the Animal (scientific procedures) Act (UK) and to the Association for Research in Vision and Ophthalmology Statement for the use of Animals in Ophthalmic and Vision Research.

EAU induction was performed as described previously [21 22 ]. Briefly, B10.RIII mice were immunized s.c. with the IRBPp 161-180 (SGIPYIISYLHPGNTILHVD; purity >85%, Sigma-Aldrich, Cambridge, UK) at 100 µg, emulsified with an equal volume of complete Freund’s adjuvant (CFA; H37Ra, Difco Laboratories, Detroit, MI, USA) in a total volume of 100 µl. Control mice were immunized with the same volume of PBS instead of IRBP in CFA. In this model, EAU normally occurred at Days 8–9 and peaked at Days 14–15 [23, 24]. EAU was graded clinically, using previous protocols [25 ], as follows: 0 = normal; 0.5 = dilated iris blood vessels or few (one or two), small peripheral focal lesions in the retina; 1 = engorged iris vessels or mild vasculitis in the retina and less than five small focal lesions; 2 = hazy anterior chamber, decreased red reflex, and multiple chorioretinal lesions and/or infiltrates; 3 = moderately opaque anterior chamber but pupil still visible and dull-red reflex; 4 = opaque anterior chamber and obscured pupil, absence of red reflex, proptosis. At Scores 3 and 4, the fundus could not be observed through the opaque anterior chamber.

Administration of JWH 133
JWH 133 was prepared as described previously [26 ]. Briefly, JWH 133 was dissolved at 0.5 mg/ml in ethanol containing 1 mg/ml Tween 80 (Sigma-Aldrich). The ethanol was removed by vacuum-drying, and samples were reconstituted with PBS to different concentrations as required. In the initial experiment, JWH 133 was administered i.p., once daily, at the doses indicated (see Results) from Days 1 to 15 postimmunization (p.i.). Six to 10 animals were used in each group. In Experiment 2, JWH 133 was administered from Days 0 to 15 p.i. at the doses of 0.015 mg/kg and 0.15 mg/kg, respectively. Five mice were used in each group. In Experiment 3, JWH 133 was administered i.p. daily for two shorter time-periods, i.e., at the disease-induction phase from Days 0 to 7 p.i. or during the effector phase from Days 7 to 15 p.i. at the dose of 1.5 mg/kg. Five mice were used in each group in this experiment. In the control groups, mice were injected with the same amount of Tween 80. JWH 133 (1.5 mg/kg) was also administered to three nonimmunized, normal B10R III mice, once daily for 3 days to test the effect of JWH 133 on immune cell function in normal physiological conditions.

Histopathology
Eyes were removed and fixed in 2.5% (w/v)-buffered glutaraldehyde (Fisher Chemicals, Loughborough, UK) and embedded in resin for standard H&E staining. The intensity of uveoretinitis was evaluated histologically and graded by two independent observers. Grading was based on the histological grading system established in this laboratory [27 ].

Leukocyte trafficking in EAU
Intravital microscopy
Leukocyte trafficking in the inflamed retina was evaluated by a custom-built scanning laser ophthalmoscopy (SLO) as detailed previously [24 28 29 30 ]. Briefly, splenocytes from EAU mice were in vitro-labeled with calcein-AM (C-AM; Molecular Probes, Leiden, Netherlands) [30 ]. Day10 p.i. EAU-recipient mice were anesthetized with an intramuscular injection of 0.4 ml/kg hypnorm (Jenssen-Cilag, High Wycombe, UK) and i.p. with 1 mg/kg Diazepam (Phoenix Pharmaceuticals, Gloucester, UK). Sodium fluorescein [50 µL 0.5% (v/v), Sigma-Aldrich] was injected through the tail vein followed by 1 x 107 C-AM-labeled splenocytes. Trafficking of injected cells in the inflamed retina was recorded for 15 min. Mice then received 1 mg/kg JWH 133 injection through the tail vein and were imaged for another 20 min. Video analysis was conducted offline. Rolling cells and those not interacting with the endothelia were counted in each venule. Rolling cells were defined as those cells with a velocity below the critical velocity [24 28 ], which was calculated as described previously [28 31 ]. The rolling efficiency was calculated as the percentage of labeled, rolling cells among the total number of labeled leukocytes that entered a venule.

Confocal microscopy of retinal flat mounts
To evaluate the effect of JWH 133 on leukocyte transendothelial migration, C-AM-labeled splenocytes were treated in vitro with 1.5 mg/ml JWH 133 for 1 h before injection into previously IRBPp-immunized mice, which had developed EAU. Control cells were treated with vehicle (DMSO). Twenty-four hours later, mice were killed by CO2 inhalation, and retinal wholemounts were prepared as described previously [22 ]. Infiltrating cells were examined by confocal microscopy of retinal wholemounts [22 ].

MLR
A modified MLR was performed using purified populations of spleen APC and allogeneic CD4 T cells.

CD4+ T cell isolation
CD4+ T cells were isolated from 10-week-old, normal BALB/c (H-2d) mice spleens using MACS column-positive selection, according to the manufacturer’s protocol. Purity of the CD4+ T cells as detected by FITC anti-mouse CD4+ antibody (BD Biosciences, Oxford, UK) in flow cytometry was over 95%.

Preparation of APC
APC were obtained from normal B10R.III (H-2r) mice spleens after depletion of CD4 and CD8 T cells. Briefly, 1 x 108 splenocytes were incubated with 20 µl antibody mix (CD4 and CD8), followed by 100 µl Dynabeads® per 108 cells, which were then incubated for 20 min and placed in the Dynal MPC®. The supernatant (negatively isolated macrophages and B cells) was removed carefully and suspended in complete medium RPMI 1640. APC were then treated with 50 ug/ml mitomycin C (MMC; Sigma-Aldrich) for 20 min at 37°C. The cells were then washed three times with PBS to remove excess MMC.

MLR
MMC-treated APC (H-2r) were cultured in a 96-well plate with CD4 T cells (H-2d; APC:T cells ratio=1:10). After a further 48-h culture, T cell proliferation was determined by [3H]thymidine incorporation assay. To evaluate the effect of JWH 133 on antigen presentation, APC or T cells were pretreated with different doses of JWH 133 overnight before they were incorporated in the coculture system.

Proliferation assay
Proliferation assay was carried out using three types of cells: unpurified splenocytes, whole lymphocyte populations from inguinal draining lymph nodes, and purified inguinal lymph node CD4 T cells. Unpurified splenocytes and lymphocytes from inguinal draining lymph nodes were collected from IRBPp-immunized mice, treated with or without JWH 133. Cells (1x105) were incubated in 96-well plates and stimulated with 5 µg/ml Con A or 20 µg/ml IRBP in the presence or absence of varying concentration of JWH 133 (for cells from JWH 133-untreated mice). After 72 h culture, the cells were pulsed with 0.5 µCi/well [3H] thymidine overnight, and radioactivity was measured.

Bone marrow dendritic cell (BM-DC) preparation
BM-DC were prepared as described previously [32 ]. Briefly, cells were aspirated from mouse femurs and filtered (75 µm mesh) to obtain a single-cell suspension. RBCs were removed by incubating the cell suspension with ammonium chloride cell lysis buffer. Cells were plated at 2.0 x 106 per dish in a 100-mm microbiology culture dish at 37°C in complete RPMI medium containing GM-CSF (20 ng/ml). Eight days later, clusters with developing DC were harvested. Over 85% of DC prepared in this way were CD11b+CD11c+ in flow cytometry (data not shown). To induce DC maturation, 1 µg/ml LPS was added into 106 DC cultures and incubated overnight at 37°C. To evaluate the effect of JWH 133 on DC activation, maturation, and TLR expression, DC were incubated with different concentrations of JWH 133 in the presence of absence of LPS. Intracellular and cell surface markers were analyzed by flow cytometry.

For detection of apoptotic cells in BM-DC cultures, BM-DC were cultured in 24-well plates in complete RPMI-1640 medium containing 10% FCS in the presence or absence of various concentration of JWH 133 for 2 h. The cells were harvested, washed twice with PBS, and stained with an Annexin V/propidium iodide (PI) kit (BD Biosciences), according to the manufacturer’s protocol. Apoptotic cells were analyzed by flow cytometry.

Flow cytometry
Cells from tissues or cultures were analyzed by flow cytometry. Cells (1x105 cells per sample) were washed with PBS, incubated with 2% (w/v) BSA/PBS to block nonspecific binding, followed by FITC-, R-PE-, APC-, or PerCP-conjugated anti-mouse antibodies to CD3, CD4, CD8, CD69, CD25, CD44, CD80 (B7-1), CD86 (B7-2), CD40, CD11a (LFA-1), CD11c, CD11b, CD162 [P-selectin glycoprotein ligand 1 (PSGL-1)], and MHC II antigen (all purchased from BD Biosciences) and TLR2, TLR3, and TLR4 (all from eBioscience, San Diego, CA, USA), and analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA) using CELLQuest Pro software (Becton Dickinson). To detect intracellular protein MyD88 in DC after cell surface CD11b, CD11c staining, DC were fixed with 1% (w/v) paraformaldehyde (Agar Scientific Ltd., Cambridge, UK) at 4°C for 30 min and then permeabilized with 0.3% (v/v) Triton at room temperature for 10 min. Cells were then stained with anti-MyD88 (Abcam Ltd., Cambridge, UK) followed by FITC-conjugated goat anti-rabbit Ig.

Cytokine measurement
Splenocytes or lymph node cells from EAU control or JWH 133-treated mice were cultured in 96-well plates in the presence or absence of 25 µg/ml IRBP 161-180 peptide for 24 h. Supernatants were collected and analyzed for TNF-{alpha}, IFN-{gamma}, IL-4, IL-6, IL-10, IL-12, and chemokine CCL2 using the FACS cytometric bead assay (CBA) kit (BD Biosciences).

Data analysis
Grading of EAU (clinical and histological grades) was analyzed using the Mann-Whitney test. Each mouse (average of both eyes) was treated as one statistical event. T cell proliferation, cytokine production, and cell surface antigen expression data were analyzed by Dunnett’s multiple comparison test (dose-response studies) or Student’s t-test (time-course studies). Probability values of P < 0.05 were considered statistically significant.

RESULTS

Effect of JWH 133 on development of EAU
The effect of JWH 133 on EAU was first tested at doses of 0.15 mg/kg, 1.5 mg/kg, and 15 mg/kg by i.p. administration from Days 0 to 15 p.i. with IRBPp. Clinical signs of EAU were markedly suppressed by JWH 133 at all doses used (Fig. 1A ). To determine the minimal effective dose of JWH 133 for suppression of EAU, a second experiment was carried out at the doses of 0.015 mg/kg and 0.15 mg/kg i.p. injection from Days 0 to 15 p.i. As shown in Figure 1B , the administration of 0.015 mg/kg JWH 133 had a weak suppressive effect. Examination of H&E-stained sections from Day 16 p.i. mice showed that all EAU control mice exhibited signs of severe inflammation, including massive inflammatory cell infiltration in all layers of eyes, retinal detachment and folding, vasculitis, and granuloma (Fig. 1C) , whereas in JWH 133 (0.015–0.15 mg/kg)-treated mice, the retinal inflammation was much less than that of control mice with only a few foci of inflammatory cell infiltration and small granulomas (Fig. 1C) . The overall retinal structure was better-preserved (Fig. 1C) . The mean infiltrative score using a histological, cellular infiltration-grading system [27 ] was reduced in a dose-dependent manner in JWH 133-treated groups (Fig. 1D) . There was no obvious side-effect noticed in any drug-treated animals during the experimental period.


Figure 1
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  		Figure 1. The effect of JWH 133 on EAU development. (A, B) Clinical score; (D) histological, infiltrative score at Day 16 p.i. Mice were treated with different doses of JWH 133 daily from Days 0 to 15 p.i. Each point represents one mouse (average of both eyes). The average of each group is denoted by a horizontal bar. *, P < 0.05; **, P < 0.01, by Mann-Whitney compared with control group; #, no animal developed detectable, clinical EAU. (C) Histopathologic changes in B10R.III mice eyes with standard H&E staining. C-i, A control EAU eye showing massive inflammatory cell infiltration, a granuloma (arrow), and vasculitis (star). C-ii, An eye from a JWH 133 (0.015 mg/kg)-treated mouse showing few cell infiltrations, a small granuloma (arrow), and relatively normal retinal structure. Vi, Vitreous; GL, ganglion layer; INL, inner nuclear layer; ONL, outer nuclear layer; Ch, choroid; Sc, sclera. Original magnification, x65. Non-Tr, no treatment.

 
To further clarify the activation phase of its immunosuppressive effect, JWH 133 was administered at two different time periods relative to immunization with IRBPp: Days 0–7 p.i., i.e., during the phase of antigen presentation (afferent phase), and Days 7–15 p.i., i.e., during the effector phase of T cell activation (efferent phase of EAU). Figure 2 shows that in both groups, the EAU was suppressed significantly but not completely (Fig. 2A and 2B) by JWH 133 treatment at a dose of 1.5 mg/kg.


Figure 2
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  		Figure 2. The suppressive effect of JWH 133 on different phases of EAU. (A) Clinical score; (B) histological, infiltrative score at Day 16 p.i. JWH 133 (1.5 mg/kg) was injected at afferent phage (Days 0–7 p.i.) or efferent phage (Days 7–15 p.i.). Each point represents one mouse (average of both eyes). The average of each group is denoted by a horizontal bar. *, P< 0.05; **, P < 0.01, by Mann-Whitney compared with control group; {dagger}, P< 0.05, compared with Days 0–7 p.i. group.

 
Effect of in vivo JWH 133 treatment on immune cell functions
Splenocytes and lymphocytes from draining lymph nodes of Day 15 p.i. EAU control mice and IRBPp-immunized, JWH 133-treated (1.5 mg/kg per day, from Days 0 to 15 p.i.) mice were stimulated in vitro with IRBPp. Figure 3A shows that lymphocytes from JWH 133-treated mice failed to respond to in vitro IRBP stimulation. However, this effect was not antigen-specific, as the response to Con A stimulation was also reduced significantly (Fig. 3A) . Splenocytes from the JWH 133-treated mice showed similar responses (data now shown). It is interesting that T cells from IRBP-immunized mice treated from Days 0 to 7 p.i. or from Days 7 to 15 p.i. with JWH 133, in which EAU was suppressed only partially, also showed significantly impaired in vitro responses to IRBPp and Con A stimulation (Fig. 3B) , although the effect was less marked in the Days 0–7-treated group to Con A stimulation (Fig. 3B) .


Figure 3
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  		Figure 3. The effect of JWH 133 on T cell proliferation. (A) IRBPp-immunized mice were treated with JWH 133 (1.5 mg/kg) from Days 0 to 15 p.i. (B) IRBPp-immunized mice were treated with JWH 133 from Days 0–7 p.i. or Days 7–15 p.i. (C) Normal, nonimmunized mice were treated with JWH 133 (1.5 mg/kg) daily for 3 days. T cell proliferation in response to IRBPp (20 µg/ml) or Con A (5 µg/ml) was tested in vitro. (D) T cells from normal, nonimmunized mice were treated in vitro with different concentrations of JWH 133 in the presence of Con A for 3 days. Cell proliferation was analyzed. *, P < 0.05, compared with the control group. Data represent one of two (C) or three (D) repeated experiments.

 
The attenuated cell proliferation was also observed in normal, nonimmunized, JWH 133-treated mice (Fig. 3C) , although the treatment did not affect the expression levels as well as the ratios of CD3, CD4, CD8, CD25, CD44, CD86 (B7-2), CD11c, CD11b, and MHC II molecules in cells isolated from the spleen and lymph nodes of JWH 133-treated, normal mice (data not shown). Reduced cell proliferation was also observed when the leukocytes were exposed to JWH 133 in vitro (Fig. 3D) .

The impaired T cell-proliferative responses in leukocytes from JWH 133-treated mice were accompanied by marked reductions in cytokine production. Splenocytes from IRBP-immunized mice treated with JWH 133 (1.5 mg/kg i.p.) from Days 0–7 p.i. or Days 7–15 p.i. produced neither Th1 (IFN-{gamma}, TNF-{alpha}) nor Th2 (IL-6, IL-10) cytokines, even with IRBP restimulation (Fig. 4 ). They also produced no chemokine CCL2 (Fig. 4) . Cells from draining lymph nodes showed similar results (data not shown).


Figure 4
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  		Figure 4. The effect of JWH 133 on leukocyte cytokine and chemokine production. Mice were immunized with IRBPp 161-180 and were treated with 1.5 mg/kg JWH 133 daily from Days 0–7 p.i. or Days 7–15 p.i. Control mice were treated with the same amount of Tween 80. Spleen cells were collected at Day 16 p.i. and cultured in vitro for 24 h in the presence or absence of 25 mg/ml IRBPp. Cytokines and chemokines in the supernatants were measured by DBA. n = 3–6 mice; *, P < 0.05; **, P < 0.01, compared with the control group; #, no detectable level of cytokines in that group.

 
Effect of JWH 133 on antigen presentation
APC–T cell interactions play a major role in autoimmunity. The impaired, immune responses in JWH 133-treated mice, particularly in the animals treated only during the afferent phase (Days 0–7 p.i.), raised the possibility that JWH 133 may suppress antigen presentation. Previous studies have shown that CB2 is expressed in all leukocytes, and the expression level is higher in APC (B cells, monocytes, and macrophages) than in T cells [33 ]. We have therefore further investigated this possibility separately at the APC and T cell level in an in vitro antigen-presentation model (MLR, see Materials and Methods). Figure 5A shows that pretreatment of B10R.III (H-2r) mouse APC with JWH 133 had a moderate but statistically significant, negative effect on their ability to induce a one-way MLR in BALB/c (H-2d) T cells. A similar reduction was observed when BALB/c T cells were pretreated with JWH 133, but the suppressive effect was not statistically significant (Fig. 5B) . The above results suggest that JWH 133 has some effect on antigen presentation via the APC.


Figure 5
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  		Figure 5. The effect of JWH 133 on antigen presentation in the MLR study. (A) APC from B10R.III (H-2r) mice were pretreated with 24 µM JWH 133 before coculturing with normal CD4 T cells from BALB/c (H-2d) mice. (B) CD4 T cells from BALB/c mice were pretreated with 24 µM JWH 133 before coculturing with APC from B10R.III mice. n = 3. *, P < 0.05, compared with control group. Student’s t-test. Data represent one of two repeated experiments.

 
The effect of JHW 133 on BM-DC cell phenotype and function
To understand the mechanism of JWH 133 on suppressing antigen presentation, we further evaluated the effect of JWH 133 on DC, the major APC in adaptive immunity. JWH 133 treatment did not affect costimulatory molecule expression in BM-DC (Supplemental Fig. 1) but suppressed LPS-induced up-regulation of TLR4, a critical receptor for DC maturation and activation [34 ] in BM-DC, significantly (Fig. 6A and 6B ). To evaluate whether JWH 133 could also suppress TLR4 signal transduction, BM-DC were incubated with different concentrations of JWH 133 together with LPS for 2 h. Expression of the intracellular signal transduction protein MyD88 was analyzed by confocal microscopy as well as by flow cytometry. Confocal microscopy shows that MyD88 proteins are granule-like and distribute in the cytoplasm (Fig. 6 C). In some cells, the protein is more pronounced at the subcytoplasm membrane area (Fig. 6C) . Statistical analysis of the MFI acquired from flow cytometry showed that short-term LPS stimulation markedly up-regulated the MyD88 protein expression in CD11c BM-DC (Fig. 6D) . JWH 133 at the concentration of 4.8 µM almost completely blocked LPS-induced MyD88 up-regulation (Fig. 6D) .


Figure 6
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  		Figure 6. The effect of JWH 133 on BM-DC TLR4 and MyD88 expression. BM-DC were incubated with different concentrations of JWH 133 in the presence or absence of 1 µg/ml LPS for 2 h. The expression of TLR4 and cellular MyD88 was analyzed by flow cytometry. (A) Histogram of TLR4 expression in BM-DC, with and without LPS stimulation and in the presence or absence of 4.8 µM JWH 133. (B) MFI of TLR4 in BM-DC. (C) Confocal image of BM-DC stained with CD11c (red) and MyD88 (green) showing granule appearance of MyD88 in cytoplasm. One CD11chigh cell expressed few MyD88 (stars), whereas another MyD88high cell expressed a low level of CD11c (arrowheads). (D) MFI of MyD88 in CD11c+ DC. n = 3; *, P < 0.05; **, P < 0.01 (in B and D), compared with LPS-only-treated controls. Dunnett’s multiple comparison test (B). Student’s t-test (D).

 
A previous investigation has shown that other cannabinoid agonists such as {Delta} 9-tetrahydrocannabinol (THC) could induce DC apoptosis through CB1 and CB2 signaling [35 ]. To investigate whether the reduced expression of TLR4 in JWH 133-treated DC is a result of DC apoptosis, the effect of JWH 133 on BM-DC apoptosis was evaluated. The results showed that JWH 133 did not induce BM-DC apoptosis at the concentrations tested (4.8–48 µM; Supplemental Fig. 2).

Effect of JWH 133 on leukocyte trafficking
The above studies indicate that the cannabinoid agonist JWH 133 has clear effects on the induction of activated, autoreactive T cells, which may contribute to its immunosuppressive effect in the initial stages of the disease. However, the initial in vivo data (Fig. 2 above) also suggested a significant role for cannabinoids in inhibiting the efferent phase, as treatment of animals with JWH 133 7 days after priming was still effective in inhibiting EAU. Although some of this effect may have been a result of continued in situ antigen presentation and T cell activation, we wished to determine whether there was an additional component as a result, predominantly, of the efferent response, particularly in the recruitment of T cells to the site of inflammation. We have therefore investigated the effect of JWH 133 on leukocyte trafficking in EAU. As shown in Figure 7A , administration of JWH 133 reduced leukocyte rolling markedly in retinal vessels in vivo in real time. Furthermore, infiltration of leukocytes into the inflamed retina was also reduced significantly by JWH 133 treatment as determined by analysis of flat-mount, retinal preparations (Fig. 7B) .


Figure 7
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  		Figure 7. The effect of JWH 133 on leukocyte trafficking. (A) Leukocyte rolling efficiency in EAU mouse retina before and after JWH 133 treatment. C-AM-labeled splenocytes were injected into Day 10 p.i. EAU mice through the tail vein. Leukocyte rolling was observed by SLO before and after JWH 133 (1 mg/kg) treatment. (B) The number of infiltrating leukocytes in EAU mouse retinae. C-AM-labeled spenocytes were treated with JWH 133 (4.8 µM) or vehicle (DMSO) and then were injected into Day 10 p.i. EAU mice. Infiltrating cells were observed by confocal microscopy of retinal wholemounts 24 h later. (C, D) Splenocytes from normal mice were treated with 4.8 µM JWH 133 in the presence or absence of 5 µg/ml Con A for 16 h; cell surface adhesion molecule expression was analyzed by flow cytometry; (C) splenocytes without Con A activation; (D) splenocytes with Con A activation. n = 3; *, P < 0.05; **, P < 0.01, compared with control cells. Student’s t-test. Data in C and D represent one of three repeated experiments. In each experiment, samples were triplicated.

 
In an attempt to understand the mechanism by which JWH 133 suppressed leukocyte trafficking, the expression of the cell surface adhesion molecule was evaluated by flow cytometry. As shown in Figure 7 , JWH 133 suppressed Con A-induced up-regulation of CD11a (LFA-1, an adhesion molecule responsible for leukocyte transendothelial migration) [28], CD162 (PSGL-1, an adhesion molecule responsible for leukocyte rolling and important for trafficking in EAU) [28 ], and CD25 (IL-2R) expression.

DISCUSSION

It is well recognized that cannabinoids have immune regulatory properties, and they have been proposed as therapeutic drugs for various autoimmune diseases [5 6 ]. However, the mechanism regarding their immune regulatory properties has not been defined fully. Although CB1 and CB2 have been detected in the immune system [6 ], CB2 is particularly abundant [36 ]. In the present study, we have evaluated the effect of CB2 activation on the immune system by using JWH 133, a synthetic, CB2-selective cannabinoid agonist [14 37 ]. Using an experimental model of EAU, we demonstrated that JWH 133 induced a strong, suppressive effect in the immune system and suppressed EAU in a dose-dependent manner. Leukocytes from JWH 133-treated mice had impaired immune responses to retinal antigen and mitogen Con A stimulations. They also had diminished ability to produce Th1 and Th2 cytokines and chemokines and to sustain capacity to migrate into the inflamed retinal tissue. JWH 133 has a high potency (inhibitor constant=3.4 nM) and selectivity (200-fold more binding affinity over CB1) to CB2 [38 ]. Previous studies have shown that the effect JWH 133 on attenuating inflammatory and neuropathic pain [39 40 ] and suppressing microglial activation [41 ] is CB2-mediated. As the CB2 is expressed predominately on immune cells, and activation of CB2 has been shown to be able to suppress inflammation in other autoimmune disease models [11 12 13 ], we believe that the immune-suppressive effect of JWH 133 on EAU observed in this study is mediated mainly by the CB2 pathway. However, as JWH 133 is CB2-selective but not CB2-specific, further experiments using CB2 and/or CB1 knockout mice are required to confirm this.

The lack of response in leukocytes from JWH 133-treated mice to retinal antigen indicated that JWH 133 inhibited the induction of activated, autoreactive T cell formation in vivo. In an attempt to understand the mechanism of this inhibitory action, we examined the effect of JWH 133 on antigen presentation, a critical step to generate autoreactive T cells. The in vitro study revealed a weak but statistically significant, suppressive effect of JWH 133 on antigen presentation at the APC level. Previous studies have shown that THC-type cannabinoids suppressed macrophage-mediated antigen presentation [42 ], mainly through reducing macrophage costimulatory activity [43 44 45 ]. A more recent study has shown that THC suppresses costimulatory molecules CD40, CD86, and MHC II expression in Legionella pneumophila-infected BM-DC and reduces their stimulatory ability in culture-primed CD4+ T cells [46 ]. In the current study, however, the costimulatory molecules CD40, CD86, and MHC II were not affected by JWH 133 in vitro; instead, TLR4 and its signaling protein Myd88, a critical receptor/molecule for DC activation and maturation, were down-regulated, which may contribute to its in vivo, immune-suppressive effect at the disease-induction stage (i.e., antigen presentation).

TLRs are critical for linking innate and adaptive immunities [47 ]. Recognition of invading pathogens by TLRs expressed on host phagocytes triggers cytokine production and up-regulation of costimulatory molecules, which ultimately leads to T cell activation. TLR4 is one of the main receptors for LPS and is critical for the recognition of gram-negative bacteria, Chlamydia, as well as some endogenous ligands such as heat shock protein (HSP60 and HSP72), fibronectins, heparan sulfate, and oligosaccharides of hyaluronic acid [47 ]. The finding that JWH 133 down-regulates TLR4 expression in DC may account for the well-recognized, decreased resistance to bacterial, protozoan, and viral infections in chronic users of cannabinoids [48 49 ] and warrants further investigation. In addition, it may account for some of the effect of JWH seen here in EAU, as in parallel studies, we have observed that treatment of mice with LPS-activated DC (mDC) can prevent induction of EAU, provided TLR4 expression is down-regulated. LPS signaling through CD14, however, remains intact (Annie Lau, manuscript in preparation).

The anti-inflammatory property of JWH 133 on the effector stage may be attributable predominantly to a direct effect on leukocyte trafficking, particularly T cells. Previous studies have shown that activation of the CB2 inhibits macrophage [50 ], Jurkat T cell [51 ], and CD8 T cell [52 ] chemotaxis in vitro. In this study, we extend these findings to the in vivo model using the SLO combined with confocal microscopy of retinal wholemounts and show that vascular rolling and infiltration of leukocytes into the inflamed retina were reduced by JHW 133 treatment. This is in line with previous studies in the brain [53 54 ], in which the authors showed that leukocyte rolling was attenuated by CB2-selective agonists (O-3853, O-1966) [54 ], as well as a CB1/CB2 agonist, Win 55212-2 [53]. In the present study, it appears that JWH 133 inhibited leukocyte trafficking through suppression of adhesion molecules PSGL-1 and LFA-1 expression. We showed previously that CD162 (PSGL-1) is important for Th1 cell trafficking in the inflamed retina [28 ]. CD162 is also a critical molecule for monocyte trafficking [55 56 ]. CD11a (LFA-1), although not involved in leukocyte rolling [28 ], is critical for transendothelial migration of activated leukocytes into normal retina [29 ] and also important for Th1 and Th2 cell transendothelial migration in inflamed retina [28 ]. However, whether the antitrafficking property of JWH 133 is retina-specific or leukocyte subset-specific requires further investigation.

In summary, in this study, we demonstrate that JWH 133 has a high in vivo, immunosuppressive effect in our EAU model. The mechanisms underlying the immunosuppressive effect include, but are not limited to, suppression of antigen presentation through TLR4 signaling down-regulation and inhibition of leukocyte trafficking. More experiments are needed to further establish underlying mechanisms for its in vivo effects and to explain its high in vivo potency. It has been shown that cannabinoids such as THC could suppress Th1-biasing activities and enhance Th2-biasing activities [57 58 59 ]. A more recent study showed that JWH 133 (10 nM–5 µM) suppressed IL-12p40 and enhanced IL-10 production in mouse macrophages induced by LPS [60 ]. As IL-12p40 is critical for the development of EAU [61], and IL-10 could suppress EAU [62 63 64 ], this may also be one of the mechanisms responsible for the effect of JWH 133 on EAU. It thus appears that cannabinoids have widespread effects at several levels of induction of the innate and the linked adaptive immune responses.

ACKNOWLEDGEMENTS

This work is supported by the Development Trust of University of Aberdeen. H. X. is a Research Council UK (RCUK) fellow supported by the Department of Trade and Industry and Office of Science and Technology (DTI/OST). The authors thank Lesley Stevenson for technical support in preparing JWH 133 for in vivo studies, Morgan Blaylock and Linda Duncan for helping with the CBA assay, and Annie Lau for some of the BM-DC cultures.

FOOTNOTES

1 These authors contributed equally to this work. Back

Received March 14, 2007; revised April 20, 2007; accepted April 23, 2007.

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