The CBR1 antagonist AM281 (1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide), the CBR2 antagonist AM630 (6-Iodo-2-methyl-1- [2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone) and the CBR2 agonist JWH015 ((2-Methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone) were obtained from Tocris, Ellisville, MI, USA. Drugs were diluted in dimethylsulfoxide (DMSO) and then in saline to a final concentration of 100 μM (5% DMSO). The final concentration of DMSO in cell culture with cannabinoid treatments was never higher than 0.05%, and this was used as the control group (medium control). JWH015 was chosen because we have shown in vivo that it selectively acts on spinal microglial CBR2 in L5 nerve transection rats and reduces spinal Iba-1 expression in association with its anti-allodynic effects . We chose the specific antagonists because we also showed in the same study that AM630 blocked JWH015's effects, while AM281 did not. JWH015 at 5 μM concentration has been shown to effectively reduce pro-inflammatory factors in microglia with CBR2 selectivity . We chose a maximum dose of 1 μM for JWH015 (and equimolar doses for the antagonists) to assure its specificity.
Since ERKs are the only known substrates of MEK, the MEK inhibitor, UO126 (Cell Signaling, Danvers, Massachusetts, USA) was used to produce ERK inactivation. PSI2106  (kindly provided by Dr. Key Brummon, University of Pittsburgh, Pennsylvania, USA), Ro-31-8220  and triptolide [28, 53, 54] were used as MKP-1 inhibitors. UO126, PSI2106 and triptolide (Sigma, St. Louis, Missouri, USA) were diluted in DMSO and then in saline to a final concentration of 100 μM (1% DMSO). The final concentration of DMSO in cell culture with these drug treatments was never higher than 0.01%, and this was used as control group (medium control). Ro-31-8220, minocycline, adenosine diphosphate (ADP) and lipopolysaccharide (LPS, 0111:B4 serotype) were diluted in saline (Sigma).
After approval by the Institutional Animal Care and Use Committee at Dartmouth College (Hanover, New Hampshire, USA), highly purified primary microglial cultures were prepared using postnatal day (P) 2–3 Harlan Sprague-Dawley pups (Indianapolis, Indiana, USA) as described previously [33, 47]. Briefly, pups were decapitated and the cerebral cortices were removed; meninges were dissected away; cortical tissue was minced with a sterile scalpel blade and digested with trypsin/EDTA 1× (Mediatech, Herndon, Virginia, USA) for 15 min at 37°C. The supernatant was discarded and 5 mL Dulbecco's modified Eagle's medium (DMEM; Mediatech) supplemented with 10% charcoal-stripped fetal bovine serum (FBS; Hyclone, Logan, Utah, USA), 1.1% GlutaMax (Gibco-Invitrogen, Carlsbad, California, USA), and 1% penicillin/streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin; Mediatech) containing 2000 U DNase (Sigma) were added to the tissue on ice. The tissue was triturated with a 5 mL pipette. The tissue clumps were allowed to settle and the supernatant was removed to a sterile 50 mL conical tube on ice between triturations. Triturations were repeated until no tissue clumps were observed. The final volume was diluted to 25 mL with media and centrifuged at 310 g for 15 min. The supernatant was discarded and the cells resuspended in media. A small aliquot of cells was stained for trypan blue (Sigma) exclusion and cells were plated at 1 × 106 cells per 75-cm2 flask. Cultures were maintained at 37°C and 5% CO2. Media was changed every 3–4 days. After 8 days in vitro (DIV 8), the flasks were confluent with astrocytes and microglia. Flasks were lightly shaken by hand for 1 min and the media containing microglia was removed and centrifuged at 310 g for 15 min. Cultures were found to be 99% microglia by staining with OX-42 antibody (generous gift from Dr William Hickey) a marker for the CR3/CD11b receptor.
Western blot analysis
Confluent DIV 8 primary microglia were shaken, counted and plated for at least 1.5 hr in serum free medium (SFM) at a density of 400 × 103 cells/mL. The cells were treated with different drugs (see below) using a time course incubation (15, 30, 60 and 120 min). Following treatments, the culture plates were briefly centrifuged; supernatants were removed and 60 μL of 1× Laemmli buffer (Bio-Rad, Hercules, California, USA) containing 2-mercaptoethanol (ME) (Sigma) was added to each well. Protein expression was assessed using western blot analysis. Briefly, samples and standard protein markers were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gels; Bio-Rad) and transferred to polyvinylidene difluoride (Bio-Rad) membranes. Non-specific binding was blocked by incubation with 5% bovine serum albumin in Tris-buffered saline-Tween 20 (0.05%; Sigma) at 22°C, then membranes were incubated overnight or for 36–40 hr at 4°C with rabbit anti-phospho-ERK 44/42 (phospho-mitogen-activated protein kinase, p-ERK, 1:500; Cell Signaling), mouse anti-total-ERK 44/42 (total mitogen-activated protein kinase, t-ERK, 1:1000; Cell Signaling), rabbit anti-MKP-1 (mitogen-activated protein kinase phosphatase-1, MKP-1, 1:400; Santa Cruz, California, USA), goat anti-MKP-3 (mitogen-activated protein kinase phosphatase-3, MKP-3, 1:400; Santa Cruz, USA) or rabbit anti-TNF (1:1000; Peprotech, Rocky Hill, New Jersey, USA). The next day (or 36–40 hr), blots were incubated for 1 h at 22°C with goat anti-rabbit, mouse or donkey anti-goat horseradish peroxidase-conjugated secondary antibodies (1:3000; Pierce, Rockford, Illinois, USA), visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) for 5 min and imaged using the Syngene G-Box (Synoptics, Frederick, Maryland, USA). After incubation with the first primary and secondary antibodies, three blots were incubated for 25 min at 37°C in stripping buffer. Blots were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) for 5 min and we observed that the antibodies were completely removed. Therefore, the same stripping procedure was used to re-probe with another primary and secondary antibodies. This methodology allowed us to observe the effects of the drug treatments in different proteins from the same treated cells. Finally, blots were subsequently stripped and re-probed with mouse anti-beta-actin antibody (1:3000; Abcam, Cambridge, Massachusetts, USA), and this was used as the protein loading control. Band intensity was assessed using the analysis software package provided with the Syngene G-Box and data were quantified as relative intensity of band of interest divided by intensity of beta-actin. Normalization of p-ERK and t-ERK were also conducted against beta-actin, then p-ERK data were quantified as relative intensity of band divided by intensity of t-ERK. Data were expressed as relative intensity normalized to beta actin control and to the control group for each experiment ± SEM. Incubation of blots with only secondary antibodies did not show any protein band.
The following treatment groups were performed. Medium control (time 0) or LPS (5 ng/ml) alone groups were used as the control groups to compare the effects of selective microglial CBR2 activation on MKP-1/3 (n = 5–7), t-ERK (n = 5–7), p-ERK (n = 5–7) and TNF (n = 4) using a group with LPS + JWH015 (1 μM). To test the specificity of the CBR2 agonist (JWH015), LPS + JWH015 (1 μM) group was compared to LPS + JWH015 (1 μM) + AM281 or AM630 (1 μM, n = 9) groups. The dependence of TNF production on p-ERK has been previously shown [21, 32]. To confirm this in our preparation, we used the MEK inhibitor, UO126 (1 μM, n = 6) in LPS-stimulated cells. Additionally, to probe that CBR2 activation reduces p-ERK by inducing MKP-1 and/or MKP-3, we challenged the effects of JWH015 (1 μM) in the presence of LPS with different drugs claimed to be MKP-1 inhibitors. Thus, the LPS + JWH015 (1 μM) group was compared with LPS + JWH015 (1 μM) + Ro-31-8220, triptolide or PSI2106 (1–10 μM, n = 3). All data were normalized to control groups which were given a value of 1.
Our laboratory has previously characterized and optimized the incubation times and chemoattractants that allow optimum cell migration susceptible to pharmacological modulation [33, 47]. We used Costar Transwell® plates (6.5 mm diameter insert, 8.0 μm pore size, polycarbonate membrane; Corning Inc., Corning, New York, USA). For this group of experiments, the bottom chamber of these plates contained 10 μM ADP, a potent microglial chemoattractant released from injured neurons [33, 41, 47, 55]. Confluent DIV 8 primary microglia were shaken, washed with PBS, counted using trypan blue, then placed in SFM (DMEM). Cells were resuspended at 100 × 103 cells in 200 μL SFM and treated with different drugs. To assess the effects of cannabinoid or other pharmacological treatments on primary microglia, cells were pre-treated with drugs for 2 h in the presence of LPS (5 ng/ml). Then, cells were centrifuged, medium containing the drugs was discarded and fresh SFM was added. Cells were counted using trypan blue to insure survival post-treatment (> 95% viability).
Cells were added to the top chamber (100 × 103 cells in 200 μL SFM) of a transwell plate with fibronectin-coated membranes with ADP (10 μM, 500 μL in SFM) in the bottom chamber. Cells were then allowed to migrate towards ADP for 1 or 2 h at 37°C and 5% CO2. Following migration, the medium in the top chamber was aspirated and the membrane gently wiped with a cotton swab to remove the cells that did not migrate. The membranes were first rinsed with PBS, the cells were then fixed with 2% formaldehyde in PBS, permeabilized with 0.01% Triton X-100 (Sigma) in PBS, and finally stained with crystal violet (Sigma). The membranes were then dried and mounted on microscope slides. Images of nine random fields (20× objective) for each membrane were captured via a Q-Fired cooled CCD camera attached to an Olympus microscope and counted by hand with aid of SigmaScan Pro imaging analysis software. Counts for all nine fields were averaged to give a mean cell count for each membrane. All experiments were completed at least three times (n = 3).
To study the effects of CBR2 activation on cell migration in LPS-stimulated microglia we used the following groups: LPS + JWH015 (0.01–1 μM, n = 9). To test the specificity of the CBR2 agonist, we challenged its effects with specific CBR1 (AM281) and CBR2 (AM630) antagonists by using the following groups: LPS + JWH015 (1 μM) + AM281 (1 μM, n = 4) or AM630 (1 μM, n = 6). To test whether p-ERK is involved in microglial migration we used a specific MEK inhibitor, UO126 (1 μM, n = 6) in LPS-stimulated cells. We also used a positive control group using LPS-stimulated microglia + minocycline (60 μM, n = 6), a dose that our laboratory has shown to be effective in reducing microglial migration in non-stimulated microglia . To further test whether JWH015's effects on MKP-1/3 were causatively linked with JWH015's effects on microglial migration, we used Triptolide (10 μM, 15 min pre-treatment, n = 6) in LPS-stimulated cells + JWH015 (1 μM) incubated for 2 hr in the migration well. It has been shown that JWH015 act as chemoattractant in human monocytes when used at 20 μM concentration in parallel to an induction in ERK phosphorylation. However, JWH015 does not induce chemotaxis at 5–10 μM concentrations, . Therefore, we chose 1 μM as our highest JWH015 dose tested.
Data are expressed as mean ± SEM. Statistical analyses were completed using GraphPad Prism 4 (GraphPad Software, Inc., San Diego, California, USA). The effects of LPS and drug on proteins measured by western blot analyses and cell migration were examined using the repetitive measurements one-way analysis of variance. If significant effects were found, Dunnett's test was conducted. When appropriate (non-normally distributed), these data were evaluated using the Friedman Repeated Measures Analysis of Variance on Rank test. If significant effects were found, non-parametric Wilcoxon signed ranks tests were conducted comparing each time point to the medium control group. Between group differences were examined using two-way analysis of variance. If differences were found, Bonferroni post test was used. When appropriate (non-normally distributed), between group differences were examined at each time period using the Kruskal-Wallis test. Significant effects were followed using the Mann-Whitney U test comparing only the novel treatment to control or agonist group. Data are presented as mean ± SEM. Significance was determined at a level of p < 0.05.