Cannabinoid inhibition of macrophage migration to the trans-activating (Tat) protein of HIV-1 is linked to the CB(2) cannabinoid receptor

Abstract

Macrophages and macrophage-like cells are important targets of HIV-1 infection at peripheral sites and in the central nervous system. After infection, these cells secrete a plethora of toxic factors, including the viral regulatory trans-activating protein (Tat). This protein is highly immunogenic and also serves as a potent chemoattractant for monocytes. In the present study, the exogenous cannabinoids delta-9-tetrahydrocannabinol (THC) and (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55940) were shown to significantly inhibit migration of human U937 macrophage-like cells to the Tat protein in a concentration-related manner. The CB(1) receptor-selective agonist N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA) had no effect on Tat-mediated migration. In contrast, the CB(2) receptor-selective agonist (1R,3R)-1-[4-(1,1-dimethylheptyl)-2,6-dimethoxyphenyl]-3-methylcyclohexanol (O-2137) exerted a concentration-related inhibition of U937 cell migration in response to Tat. Pharmacological blockage of CB(1) receptor signaling using the antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide hydrochloride (SR141716A) had no effect on CP55940-mediated inhibition of macrophage migration to Tat, whereas treatment with the CB(2) receptor antagonist (1S-endo)-5-(4-chloro-3-methylphenyl)-1-((4-methylphenyl)methyl)-N-(1,3,3-trimethylbicyclo(2.2.1)hept-2-yl)-1H-pyrazole-3-carboxamide (SR144528) reversed the CP55940-mediated inhibition of migration. In addition, THC had no inhibitory effect on U937 migration to Tat after small interfering RNA knockdown of the CB(2) receptor. Collectively, the pharmacological and biochemical knockdown data indicate that cannabinoid-mediated modulation of macrophage migration to the HIV-1 Tat protein is linked to the CB(2) cannabinoid receptor. Furthermore, these results suggest that the CB(2) cannabinoid receptor has potential to serve as a therapeutic target for ablation of HIV-1-associated untoward inflammatory response.

Fig. 1.

U937 human macrophage-like cells express cannabinoid receptor CB₂. A, products from real-time SYBR Green RT-PCR showing the presence of message for the CB₂ receptor (middle) and the absence of message for the CB₁ receptor (top) in U937 cells. An amplification product of 185 bp was generated for the CB₂ receptor. Constitutively expressed GAPDH product was used as a positive internal control (bottom). B, Western immunoblot analysis (top) illustrating that CB₂ receptor protein levels are not altered by treatment with Tat (50 nM), THC (1 μM), or THC + Tat (4 h). Bottom, the 43-kDa actin band on the Coomassie-stained gel used for electroblotting that served as a loading control.

Fig. 2.

HIV-1 protein Tat induces migration of U937 human macrophage-like cells. Migration of U937 cells to Tat (25–100 nM) was assessed in vitro (2 h) using Transwell tissue culture inserts. Results are representative of three experiments and are presented as the mean ± S.D. **, p < 0.01.

Fig. 3.

Treatment in vitro with THC results in inhibition of macrophage migration to the HIV-1 Tat protein. Migration of U937 human macrophage-like cells to Tat (50 nM) was assessed using Transwell migration assay after in vitro treatment (3 h) with THC (1 μM–10 nM) or vehicle (0.01% ethanol). Statistical analysis was performed by comparing the number of migrating cells in THC treatment conditions with vehicle-treated cells exposed to Tat in the bottom chamber (positive migration control, designated by solid horizontal line). Solid box at abscissa = 0 represents the baseline migratory control of vehicle-treated cells exposed to serum-free complete assay medium in the bottom chamber. Results are representative of three experiments and are presented as the mean ± S.D. *, p < 0.05.

Fig. 4.

Treatment in vitro with CP55940 results in inhibition of macrophage migration to the HIV-1 Tat protein. A, migration of U937 human macrophage-like cells to Tat (50 nM) was assessed using Transwell migration assay after in vitro treatment (3 h) with CP55940 (1 μM–1 nM) or vehicle (0.01% ethanol). Statistical analysis was performed by comparing the number of migrating cells treated with CP55940 to vehicle-treated cells exposed to Tat in the bottom chamber (positive migration control, designated by the solid horizontal line). Solid box at abscissa = 0 represents the baseline migratory control of vehicle-treated cells exposed to serum-free complete assay medium in the bottom chamber. Results are representative of three experiments and are presented as the mean ± S.D. *, p < 0.05; **, p < 0.01. B, the EC₅₀ of THC and CP55940 was determined by subtracting the baseline migratory control from each group, followed by normalizing each drug treatment group to represent percentage of maximal migration designated by vehicle-treated cells exposed to Tat in the bottom chamber.

Fig. 5.

Effect of CB₁ and CB₂ receptor-selective agonists on macrophage migration to Tat. A, treatment with the CB₂ receptor-selective agonist O-2137 (3 h) significantly inhibits U937 migration to Tat. Migration of U937 human macrophage-like cells to Tat (50 nM) was assessed using Transwell migration assay after in vitro treatment (3 h) with O-2137 (1 μM–1 nM) or vehicle (0.01% ethanol). Results are representative of three experiments and are presented as the mean ± S.D. B, treatment with the CB₁ receptor-selective ligand ACEA has no effect on Tat-induced macrophage migration. Statistical analysis was performed by comparing the number of migrating cells treated with cannabinoid to vehicle-treated cells exposed to Tat in the bottom chamber (positive migration control, designated by solid horizontal line). Solid box at abscissa = 0 represents the baseline migratory control of vehicle-treated cells exposed to serum-free complete assay medium in the bottom chamber. Results are representative of three experiments and are presented as the mean ± S.D. *, p < 0.05; **, p < 0.01.

Fig. 6.

Effect of cannabinoid receptor antagonists on migration to Tat. A, treatment with the CB₁ receptor antagonist SR1 has no effect on CP55940-mediated inhibition of macrophage migration to Tat. U937 macrophage-like cells were pretreated with antagonist (1 μM, 30 min) before being treated with CP55940 (1 μM–1 nM; 3 h). Migration to Tat (50 nm) was assessed using Transwell migration assay (2 h). Results are representative of three experiments and are presented as the mean ± S.D. B, the CP55940-mediated inhibition is reversed by treatment with the CB₂ receptor antagonist SR2. Statistical analysis was performed by comparing the number of migrating cells treated with cannabinoid (●; significant difference designated by asterisks), antagonist alone (▴), or cannabinoid plus antagonist (■; significant difference designated by δ) to vehicle-treated cells exposed to Tat in the bottom chamber (positive migration control, designated by solid horizontal line), as well as comparing the number of cannabinoid-treated migrating cells (●) with those pretreated with antagonist plus cannabinoid (□, SR1; ■, SR2; significant difference designated by α). Inverted solid triangle (▾) at abscissa = 0 represents the baseline migratory control of vehicle-treated cells exposed to serum-free complete assay medium in the bottom chamber. Results are representative of three experiments and are presented as the mean ± S.D. *, α, δ, p, < 0.05; **, αα, δδ, p < 0.01.

Fig. 7.

RNA interference of the CB₂ receptor blocks THC-mediated inhibition of migration to Tat. A, knockdown of CB₂ receptor protein expression. U937 cells were transfected with CB₂ receptor (ΔCB₂) or negative control (Neg) siRNA (25 and 50 nM) and assessed for CB₂ receptor protein expression by Western immunoblot analysis at 48 h post-transfection (top blot). Cells treated with serum-free medium (Veh) or transfection reagent (TKO) were used to establish baseline CB₂ receptor protein expression. The middle blot designates the 43-kDa actin band on the Coomassie-stained gel used for electroblotting that served as a loading control. The bottom blot designates the actin band identified on the same Western immunoblot that was used to identify immunoreactive product for the CB₂ receptor protein that served as an additional loading control. B, knockdown of CB₂ receptor protein expression reverses THC-mediated inhibition of macrophage migration to Tat. U937 cells were transfected (48 h) with CB₂ receptor (ΔCB₂) (top) or negative control (Neg) siRNA (25 nM) (bottom) and assessed for migration to Tat (50 nM) using the Transwell migration assay. Macrophages were pretreated (3 h) with THC (1 μM–10 nM) or vehicle (0.01% ethanol) and then migration to Tat was analyzed. Statistical analysis was performed by comparing the number of migrating cells in THC treatment conditions with vehicle-treated cells exposed to Tat in the bottom chamber (positive migration control, designated by solid horizontal line). Solid box at abscissa = 0 represents the baseline migratory control of vehicle-treated cells exposed to serum-free complete assay medium in the bottom chamber. Results are representative of three experiments and are presented as the mean ± S.D.