Identification of a functionally relevant cannabinoid receptor on mouse spleen cells that is involved in cannabinoid-mediated immune modulation

Abstract

Extensive behavioral and biochemical characterization of cannabinoid-mediated effects on the central nervous system has revealed at least three lines of evidence supporting the role of a putative guanine nucleotide-binding protein-coupled cannabinoid receptor for cannabimimetic effects, (i) stereoselectivity, (ii) inhibition of the adenylate cyclase/cAMP second messenger system, and (iii) radioligand-binding studies with the synthetic cannabinoid [3H]CP-55,940 indicating a high degree of specific binding to brain tissue preparations. Based on recent findings from our laboratory demonstrating that delta 9-tetrahydrocannabinol markedly inhibited forskolin-stimulated cAMP accumulation in mouse spleen cells, the presence of a guanine nucleotide-binding protein-coupled cannabinoid receptor associated with mouse spleen cells and its functional role in immune modulation were investigated. In the present studies, stereoselective immune modulation was observed with the synthetic bicyclic cannabinoid (-)-CP-55,940 versus (+) CP-56,667 and with 11-OH-delta 8-tetrahydrocannabinol-dimethylheptyl, (-)-HU-210 versus (+)-HU-211. In both cases, the (-)-enantiomer demonstrated greater immunoinhibitory potency than the (+)-isomer, as measured by the in vitro sheep red blood cell antibody-forming cell response. Radioligand binding studies produced a saturation isotherm exhibiting approximately 45-65% specific binding to mouse spleen cells. Scatchard analysis demonstrated a single binding site on spleen cells, possessing a Kd of 910 pM and a Bmax of approximately 1000 receptors/spleen cell. RNA polymerase chain reaction of isolated splenic RNA using specific primers for the cannabinoid receptor resulted in the amplification of a 854-kilobase predicted product that hybridized with cannabinoid receptor cDNA, demonstrating the presence of cannabinoid receptor mRNA in mouse spleen. Together, these findings strongly support the role of a cannabinoid receptor in immune modulation by cannabimimetic agents.

Fig. 1

Direct addition of stereoisomers CP-55,940 and CP-56,667 to naive spleen cell culture and their effect on the in vitro sRBC AFC response. Spleens from naive female B6C3F, mice were isolated aseptically and made into single-cell suspensions. The splenocytes were washed, adjusted to 1.0 × 10⁷ cells/ml, and transferred in 500-µl aliquots to wells of a 48-wel culture plate. Quadruplicate cultures were prepared with vehicle [0.01% dimethylsulfoxide (DMSO) or 0.1% ethanol (EtOH) final concentration in culture], CP-55,940, or CP-56,667 and were sensitized with sRBC. Cultures were subsequently assayed, using sRBC, for their day 5 IgM antibody response by enumerating the number of AFC, spleen cell viability, and total recovered cells/culture. Bars, mean ± standard error as determined for each group. * p < 0.05, as determined by Dunnett’s t test, compared with the vehicle group.

Fig. 2

Direct addition of stereoisomers HU-210 and HU-211 to naive spleen cell culture and their effect on the in vitro sRBC AFC response. Spleens from naive female B6C3F₁ mice were isolated aseptically and made into single-cell suspensions. The splenocytes were washed, adjusted to 1.0 × 10⁷ cells/ml, and transferred in 500-µl aliquots to wells of a 48-well culture plate. Quadruplicate cultures were prepared with vehicle (0.1 % ethanol, final concentration in culture). HU-210, or HU-211 and were sensitized with sRBC. Cultures were subsequently assayed, using sRBC, for their day 5 IgM antibody response by enumerating the number of AFC, spleen cell viability, and total recovered cells/culture. Bars, mean ± standard error, as determined for each group. *, p < 0.05, as determined by Dunnett’s t test, compared with the vehicle group.

Fig. 3

In vitro radioligand binding of [³H]CP-55,940 to mouse spleen cells. Spleens from naive female B6C3F₁ mice were isolated aseptically and made into single-cell suspensions. The splenocytes were washed, adjusted to 2.0 × 10⁶ cells/ml in buffer (50 mm Tris · HCl, pH 7.4, 1 mm EDTA, 3 mm MgCl₂, 5 mg/ml BSA), and transferred in 1-ml aliquots to silanized glass culture tubes. Spleen cells were incubated in the presence of [³H]CP-55,940 at 30° for 1 hr. The reaction was terminated by addition of cold 50 mm Tris · HCl plus 5 mg/ml BSA (pH 7.4) and was then rapidly filtered through Whatman OF/C glass fiber filters. Specific binding was defined as the difference between the binding that occurred in the presence and in the absence of 1 µm unlabeled ligand. A, Saturation isotherm; B, Scatchard plot, for which EBDA analysis was used to determine K_d and B_max values.

Fig. 4

Reverse transcription-PCR of mouse spleen RNA. RNA from splenocytes and rat brain cerebellum was amplified as described in Materials and Methods. A, Agarose gel Lanes 1–4, PCR products using the cannabinoid receptor primers (bp 1–21 and 822–843 on the opposite strand). Lane 1,2 µl from the RNA PCR reaction using splenocyte RNA, which is the predicted size of 835 bp. Lane 2, 2 µl from the DNA PCR reaction using splenocyte RNA, indicating the absence of DNA contamination of the RNA. Lane 3, 2 µl from the RNA PCR reaction using rat cerebellar RNA. Lane 4, product from the DNA PCR reaction with no added RNA. Lane 5, 2 µl of the PCR product from the RNA PCR reaction using the c-fos primers. These primers were based on bp 232–252 and 322–342 on the opposite strand of the c-fos gene. The predicted size of 110 bp, as seen, indicates the absence of DNA contamination (which would be seen as a 800-bp product). The gel was transferred to nylon membranes and hybridized with the cannabinoid cDNA probe. B, Resulting autoradiogram.