Changes effected through cannabinoid receptors

THC at lower concentrations, and at sites distal to the lung, may affect immune cell functions by signaling through cannabinoid receptors. Cannabinoid receptors have been identified both within the brain and cells of the immune system (Figure 9.5). Two cannabinoid receptors, CBX and CB2, have been identified. The CBX has been localized to neuronal tissues (Matsuda et al., 1990) and testis (Galiègue et al., 1995), and to a lesser extent to immune cells. In contrast, the CB2 has been observed only in cells of the immune system. Both receptors are coupled to a pertussis toxin-sensitive Gi/Go protein (Howlett et al., 1986; Matsuda et al., 1990) to

Cannabinoid Membrane Interactions

Figure 9.4 Transmission electron micrograph demonstrating that exposure to high concentrations of A9-tetrahydrocannabinol (THC) results in membrane perturbation. Murine peritoneal macrophages were exposed to vehicle (0.1% ethanol) or THC (I0-5M) for 24 h. (A) Cells treated with vehicle. (B) Cells treated with THC exhibiting large intracytoplasmic vacuoles (arrows). (C) Cells treated with THC exhibiting large intracytoplasmic membranous whorls (arrow) indicative of membrane damage. The bars represent I |j,m.

Figure 9.4 Transmission electron micrograph demonstrating that exposure to high concentrations of A9-tetrahydrocannabinol (THC) results in membrane perturbation. Murine peritoneal macrophages were exposed to vehicle (0.1% ethanol) or THC (I0-5M) for 24 h. (A) Cells treated with vehicle. (B) Cells treated with THC exhibiting large intracytoplasmic vacuoles (arrows). (C) Cells treated with THC exhibiting large intracytoplasmic membranous whorls (arrow) indicative of membrane damage. The bars represent I |j,m.

Figure 9.5 Identification of CB, and CB2 mRNA by Mutagenic Reverse Transcription-Polymerase Chain Reaction (MRT-PCR) (Dove Pettit et al., 1996). For detection of CB, mRNA, total RNA was subjected to reverse transcription using an oligonucleotide primer containing a single base mismatch generating a unique MspI restriction site. The reverse transcription products then were amplified by PCR using a pair of highly conserved oligonucleotide primers specific for human, rat, and mouse CB, sequences. The PCR amplification products were digested with MspI and subjected to electrophoretic separation through a 3% agarose gel and the DNA was transferred to nylon membrane. A similar approach was applied for detection of CB2 mRNA, except that a primer conserved for mouse and rat CB2 sequence and containing an unique Hindlll restriction site was used for reverse transcription. PCR was performed using conserved primers for mouse and rat CB2, and PCR products were digested with Hindlll. The blots were hybridized with a 32P-labeled random-primed rat CB, (for mouse or rat amplicons) or a mouse or rat CB2 fragment. The CB, cDNA fragment was generated by amplification from a pCD-SKR6 template (Matsuda et al., !990) using the CB, primers. The murine and rat CB2 cDNA fragments were generated by amplification from a mouse and a rat CB2 template (gift from T. l. Bonner, NlMH Bethesda MD) using the CB2 primers. (Top panel) Southern blot analysis of MRT-PCR products amplified from total RNA from rat brain, rat Bi03 neuroblastoma cells, murine P388Di and RAW264.7 macrophage-like cells, rat spleen, and thioglycolate-elicited murine peritoneal macrophages. CB, mRNA was detected in rat brain and rat B,03 neuroblastoma cells as demonstrated by the presence of two products following digestion with Mspl. The upper band (i.e. larger digestion product) represents product derived from genomic DNA templates, while the lower band (i.e. smaller digestion product) represents that derived from mRNA templates. (Bottom panel) CB2 mRNA was detected in total RNA from murine P388D, and RAW264.7 macrophage-like cells, rat spleen, and murine peritoneal macrophages as demonstrated by the presence of two products following digestion with Hind lll. No CB2 mRNA was detected in total RNA from rat brain or rat B,03 neuroblastoma cells.

inhibit adenylate cyclase activity resulting in decreases in levels of cAMP (Howlett etal., 1990; Felder et al., 1992; Felder et al., 1995) and initiate mitogen-activated protein kinase (MAPK) and immediate early gene signaling pathways (Bouaboula et al., 1993, 1995, 1996). However, in contrast to the CBj, no modulation of N-type calcium channels (Mackie and Hille, 1992) and G-protein regulated inwardly rectifying K+ channels (Childers and Deadwilder, 1996) has been observed for the CB2 (Felder et al., 1995).

The presence of CB2 receptors exclusively within immune cells suggests a role for these receptors in their activities. Transcripts (i.e. mRNAs) for the CB2 have been found in spleen and tonsils (Galiegue et al, 1995; Munro et al, 1993) and other immune tissues (Munro et al., 1993; Bouaboula et al., 1993). However, in all cases reported to date, levels of message for the CB2 exceed those for the CBj. The distribution pattern of levels of CB2 mRNA displays major variation in human blood cell populations with a rank order of B lymphocytes > natural killer (NK) cells >> monocytes > polymorphonuclear neutrophils > T8 lymphocytes > T4 lymphocytes (Galiegue et al., 1995). A rank order for levels of CB2 transcripts similar to that for primary human cell types has been recorded for human cell lines belonging to the myeloid, monocytic, and lymphoid lineages (Galiegue et al., 1995). In addition, the presence of cognate protein has been demonstrated in rat lymph nodes, Peyer's Patches, and spleen (Lynn and Herkenham, 1994). Table 9.1 summarizes the reported distribution of cannabinoid receptors in immune cells and tissues.

Kaminski et al. (1992, 1994) provided evidence which implicated a functional linkage between cannabinoid receptors and cannabinoid-mediated alterations in the activities of immune cells. It was noted that suppression of the humoral immune response by cannabinoids was mediated partially by inhibition of adeny-late cyclase through a pertussis toxin sensitive Guanine nucleotide binding protein (G protein) coupled mechanism. THC and the synthetic bicyclic cannabinoid CP55940 inhibited the lymphocyte proliferative response and the sheep erythrocyte IgM antibody-forming cell response of murine splenocytes to phorbol-12-myristate-13-acetate (PMA) plus the calcium ionophore ionomycin. Also, Jeon et al. (1996) indicated that LPS-inducible NO release by the murine macrophage-like cell

Table 9.1 Distribution of cannabinoid receptors in immune cells and tissues

Cell Type/Tissue

Species

Receptor

Reference

B lymphocytes

Human

CB2

Galiegue et al. (1995)

T4 lymphocytes

Human

CB2

Galiegue et al. (l995)

T8 lymphocytes

Human

CB2

Galiegue et al. (l995)

Leukocytes

Human

CB2

Bouaboula et al. (1993)

Macrophages

Human

CB2

Galiegue et al. (1995)

Microglia

Rat

CBj

Waksman et al. (1999)

Mononuclear Cells

Human, rat

CB2

Galiegue et al. (1995)

Facci et al. (1995)

Mast cells

Rat

CB2

Facci et al. (l995)

Natural Killer Cells

Human

CB2

Galiegue et al. (1995)

Peyer's Patches

Rat

CB*

Lynn and Herkenham (1994)

Spleen

Human,

CBj, CB2

Kaminski et al. (1992)

Mouse, rat

CB*

Munro et al. (1993)

Galiegue et al. (1995)

Facci et al. (1995)

Lynn and Herkenham (1994)

Thymus

Human

CB2

Galiegue et al. (1995)

Tonsils

Human

CB2

Galiegue et al. (l995)

Lymph Nodes

Rat

CB*

Lynn and Herkenham (1994)

* Cannabinoid receptor subtype not discriminated.

* Cannabinoid receptor subtype not discriminated.

line RAW264.7 was suppressed by THC and other agonists by mechanisms which implicated the involvement of cannabinoid receptors. It was indicated that the attenuation of inducible NO gene expression by THC was mediated through the inhibition of nuclear factor-fiB/Rel activation. In addition, Burstein et al. (1994) have presented evidence that indicates that THC-induced arachidonic acid release from mouse peritoneal cells occurs through a series of events that are consistent with a receptor-mediated process involving the stimulation of one or more phospholipases.

Recently, Waksman et al. (1999) provided evidence that cannabinoids can affect immune cell function through the CBX. The synthetic cannabinoid CP55940 inhibited the production of inducible NO by neonatal rat microglial cells (Figure 9.6), cells which constitute the resident macrophages of the brain. The inhibitory effect was stereoselective in that the dose-dependent inhibition of NO release was exerted by the cannabinoid receptor high affinity binding enantiomer CP55940 while a minimal effect was exerted by the low affinity binding paired enantiomer CP56667. Furthermore, reversal in CP55940-mediated inhibition of NO release was effected when microglial cells were pretreated with the CBrselective antagonist SR141716A. In contrast, Stefano et al. (1996) demonstrated that cannabinoid receptor agonists exerted an opposite effect on the production and/or release of constitutively expressed NO. Cannabinoid receptor agonists increased constitutive NO levels in cultures of human monocytes. As in the case of inducible NO, this effect was inhibited by the CBX antagonist SR141716A suggesting that the CBX was involved in the augmentation process. On the other hand, McCoy et al. (1999) implied that a functional linkage existed between cannabinoid mediated inhibition of the processing of hen egg lysozyme(HEL) by macrophages and the CB2. In their studies, processing of HEL was inhibited by THC and other cannabinoid agonists. Stereoselective cannabinoid enantiomers showed a differential inhibitory effect for the bioactive enantiomer CP55940 versus that of its less bioactive enantiomeric pair CP56667. Furthermore, the CBrselective antagonist SR141716A did not block the inhibitory effect of the cannabinoid agonist while the CB2-selective antagonist SR144528 did. More recently, Massi et al. (2000) reported that both types of cannabinoid receptors are involved in mediating NK cell cytolytic activity. Inhibition of NK cell activity by THC was partially reversed by both the CBX and the CB2 antagonists, although the CBX antagonist was more effective. In addition, the CBX and the CB2 antagonists completely reversed THC-mediated inhibition of IFN7 production.

Few studies have addressed the issue of the effects of cannabinoids on infectious processes in the context of a functional linkage to a cannabinoid receptor. Noe et al. (1998), using syncytial formation as a barometer of infection, reported that cannabinoid receptor agonists enhanced syncytia formation in MT-2 cells infected with cell free Human Immunodeficiency virus MN strain (HIV-1MN). Recently, Gross et al. (2000) implicated the CBX receptor as linked functionally to cannabinoid effects on Brucella suis growth within macrophages. The CBrselective antagonist SR141716A effected a dose-dependent inhibition of the intracel-lular multiplication of this Gram-negative bacterium. However, the nonselective CBx/CB2 cannabinoid receptor agonists CP55940 or WIN55212-2 reversed the SR141716A-mediated effect. These results suggested a beneficial application of a CBj antagonist as an inhibitor of macrophage infection by the intracellular

Los Receptores Cbj

Figure 9.6 Inhibition of neonatal rat cortical microglial inducible nitric oxide release by the synthetic cannabinoid agonist CP55940. (A) Differential inhibition of NO release by the cannabinoid agonist CP55940 versus its paired enantiomer CP56667. Microglial cells were pretreated with drug or vehicle for 8 h, treated with 20 |ag/ml LPS plus I0U/ml IFN7, and culture supernatants were assayed for nitrite 24 h later. Nitrite release from vehicle-treated cultures was 25.4 ± 3.3 [ ^M/106 cells/ml]. Results are expressed as percent inhibition versus vehicle and are the mean ± S.E.M. of triplicate wells. The high affinity cannabinoid CP55940 exerted a dose-dependent inhibition of NO release from rat microglial cells. The drug dose-dependent inhibition was significantly greater (p< 0.05, Student's t-test) than that exerted by its paired enantiomer CP56667 at each comparable concentration. (B) Reversal of CP55940-mediated inhibition of NO release by the CBI-specific antagonist SRI4I7I6A. Microglial cells were pretreated with 5 x I0-7M SRI4I7I6A prior to exposure to 5 x I0-6M CP55940 or CP56667 and LPS/IFN7 activation. Results (mean ± S.E.M. of triplicate wells) are expressed as percent inhibition versus vehicle control (**p< 0.0I versus SRI4I7I6A). Nitrite accumulation in vehicle-treated cultures was 29.3 ± 3.5 [|J.M/I06 cells]. Collectively, these results are consistent with a functional linkage between the CBI receptor and cannabinoid-mediated inhibition of inducible NO production by neonatal rat cortical microglial cells.

pathogen Brucella suis. Thus, data from several laboratories suggest that both CBX and CB2 receptors may be linked functionally to cannabinoid agonist-mediated effects on immune cells. However, the relative contributions of the two canna-binoid receptors to individual functional activities within specified immune cell types, or within individual cells co-expressing CBX and CB2, remain to be defined. The present availability of CBX and CB2 specific antagonists (Rinaldi-Carmona et al., 1994; Rinaldi-Carmona et al., 1998), as well as of CBX and CB2 knock-out mice which already have provided valuable insight regarding functional activities such as enhancement of memory, hypoalgesia, vasodilation, and embryonic development (Reibaud et al., 1999; Zimmer et al., 1999; Jarai et al., 1999; Buckley et al., 1998), affords the opportunity to systematically identify signal transductional systems within immune cells which serve as specific targets of cannabinoids.

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