Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB2 receptor

https://doi.org/10.1016/S0014-2999(00)00211-9Get rights and content

Abstract

Cannabinoids have immunomodulatory as well as psychoactive effects. Because the central cannabinoid receptor (cannabinoid CB1 receptor) is highly expressed in many neuronal tissues and the peripheral cannabinoid receptor (cannabinoid CB2 receptor) is highly expressed in immune cells, it has been suggested that the central nervous system effects of cannabinoids are mediated by cannabinoid CB1 receptors and that the immune effects are mediated by cannabinoid CB2 receptors. To test this hypothesis, we have generated the first mouse strain with a targeted mutation in the cannabinoid CB2 receptor gene. Binding studies using the highly specific synthetic cannabinoid receptor agonist (−)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]CP 55,940) revealed no residual cannabinoid binding sites in the spleen of the cannabinoid CB2 receptor knockout mice, while binding in the central nervous system was unchanged. Cannabinoid CB2 receptor knockout mice, which appear healthy, are fertile and care for their offspring. Fluorescence activated cell sorting (FACS) analysis showed no differences in immune cell populations between cannabinoid CB2 receptor knockout and wildtype mice. We investigated the immunomodulatory effects of cannabinoids in cannabinoid CB2 receptor deficient mice using a T cell co-stimulation assay. Δ9Tetrahydrocannabinol inhibits helper T cell activation through macrophages derived from wild type, but not from knockout mice, thus indicating that this effect is mediated by the cannabinoid CB2 receptor. In contrast, central nervous system effects of cannabinoids were not altered in these mice. Our results suggest that cannabinoid CB2 receptor-specific ligands may be clinically useful in the modulation of macrophage immune function while exhibiting no central nervous system activity. Furthermore, we conclude that the cannabinoid CB2 receptor knockout mouse is a useful animal model in which to study the role of the cannabinoid system in immunoregulation.

Introduction

Cannabinoids have been shown to modulate immune functions. The most widely studied cannabinoid is Δ9tetrahydrocannabinol, the major psychoactive compound of marijuana. Within the immune system, Δ9tetrahydrocannabinol and other cannabinoids affect lymphocytes, macrophages, and natural killer cells in a variety of functions such as cellular proliferation, cytokine activity, humoral responses, and cell mediated immunity (reviewed in Klein et al., 1998a, Klein et al., 1998b, Klein et al., 1998c).

Although these observations suggest a role for cannabinoids in immunomodulation, the mechanisms by which cannabinoids induce these immunomodulatory effects has remained elusive. However, the discovery of two cannabinoid receptors Matsuda et al., 1990, Munro et al., 1993 has shed some understanding on the mechanisms of cannabinoid action. The central cannabinoid receptor (cannabinoid CB1 receptor), cloned in 1990 by Matsuda et al. (Matsuda et al., 1990), is predominantly found in the central nervous system (Herkenham et al., 1991). This receptor is found to a much lesser degree in cells of the immune system Bouaboula et al., 1993, Galiegue et al., 1995, Daaka et al., 1995, Schatz et al., 1997. The peripheral cannabinoid receptor (cannabinoid CB2 receptor) (Munro et al., 1993) is predominantly expressed in cells of the immune system, such as B cells, T cells, and macrophages Munro et al., 1993, Galiegue et al., 1995, Schatz et al., 1997. This latter finding has led investigators to postulate that the immunoregulatory effects of cannabinoids may be cannabinoid CB2 receptor mediated, although very few studies support this notion.

Recently, there has been direct evidence indicating that cannabinoids act through cannabinoid CB2 receptor to alter immune function. Using the newly developed cannabinoid CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) (Rinaldi-Carmona et al., 1998), cannabinoid-induced B cell proliferation (Derocq et al., 1995) was shown to be dependent on activation of the cannabinoid CB2 receptor (Carayon et al., 1998). Furthermore, the cannabinoid CB2 receptor is down-regulated during B cell differentiation as assessed by cannabinoid CB2 receptor mRNA expression and anti-cannabinoid CB2 receptor antibody binding (Carayon et al., 1998).

Here we report the development of another useful research tool to study the role of cannabinoid CB2 receptor activation in the immune system. Using homologous recombination, we created the first mouse deficient for the cannabinoid CB2 receptor (cannabinoid CB2 receptor knockout mice or CB2−/− mice). Here, we demonstrate that this mouse model is an effective system in which to investigate the role of the cannabinoid CB2 receptor in cannabinoid-induced immunomodulation. In the present study, we have investigated the role of the cannabinoid CB2 receptor on Δ9tetrahydrocannabinol inhibition of macrophage co-stimulatory activity.

Previously, McCoy et al. observed that Δ9tetrahydrocannabinol, at nanomolar concentrations, inhibits helper T cell activation by a macrophage cell line McCoy et al., 1995, McCoy et al., 1999, Clements et al., 1996, Clements et al., 1998. T cell stimulation by macrophages or dendritic cells is one of the first and most important steps in initiating an acquired immune response. Activation of T cells requires their physical interaction with an antigen-presenting cell or accessory cell. This requirement is partially due to the specificity of the T cell receptor (TCR), because its ligand is a molecular complex composed of a peptide fragment of the antigen bound to major histocompatibility complex molecules expressed on the surface of accessory cells Germain, 1994, Watts, 1997. The cellular interaction between T cells and accessory cells also involves several cell adhesion molecules and serves to prolong the length of time of the primary signal through the TCR Altman et al., 1990, Hynes, 1992. A second co-stimulatory signal delivered by accessory cells, which is independent of the TCR, is also critical for maximal proliferation and cytokine secretion by T cells Linsley and Ledbetter, 1993, Janeway and Bottomly, 1994, Mondino and Jenkins, 1994. Once activated, the T cell proliferates and produces cytokines (most notably interleukin-2). These cytokines can then act on the same T cells or on other immune cells such as B cells and macrophages to induce different immune functions.

We have utilized macrophages derived from the cannabinoid CB2 receptor knockout mice to investigate whether cannabinoids affect macrophage co-stimulatory functions via the cannabinoid CB2 receptor. Our results show that cannabinoids inhibit macrophage co-stimulatory activity and hence T cell-activation via the cannabinoid CB2 receptor.

Section snippets

Homologous recombination

The targeting vector was derived from the plasmid pPNT (Tybulewicz et al., 1991) by introducing upstream of the neomycin gene, 731 base pairs (bp) of the cannabinoid CB2 receptor genomic fragment containing the 5′ region of the exon which has the entire coding sequence. Downstream of the neomycin gene, we introduced 5.4 kb of the non-coding cannabinoid CB2 receptor gene sequence. Upon homologous recombination, 341 bp of the cannabinoid CB2 receptor gene is replaced by the neomycin gene (Fig. 1A)

Homologous recombination

All of the cannabinoid CB2 receptor gene coding sequence is contained within a single exon. To inactivate the gene, we replaced the 3′ region of this coding exon with the phosphoglycerate kinase (PGK)-neomycin sequences through homologous recombination in embryonic stem cells (Fig. 1A). This mutation eliminates part of intracellular loop 3, transmembrane domains 6 and 7, and the carboxy terminus. Homozygous CB2tm1/CB2tm1 mice (henceforth referrred to as CB2−/− mice) were obtained with the

Discussion

During the last decade, interest in the potential medicinal use of cannabinoid drugs has greatly increased. This renewed interest was stimulated in part by the molecular cloning of two cannabinoid receptors, the cannabinoid CB1 and the cannabinoid CB2 receptors. These two receptors, which belong to the large family of G-protein coupled receptors, are quite divergent in structure (44% overall homology) and in their pattern of expression. The cannabinoid CB1 receptor is thought to mediate most of

Acknowledgements

The authors would like to thank Dr. Ronald H. Schwartz for providing 2B4.11 cells and Dr. Thomas Giese for performing the FACS analysis. Part of this work was supported by the P50 DA05274 grant from the National Institutes of Health.

References (39)

  • T.W. Klein et al.

    Cannabinoid receptors and immunity

    Immunol. Today

    (1998)
  • Y.D. Luo et al.

    Effects of cannabinoids and cocaine on the mitogen-induced transformation of lymphocytes of human and mouse origins

    Int. J. Immunopharmacol.

    (1992)
  • S.H. Pross et al.

    Differing effects of Δ9-tetrahydrocannabinol (THC) on murine spleen cell populations dependent upon stimulators

    Int. J. Immunopharmacol.

    (1992)
  • A.R. Schatz et al.

    Cannabinoid receptors CB1 and CB2: a characterization of expression and adenylate cyclase modulation within the immune system

    Toxicol. Appl. Pharmacol.

    (1997)
  • V.L. Tybulewicz et al.

    Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene

    Cell

    (1991)
  • M. Bouaboula et al.

    Cannabinoid-receptor expression in human leukocytes

    Eur. J. Biochem.

    (1993)
  • M. Bouaboula et al.

    Gi protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2: implication for intracellular signalization cross-regulation

    Mol. Pharmacol.

    (1999)
  • D.J. Clements et al.

    Delta 9-tetrahydrocannabinol selectively inhibits macrophage costimulatory activity and down-regulates heat-stable antigen expression

    J. Pharmacol. Exp. Ther.

    (1996)
  • Y. Daaka et al.

    Expression of cannabinoid receptor mRNA in murine and human leukocytes

    Adv. Exp. Med. Biol.

    (1995)
  • Cited by (475)

    • Cannabinoids

      2022, Advances in Neurotoxicology
      Citation Excerpt :

      There has been little evidence that neurons express CB2Rs under normal conditions with a demonstrated low-level expression in midbrain dopaminergic neurons in the ventral tegmental area, the prefrontal cortex, cerebellum, dorsal striatum, and nucleus accumbens (Zhang et al., 2014, 2017); however, upon injury, specific neurons may display functional receptors (Van Sickle et al., 2005; Viscomi et al., 2009). The presence on immune cells allows CB2Rs to modulate responses to inflammatory stimuli (Buckley et al., 2000; Maresz et al., 2005; Ramirez et al., 2012; Walter et al., 2003). CB2Rs can be induced in the CNS in response to injury or pathophysiological states (Guindon and Hohmann, 2009; Jordan and Xi, 2019; Roche and Finn, 2010; Woodhams et al., 2017).

    View all citing articles on Scopus
    1

    These two authors contributed equally to the value of this work.

    2

    Requests for materials and animals: Tel.: +1-301-496-8160; fax: +1-301-435-5465.

    View full text