Regulation of the cAMP cascade, gene expression and immune function by cannabinoid receptors

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Abstract

The objective of this article is to discuss the putative role of cannabinoid receptors in immune modulation by cannabinoid compounds. The primary focus is on the signal transduction events that are initiated following ligand binding to cannabinoid receptors and how these events lead to detrimental effects on the normal responsiveness of immunocompetent cells. Toward this end, signalling events are traced from the cannabinoid receptor to the transcription factors which are adversely regulated in the presence of cannabinoid compounds during leukocyte activation. Moreover, this aberrant regulation of transcription factors is discussed in the context of altered gene expression and the impact this has on leukocyte function. Lastly, an important goal of this article is to dispel a long standing myth that the cyclic adenosine 3′:5′-monophosphate (cAMP) cascade is a negative regulatory pathway for immunocompetent cells. This chapter examines two major immunologic cell-types which are well established as exhibiting altered function following cannabinoid treatment, helper T-cells and the macrophage. Not discussed are the effects of cannabinoids on B-cell function. This is primarily due to the rather refractory nature of B-cells to inhibition by cannabinoids in spite of the fact that this cell-type expresses functional cannabinoid receptors [Schatz, A.R., Koh, W.S., Kaminski, N.E., 1993. Δ9-tetrahydrocannabinol selectively inhibits T-cell dependent humoral immune responses through direct inhibition of accessory T-cell function. Immunopharmacol., 26, pp. 129–137.]. One cautionary note, although the focus of this article is on cannabinoid receptor mediated signalling events, immune modulation by cannabinoid compounds is likely multi-factorial presumably involving receptor as well as receptor-nonrelated events. Effects on leukocytes by cannabinoids which are believed to be mediated by receptor-nonrelated events are outside the scope of this paper and will not be discussed. One last introductory point is that even though their is presumably little overlap in the genes which are regulated by cannabinoids in leukocytes as compared to other cell-types (e.g., neural cells), the major signalling pathways involved in cellular regulation are ubiquitous. With that in mind, it is likely that their is a considerable amount of similarity in the signalling pathways regulated by cannabinoids in cell-types of different lineage, given that they express cannabinoid receptors. In this context, signalling events observed in leukocytes can provide important insight into which genes may be modulated by cannabinoid in other cell types.

Introduction

Historically, the mechanism by which cannabinoids produce their broad array of physiological effects has been attributed to nonspecific intercalation of these lipophilic compounds into the lipid bilayer of the cell membrane resulting in the disruption of membrane processes. Although the specific mechanism(s) of action for cannabinoids still remains to be fully elucidated, a number of distinct lines of evidence have supported the involvement of receptors. Many of the early observations were made by using the central nervous system (CNS) and CNS-derived cell-lines as models. One of the most compelling is the general lack of correlation between the degree of lipophilicity of specific cannabinoid congeners and their biologic activity (Thomas et al., 1990). Moreover, early studies demonstrated negative regulation of adenylate cyclase following exposure to cannabinoids, an enzyme almost exclusively associated in mammalian systems with membrane bound receptors (Howlett, 1985; Howlett and Fleming, 1984). The above also correlates well with binding studies which demonstrated specific and saturable binding by cannabinoids in brain synaptosome preparations (Harris et al., 1978). Lastly, a novel guanine-nucleotide-binding protein (G-protein) coupled receptor was cloned from a rat brain cDNA library which exhibited stereospecific cannabinoid binding and negative regulation of adenylate cyclase when transfected into chinese hamster ovary cells (CHO) cells in the presence of cannabinoid compounds (Matsuda et al., 1990). Since the identification of a cannabinoid receptor in neuronal tissues (cannabinoid receptor type 1 (CB1)), a second major form of the receptor (cannabinoid receptor type 2 (CB2)) has been isolated and cloned from the promyelocytic line HL60 (Munro et al., 1993). The two receptors share approximately 68% identity within their transmembrane regions, that portion of the receptor believed to possess the ligand binding domain (Munro et al., 1993). In spite of the marked differences in identity, most cannabinoid compounds bind with similar affinity to both CB1 and CB2. One exception to this rule is the plant derived cannabinoid, cannabinol, which possesses significantly greater binding affinity for CB2 than for CB1 (Munro et al., 1993); exhibits good binding affinity to mouse spleen cells (Schatz et al., 1997); and has immunomodulatory activity in a number of leukocyte preparations (Condie et al., 1996).

Section snippets

CB1 and CB2 receptor distribution

Shortly following the identification of CB1 in rat brain, CB1 was also identified in human brain (Gerard et al., 1990) and human testis (Gerard et al., 1991) and found to be highly conserved between these two as well as other species. The first identification of cannabinoid receptors within the immune system was in mouse spleen cells (Kaminski et al., 1992). This cellular preparation exhibited: (a) saturable specific binding of the high affinity cannabinoid receptor radioligand, [3H]-CP-55940

Regulation of adenylate cyclase by cannabinoid compounds

As initially suggested by Howlett et al. (1986)from studies in neuronal cell lines prior to the identification of cannabinoid receptors, both CB1 and CB2 negatively regulate adenylate cyclase activity through a pertussis toxin sensitive GTP-binding protein (Kaminski et al., 1992). Modulation of adenylate cyclase by cannabinoid compounds has been compellingly demonstrated in virtually every tissue and cell-line shown to express functional cannabinoid receptors as well as in cell-lines devoid of

Does the inhibition of adenylate cyclase by cannabinoids have any relevance to immune function?

As discussed above, one of the earliest signalling events initiated by ligand binding to cannabinoid receptors is the inhibition of adenylate cyclase which leads to a decrease in the production and accumulation of intracellular cyclic adenosine 3′:5′-monophosphate (cAMP). That this, in turn, leads to an inhibition of immune function is contrary to a long held immunologic axiom, that the primary role of the cAMP signalling cascade in immune function is to serve as a negative regulatory pathway.

The role of the cAMP signalling cascade in T-helper cell function

In the remainder of this chapter, the discussion will primarily focus on the role of the cAMP signalling cascade in cannabinoid-mediated disruption of two immunological responses: (a) interleukin-2 (IL-2) expression by T-cells; and (b) inducible nitric oxide synthase (iNOS) expression by macrophages. We will first focus on the disregulation of interleukin-2 (IL-2) by cannabinoids in helper T-cells.

Recently, studies have focused on the effects exerted by cannabinoids, through an inhibition of

The role of the cAMP signaling cascade in the regulation of iNOS in macrophages

Recently, it has been demonstrated that cannabinoids inhibit the induction of iNOS gene expression in LPS-stimulated macrophages (Jeon et al., 1996). The iNOS catalyzes the production of large amounts of NO from l-arginine and molecular oxygen (Palmer et al., 1988). Expression of iNOS is rapidly induced in macrophages by LPS which is a major constituent of gram negative bacterial cell membranes. The production of NO in turn contributes to the cytolytic function of macrophages and is believed to

Conclusions

Cannabinoids are well established as being immune modulators. Elucidation of the specific mechanisms responsible from this biologic effect has a number of significant implications. First, from a basic science standpoint, cannabinoids are a very useful class of pharmacologic probes that can be utilized to characterize the signaling events within the cAMP cascade and its role in a variety of biological processes. As discussed above, much has already been gleaned from the use of cannabinoids as

Acknowledgements

This work was supported by funds from NIDA Grants DA07908 and DA09789.

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