INVITED REVIEW

Cannabinoid-Induced Immune Suppressionand Modulation of Antigen-Presenting Cells

Thomas W. Klein & Guy A. Cabral

Published online: 22 February 2006# Springer Science+Business Media, Inc. 2006

Abstract The study of marijuana cannabinoid biology

has led to many important discoveries in neuroscience

and immunology. These studies have uncovered a new

physiological system, the endocannabinoid system,

which operates in the regulation of not only brain

function but also the regulation of the immune system.

Studies examining the effect of cannabinoid-based

drugs on immunity have shown that many cellular

and cytokine mechanisms are suppressed by these

agents leading to the hypothesis that these drugs may

be of value in the management of chronic inflamma-

tory diseases. In this report, we review current in-

formation on cannabinoid ligand and receptor biology,

mechanisms involved in immune suppression by can-

nabinoids with emphasis on antigen-presenting cells,

and preclinical and clinical models analyzing the

therapeutic potential of cannabinoid-based drugs.

Introduction

Cannabis sativa or marijuana has been studied over the

years because of its potential as both a therapeutic in

the management of a variety of conditions ranging

from rheumatism to epilepsy (see review in Tomida

et al. 2004) and a drug of abuse especially among

young people. The chemicals in marijuana are well

known to have behavioral and analgesic effects and

therefore known to affect brain function; however,

what is less appreciated are the immunomodulatory

and anti-inflammatory effects of these compounds and

the associated cellular targets of action (Klein 2005).

Work in the immune area has shown that just as the

brain has an endocannabinoid system of receptors and

endogenous ligands, the immune system also contains

these system components that are subject to modula-

tion by natural and synthetic agonists derived from

marijuana cannabinoids. Here we will focus on some

of the cellular immune mechanisms affected by

cannabinoids as well as the therapeutic potential of

these drugs in the treatment of immune diseases.

Endocannabinoid system

Cannabinoid-based ligands and analogues

The term cannabinoid comes from the chemical char-

acterization of the major psychoactive component of

marijuana, D9-tetrahydrocannabinol or THC (Gaoni

and Mechoulam 1964). Marijuana cannabinoids are

tricyclic ring structures containing a phenol ring with

attached 5-carbon alkyl chain, a central pyran ring, and

a monounsaturated cyclohexyl ring; the structure and

function of THC and related compounds has been

recently and extensively reviewed (Howlett et al. 2002,

J Neuroimmune Pharmacol (2006) 1: 50–64

DOI 10.1007/s11481-005-9007-x

T. W. Klein (*)Department of Medical Microbiology and Immunology,University of South Florida College of Medicine,MDC Box 10, 12901 Bruce Downs Boulevard,Tampa, FL 33612, USAe-mail: tklein@hsc.usf.edu

G. A. CabralDepartment of Microbiology and Immunology,Virginia Commonwealth University, School of Medicine,Richmond, VA 23298, USA

2004). Besides THC, other natural cannabinoid products

of marijuana have been isolated and studied for biological

activity such as D8-THC (also psychoactive), cannabinol,

and cannabidiol (CBD) (both nonpsychoactive). Fur-

thermore, synthetic cannabinoid derivatives such as

CP55940, HU-210, HU-211, ajulemic acid, and abnormal

CBD have been synthesized and studied (Fig. 1). In

addition to the natural cannabinoids and derived an-

alogues, the first of several endogenous ligands operat-

ing in the endocannabinoid system was isolated and

characterized in the early 1990s (Fig. 1; Devane et al.

1992). This so-called endocannabinoid was demonstrat-

ed to be the arachidonic acid derivative, N-arachidonyl

ethanolamide (anandamide or AEA), and since its

discovery several other endocannabinoids have been

reported and extensively studied for biological activity,

including 2-arachidonoyl-glycerol (2-AG; Mechoulam

et al. 1995), 2-arachidonylglycerylether (noladin ether;

Hanus et al. 2001), and O-arachidonoyl ethanolamine

(virodhamine; Porter et al. 2002). The last group of

ligands has inverse agonist/antagonist activity. Several of

these have played a central role in defining the biology

of the endocannabinoid system such as SR141716A

(rimonabant), AM251, and SR144528, and their activity

has recently been reviewed (Fowler et al. 2005). What is

interesting about these many cannabinoid-related com-

pounds is that not all of them appear to exert their effect

through cannabinoid receptors. Many with biological

activity have little or no affinity for either of the two

known cannabinoid receptors, suggesting that other

receptors and mechanisms are involved in the action of

these agents (see below).

Receptors

A number of pharmacological studies with high-

affinity ligands strongly supported the existence of

cannabinoid receptors (Devane et al. 1988), and the

gene for the first of these (CB1) was eventually cloned

from brain in 1990 (Matsuda et al. 1990) with a second

receptor (CB2) cloned a few years later from immune

cells (Munro et al. 1993). Cannabinoid receptors are

seven-transmembrane G-protein-coupled receptors

and are linked primarily to Gi and thus inhibit adenylyl

cyclase (Howlett et al. 1988); however, there is some

evidence that at least CB1 also stimulates cAMP,

suggesting it may be linked to Gs (Glass and Felder

1997). In addition to G proteins, cannabinoid receptors

are also linked to a variety of other second messengers

and signaling components such as phospholipase C,

calcium channels, nitric oxide synthase, MAP kinases,

and signaling mechanisms important in immune cell

activation (Klein et al. 2003; Howlett et al. 2004). CB1

receptors are expressed in brain and peripheral tissues

such as blood vessels (Wagner et al. 2001), testis

(Gerard et al. 1991), and immune tissues (Kaminski

et al. 1992); CB2 receptors should be expressed most

notably in immune tissues (Bouaboula et al. 1993;

Galieque et al. 1995). Besides CB1 and CB2, endocan-

nabinoids such as AEA (Zygmunt et al. 1999) and

natural cannabinoids such as CBD (Bisogno et al.

2001) can activate vanilloid receptors in vascular

tissues as well as certain neural circuits (Veldhuis et

al. 2003). Other unidentified receptors might also be

involved. For example, the antinociceptive response of

mice to AEA was less susceptible to attenuation by the

CB1 antagonist, SR141716A, than was the response

induced by the other cannabinoid agonists, CP55940

and D9-THC (Welch et al. 1998). Furthermore, G

protein signaling in response to AEA and WIN55212-

2, but not to other CB1 agonists such as CP55940, HU-

210, and D9-THC, was shown to occur equally well in

mouse brain preparations from CB1+/+ and CB1

j/j

mice and the effect was inhibited by only relatively

CANNABINOID-BASED LIGANDS

ENDOCANNABINOIDS

CANNABINOIDS ARACHIDONIC ACID METABOLITES

N-arachidonyl ethanolamide2-arachidonoyl-glycerol2-arachidonylglyceryletherO -arachidonyl ethanolamine

NATURAL SYNTHETIC

MARIJUANA

∆9-THC∆8-THCcannabinolcannabidiol

CP55940HU-210HU-211ab-cannabidiolAjulemic acid

Fig. 1. The classification ofcannabinoid-based ligandsevolved from studies on mar-ijuana cannabinoids. Thereare currently two main groupsof these agents with widelyvarying affinities for currentlyrecognized cannabinoidreceptors. These two groupsare those based on the struc-ture of marijuana cannabi-noids and the arachidonicacid metabolites, the endo-cannabinoids

51

high concentrations of SR141716A (Breivogel et al.

2001); these and other reported findings led to the

hypothesis that certain areas of the brain contained an

unknown cannabinoid receptor subtype (Breivogel et

al. 2001). Receptor subtypes have also been implicated

in the cannabinoid-induced vasodilation response and

the functioning of immune cells. For example, AEA

induced a vasodilation in rat mesenteric arteries that

was sensitive to SR141716A inhibition but the

response was not observed with other CB1 agonists

(Jarai et al. 1999). In addition, the nonpsychoactive

cannabinoid, abnormal CBD, caused a similar

SR141716A-sensitive vasodilation in vessels from

both wild-type mice and knockout mice lacking

cannabinoid receptors, and this effect was not

mediated by cyclooxygenase products or vanilloid

receptors (Jarai et al. 1999). These and other studies

suggested that this new receptor resides in the vascular

endothelial cell (Offertaler et al. 2003) and that the

endogenous ligand for this receptor might be the

endocannabinoid, virodhamine (Ho and Hiley 2004).

In addition to these vascular effects, cannabinoid

effects on immune cells have also been shown to

involve other receptors or mechanisms. For example,

inhibition of IL-2 production in splenocytes by

cannabinol was shown by various methods not to be

mediated by CB1 or CB2, but the elevation in

intracellular calcium concentration in these cells was

attenuated by treatment with both SR141716A and

SR145428, suggesting receptor involvement (Kaplan et

al. 2003). In addition, we recently observed that

suppression of IL-12 in stimulated dendritic cell

cultures was only partly mediated by CB1 and CB2.

Figure 2 shows that suppression of the cytokine

response by THC is only partly attenuated by

receptor antagonists added to DC cultures from

cannabinoid receptor single-knockout mice. Thus, as

in the above studies, at least a portion of the

cannabinoid effect persists in the functional absence

of both CB1 and CB2.

Endocannabinoid metabolism

Thus far, the endogenous ligands for the endocanna-

binoid system are AEA, 2-AG, noladin ether, and

virodhamine (see above). Some information is avail-

able on the metabolism of the first two, AEA and

2-AG. These compounds are arachidonic acid deriva-

tives arising from the membrane fatty acid matrix of

various cells including neurons and immune cells

(Sugiura et al. 2002; Di Marzo et al. 2004). AEA

synthesis starts with the combination of arachidonoyl

phospholipid and phosphatidylethanolamine by the

action of an acyltransferase (Sugiura et al. 1996) and

the subsequent hydrolysis by N-acylphosphatidyletha-

nolamine phospholipase D to yield AEA (Okamoto

et al. 2004). 2-AG synthesis, on the other hand, in-

volves the hydrolysis of membrane-associated diacyl-

A

0 3 6 10 0 3 6 100

5

10

15

20

25

IL-1

2p40

(ng

/ml)

*Lp only

CB1-/-

B

0

5

10

15

20

25

IL-1

2p40

(ng

/ml)

*Lp only

CB2-/-

Fig. 2. THC-induced suppression of IL-12p40 in cultureddendritic cells is only partially mediated through CB1 and CB2.Bone-marrow-derived dendritic cells were harvested and cul-tured from either CB1

j/j (A) or CB2j/j (B) knockout mice,

infected with L. pneumophila, and treated with either THC only

at increasing concentrations or treated with THC plus eitherSR2 (SR144528; CB2 antagonist) or SR1 (SR141716A; CB1

antagonist) at increasing concentrations to block remainingcannabinoid receptor activity. After 24 h, culture supernatantswere harvested and analyzed for IL-12p40 protein by ELISA

52

glycerol by specific diacylglycerol lipases (Bisogno

2003). Besides synthesis, some of the mechanisms

involved in intracellular degradation of endocannabi-

noids have been described. For AEA, the major

degradative enzyme is the serine hydrolase, fatty acid

amide hydrolase (Deutsch et al. 2002). This enzyme

has some activity for 2-AG but a more specific enzyme

appears to be the monoglyceride lipase (Saario et al.

2004), which has a higher activity for 2-AG. In

addition to these degradative enzymes, the endocan-

nabinoids may be inactivated in the vicinity of the

receptor by either passive diffusion across membranes

into cells or by plasma membrane transporter mole-

cules (Di Marzo et al. 2004; Ligresti et al. 2004);

however, to date no transporter protein has been

cloned. For many of the studies involving endocanna-

binoid metabolism and function, CNS mechanisms

have been emphasized; however, it is becoming clear

that these fatty acid ethanolamide mediators are also

produced and active in immune cells (Maccarrone

et al. 2001).

Immune suppression—effects on cell-mediated

and humoral immunity

Marijuana and various cannabinoids have been shown

to affect the functional activities of immune cells from

rodents and humans including B lymphocytes, large

granular lymphocytes (LGL), T lymphocytes, macro-

phages, and natural killer cells (Cabral and Dove Pettit

1998; Klein et al. 1998a; Cabral and Staab 2005). THC

was noted (Klein et al. 1985) to suppress mouse

splenocyte T-lymphocyte proliferation in response to

the T-cell mitogens ConA and PHA as well as that of

B lymphocytes induced by bacterial lipopolysaccharide

(LPS), a B-cell mitogen. Additional reports have

confirmed that cannabinoids suppress the antibody

response of humans and animals (Friedman et al.

1991; Klein et al. 1998b). It was reported (Kaminski

et al. 1994) that suppression of the humoral immune

response by cannabinoids was mediated, at least in

part, through the inhibition of adenylate cyclase by a

pertussis-toxin-sensitive G-protein-coupled mecha-

nism. In contrast, THC, as well as the potent synthetic

cannabinoid agonists CP55940 and WIN55212-2, was

reported to enhance human tonsillar B-cell growth

when used at low nanomolar concentrations (Derocq

et al. 1995). The investigators proposed that the

enhancing activity on B cells was mediated through

the CB2 receptor. Carayon et al. (1998) reported that

the CB2 receptor was downregulated at the mRNA

and protein levels during B-cell differentiation and

that the CB2 receptor antagonist SR144528 reversed

the stimulating effects of CP55940 on human tonsillar

B-cell activation. These investigators suggested a

functional involvement of CB2 receptors during B-cell

differentiation. Cannabinoids also have been reported

to suppress a variety of the activities of T lymphocytes.

For example, it was reported that the cytolytic activity

of murine cytotoxic T lymphocytes (CTLs) generated

by co-cultivation with either allospecific or TNP-

modified self-stimulators was suppressed by THC and

11-hydroxy-THC (Klein et al. 1991). Allospecific CTLs

generated in vivo also were inhibited by in vitro

exposure to these cannabinoids. In addition, the effect

of THC on CTL response to herpes simplex virus type

1 (HSV1) was examined (Fischer-Stenger et al. 1992).

THC decreased the CTL activity to virus-infected cells

and inhibited CTL cytoplasmic polarization toward the

virus-infected target cell, suggesting a mode by which

it exerted antiviral activity. Yebra et al. (1992) ex-

amined effects of THC on one of the earliest events in

T-cell activation, the mobilization of cytosolic free

calcium [Ca2+]. It was reported that a portion of the

proliferation defect in THC-treated lymphocytes could

be related to a drug-induced inhibition of [Ca2+]

mobilization that normally occurs following mitogen

treatment. More recently, the effects of cannabinoids

on adenylate-cyclase-mediated signal transduction and

interleukin(IL)-2 expression in the murine thymoma-

derived T-cell line was examined (Condie et al. 1996).

Treatment of cells with cannabinol or THC disrupted

the adenylate cyclase signaling cascade by inhibiting

forskolin-stimulated cAMP accumulation, an inhibi-

tion that led to a decrease in protein kinase A activity

and binding of transcription factors to a CRE consen-

sus sequence. These and extended findings suggested

that inhibition of signal transduction via the adenylate

cyclase/cAMP pathway induces T-cell dysfunction by

diminution in IL-2 gene transcription. Furthermore,

these studies indicated that cannabinoids such as can-

nabinol that are not high-affinity ligands for the CB1

and CB2 receptors can exert effects on immune cells.

THC and other cannabinoids also have been shown

to affect the functionality of natural killer (NK) cells.

For example, the IL-2-induced killing activity and

proliferation on murine NKB61A2 natural killer-like

cells was suppressed by THC and 11-hydroxy-THC

(Kawakami et al. 1988). Similarly, THC suppressed

proliferation of murine spleen cells stimulated with

recombinant human IL-2 and the appearance of

lymphocyte-activated killer (LAK) cells. In addition,

spleen cells stimulated with IL-2, and then incubated

with THC prior to addition of target cells, displayed

suppressed cytolytic activity against both YAC-1 and

53

EL-4 tumor targets. The mechanism of this suppres-

sion was attributed as due partly to a drug-induced

decrease in the number of high- and intermediate-

affinity IL-2 binding sites, suggesting suppression in

the expression of IL-2 receptor (IL-2R) proteins (Zhu

et al. 1995). These studies were extended indicating a

link to cannabinoid receptors for these effects (Daaka

et al. 1997). These investigators concluded that in the

NK-like cell line used in the studies, a signaling path-

way existed that was composed of the CB1 receptor,

the transcriptional factor NF-kB, and the IL-2Rareceptor gene. Recently, it was reported that in vivo

administration of THC to mice significantly inhibited

NK cytolytic activity without affecting ConA-induced

splenocyte proliferation (Massi et al. 2000). The

parallel measurement of IFNg revealed that THC sig-

nificantly reduced production of this cytokine and that

CB1 and CB2 receptor antagonists completely reversed

this reduction. These results suggested that both can-

nabinoid receptors were involved in the network medi-

ating NK cytolytic activity.

Although cannabinoids exert direct effects on

immune cell types, they also alter the expression of

chemokines and cytokines, which cross-signal among

immune cells and play a critical role in pro-inflamma-

tory vs. anti-inflammatory activities. The induction of

interferon (IFN)-a/b was reported to be suppressed by

chronic treatment of mice with THC (Blanchard et al.

1986; Cabral et al. 1986). Watzl et al. (1991) indicated

that cytokine activity was modulated in human pe-

ripheral blood mononuclear cell cultures by THC.

However, it was shown that the nonpsychoactive can-

nabinoid CBD also modulated cytokine production

and/or secretion suggesting that a noncannabinoid-

receptor-mediated mode of action also could be in-

volved. The effect of THC and CBD on cytokine

production by human leukemic T, B, eosinophilic, and

CD8+ natural killer lines was examined (Srivastava

et al. 1998). These investigators indicated that THC

and CBD could alter production of a multiplicity

of cytokines across a diverse array of immune cell

lineages. Smith et al. (2000) evaluated the effects of

cannabinoid receptor agonists and antagonists on the

production of inflammatory cytokines and the anti-

inflammatory cytokine IL-10 in endotoxemic mice.

Administration of the cannabinoid receptor agonists

WIN 55212-2 and HU-210 before exposure to LPS re-

sulted in decrease in levels of the proinflammatory

cytokines tumor necrosis factor (TNF)-a and IL-12 in

serum concomitant with increased levels of the anti-

inflammatory cytokine IL-10 in mice. The cannabi-

noids also protected C. parvum-primed mice (but not

unprimed mice) against the lethal effects of LPS. The

investigators concluded that both cannabinoid agonists

modulated LPS responses through the CB1 receptor.

In another infection model, it was reported that THC

treatment of BALB/c mice resulted in diminution of

levels of IFNg and IL-12 and of IL-12 receptor b2

expression in response to Legionella pneumophila in-

fection (Klein et al. 2000). Studies using receptor

antagonists suggested that both the CB1 and CB2 were

linked functionally to the suppression of Th1 immunity

to Legionella, resulting in a decrease in IFNg and

IL-12. On the other hand, in a tumor model it was

reported that THC inhibited immunity by a CB2

receptor-mediated, cytokine-dependent pathway (Zhu

et al. 2000). Using two different weakly immunogenic

murine lung cancer models, it was shown that THC

decreased tumor immunogenicity. Levels of the im-

mune inhibitory Th2 cytokines, IL-10 and transforming

growth factor (TGF) were augmented, whereas those

of the immune stimulatory Th1 cytokine IFNg were

down-regulated, at both the tumor site and in spleens

of THC-treated mice. In vivo administration of the

CB2 antagonist SR144528 blocked the effects of THC,

suggesting that THC promoted tumor growth by

inhibiting antitumor immunity through a CB2 recep-

tor-mediated, cytokine-dependent pathway. Thus, the

collective data that have been obtained to date suggest

that cannabinoid effects on cytokine-dependent path-

ways correlate with a shift in cytokine expression pro-

file from that of Th1 proinflammatory to that of Th2

anti-inflammatory.

It is now recognized that endogenous cannabinoids

(endocannabinoids) such as AEA and 2-AG also af-

fect immune function. Schwarz et al. (1994) reported

that AEA inhibited mitogen-induced proliferation of

T and B lymphocytes and induced apoptosis at low

doses. Furthermore, it was reported that anandamide

inhibited macrophage-mediated killing of TNF-sensi-

tive cells, implicating the cognate proinflammatory cy-

tokine as affected by this endocannabinoid (Cabral

et al. 1995). Berdyshev et al. (1997) examined the ef-

fects of AEA, palmitoylethanolamide, and THC on

the production of TNF-a, IL-4, IL-6, IL-8, IL-10, IFNg,

p55, and p75 TNF-a soluble receptors expressed by

stimulated human peripheral blood mononuclear cells

as well as [3H]arachidonic acid release by nonstimu-

lated and N-formyl-Met-Leu-Phe (fMLP)-stimulated

human monocytes. AEA at low nanomolar concentra-

tions diminished production of IL-6 and IL-8 and

inhibited that of TNF-a, IFNg, IL-4, and p75 TNF-asoluble receptors at micromolar concentrations. Palmi-

toylethanolamide inhibited IL-4, IL-6, and IL-8 synthesis

and the production of p75 TNF-a soluble receptors at

concentrations similar to those of anandamide, but did

54

not affect TNF-a and IFNg production. Neither AEA

nor palmitoylethanolamide influenced IL-10 synthesis

but THC exerted a biphasic effect on the production

of proinflammatory cytokines. TNF-a, IL-6, and IL-8

synthesis was inhibited maximally at nanomolar levels

by THC but stimulated by this cannabinoid at mi-

cromolar levels. A similar effect was observed for

IL-8 and IFNg. The level of IL-4, IL-10, and p75

TNF-a soluble receptors was diminished by micro-

molar THC. [3H]Arachidonate release was stimulated

only by high THC and AEA concentrations. Based on

these observations, the investigators suggested that the

inhibitory properties of AEA, palmitoylethanolamide

and THC are determined by the activation of the

CB2 receptor, and that various endogenous fatty acid

ethanolamides also participate in the regulation of

the immune response. Recently, AEA has been

shown to exert an inhibitory effect on chemokine-

elicited lymphocyte migration (Joseph et al. 2004). The

inhibition of stromal derived factor 1 (SDF-1)-induced

migration of CD8+ T lymphocytes was found to be

mediated through the CB2 receptor. AEA also has

been reported to exert potentiating effects. Valk et al.

(1997) indicated that it acted as a synergistic growth

factor for primary murine marrow cells and hema-

topoietic growth factor (HGF)-dependent cell lines.

In addition, it was found that AEA augmented pro-

duction of IL-6 by astrocytes that had been in-

fected with Theiler’s murine encephalomyelitis virus

(Molina-Holgado et al. 1998). The enhancing effect of

AEA was blocked by the CB1 receptor antagonist

SR1417161A suggesting involvement of the cognate

receptor-mediated pathway in elevation of levels of

this pleiotropic cytokine.

In contrast to AEA, 2-AG has been associated

primarily with augmentation of immune responses. For

example, it was reported that 2-AG stimulated the

release of nitric oxide (NO) from human immune and

vascular tissues and from invertebrate immunocytes by

a mode that was linked to the CB1 receptor (Stefano

et al. 2000) and hematopoietic cells expressing the CB2

receptor migrated in response to 2-AG (Jorda et al.

2002). Distinct profiles for CB2 receptor expression in

lymphoid tissues was reported that was dependent on

the state of receptor activation, and it was proposed

that cell migration constituted a major function of the

CB2 receptor upon stimulation with 2-AG (Rayman

et al. 2004). Furthermore, it was demonstrated that

2-AG induced the migration of human promyelocytic

leukemia HL60 cells that had been differentiated into

macrophage-like cells and of human peripheral blood

monocytes and it was proposed that activity occurred

through a CB2-dependent mechanism (Kishimoto et al.

2003). It was demonstrated subsequently that 2-AG

caused accelerated production of chemokines by the

HL-60 cells (Kishimoto et al. 2004). In addition, rat

microglia have been reported to synthesize 2-AG

in vitro, an event that was proposed as linked to

increased proliferation through a CB2 receptor-depen-

dent mechanism (Carrier et al. 2004). In view of these

effects on the immune system, it is not surprising that

marijuana, THC, and select cannabinoids have been

reported to alter resistance to bacterial, protozoan,

and viral infections in experimental animals (Morahan

et al. 1979; Mishkin and Cabral 1985; Marciano-Cabral

1988; Cabral and Vasquez 1992; Klein et al. 1994;

Cabral and Marciano-Cabral 2004). However, a direct

link between susceptibility to infection with the human

immunodeficiency virus (HIV) or progression of HIV

infection to acquired immune deficiency syndrome

(AIDS) has yet to be established.

Modulation of antigen-presenting cells

A major target of the action of exogenous and endo-

genous cannabinoids appears to be cells of macro-

phage lineage. THC and other cannabinoids have been

shown to suppress macrophage functions such as

phagocytosis, bactericidal activity, and spreading

(Klein and Friedman 1990; Friedman et al. 1991). In

addition, THC has been shown to interfere with mac-

rophage cell contact-dependent lysis of tumor cells,

herpesvirus-infected cells, and amebae, and to deplete

soluble tumoricidal activity elicited by macrophages

exposed in vivo to the drug (Burnette-Curley et al.

1993; Burnette-Curley and Cabral 1995). These obser-

vations are consistent with reports that THC inhibits

the synthesis of proteins associated with primed and

activated macrophages (Cabral and Mishkin 1989),

alters cytokine secretion by activated macrophages

(Watzl et al. 1991; Nakano et al. 1992), and inhibits

cytokine gene expression by microglia (Puffenbarger

et al. 2000) (Fig. 3) resident macrophages within the

central nervous system (CNS).

Cannabinoids also have been found to affect the

production of NO by macrophages and macrophage-

like cells. Coffey et al. (1996) indicated that an early

step in NO production, such as NOS gene transcription

or NOS synthesis, rather than NOS activity was

affected by THC. The investigators concluded that

inhibition of NO was mediated by a process that de-

pended partly on a stereoselective cannabinoid receptor/

cAMP pathway and partly on a nonselective molecular

process. Using the murine RAW264.7 macrophage cell

line, it was demonstrated that THC inhibited NOS

55

transcription factors such as NF-kB/RelA, suggesting a

mode by which this cannabinoid affected NO production

(Jeon et al. 1996). Furthermore, it was reported that

CP55940 mediated the inhibition of inducible NOS

(iNOS) produced by neonatal rat brain cortical microglia

in a mode that was linked functionally to the CB1 receptor

(Waksman et al. 1999). On the other hand, other work

indicated that the synthetic cannabinoid, WIN55212-2,

had an opposite effect on constitutive NO and increased

its release from human monocytes and vascular tissues

through the CB1 receptor (Stefano et al. 2000).

Although it is now evident that cannabinoids exert a

variety of effects on a plethora of macrophage and

macrophage-like cell activities, a picture is emerging as

to the role of cannabinoid receptors in these processes

and the state of cell activation under which they occur.

Macrophages undergo a process of multistep activation

in response to multiple signals and to infection. These

cells progress successively from Bresting^ to Bresponsive,^Bresponsive^ to Bprimed,^ and Bprimed^ to Bfully ac-

tivated^ states. Each state in this multistep process is

characterized by differential gene expression and correl-

ative functional activities, suggesting a linkage of newly

expressed proteins to these acquired activities. This

differential expression of gene products in relation to

macrophage activation state applies also to the CB2

receptor. For these cell types, maximal levels of this

receptor are found for Bresponsive^ and Bprimed^ states

of activation, whereas minimal levels are detected for

Bresting^ and Bfully activated^ cells. This pattern of CB2

receptor expression applies also to microglia in the CNS

that also express the CB1 receptor at constitutive and

relatively low levels through all states of activation

(Carlisle et al. 2002). These collective observations sug-

gest the existence of an activation state Bwindow^ of

functional relevance for the CB2 receptor in cannabi-

noid-mediated alterations of macrophage activities.

Functional activities attributed to macrophages and

macrophage-like cells when in Bresponsive^ and

Bprimed^ states of activation include chemotaxis, phago-

cytosis, and antigen processing and presentation.

Sacerdote et al. (2000) reported that in vivo and

in vitro treatment with CP55940 decreased the in vitro

migration of macrophages in the rat and that this effect

involved both CB1 and CB2 receptors. THC also has

been reported to alter the gene expression, processing,

and secretion of an array of macrophage proinflamma-

tory and anti-inflammatory factors. Studies have dem-

onstrated that THC can differentially affect macrophage

processing and presentation of soluble protein antigens

that is necessary for the activation of CD4+ T lym-

phocytes (McCoy et al. 1995). It was shown that the

biochemical and biophysical nature of the antigen

apparently dictated processing outcomes to drug expo-

sure, since THC inhibited the processing of hen egg

lysozyme (HEL) (Fig. 4), had no effect on the process-

ing of chicken ovalbumin, and augmented that of pigeon

cytochrome c. This suggested that specific proteases,

whose function is requisite for processing of specific

antigens, were affected by THC (McCoy et al. 1995).

For example, cytochrome c has a heme group and a net

positive charge, is not glycosylated, and has no disulfide

IL-1α

IL-6

IL-1β

TNF-α

L32

GAPDH

VEH 1 5 10

CP55940 (µM)

80

60

40

20

0

% V

ehic

le C

on

tro

l

1 µM5 µM

10 µM

IL-1α IL-1β IL-6 TNF-α

A

B

Fig. 3. The potent synthetic cannabinoid agonist CP55940inhibits proinflammatory cytokine gene expression by microglia(Puffenbarger et al. 2000). Purified neonatal rat brain corticalmicroglia (106 cells) were treated with vehicle (0.01% ethanol)or CP55940 for 1 h followed by exposure to LPS (10 ng/ml) for6 h. Total RNA was then isolated from cultures and LPS-inducible cytokine mRNA species were detected using theRiboQuanti rCK-1 template set RNase protection assay(PharMingen, San Diego, CA) according to the manufacturer’sinstructions. The probes were made with [32P]UTP with a specificactivity of greater than 3000 Ci/mmol. The RNA samples werehybridized to the rCK-1 probe set overnight at 56-C. Theprotected fragments were subjected to RNase digestion,resolved on a 6% polyacrylamide gel containing 6 M urea, andimaged using XOMAT-AR film (Rochester, NY). The pixelintensity of each band was quantified using a Molecular Dynamics445SI Phosphorimager with the Image Quant 4.1 software(Molecular Dynamics, Sunnyvale, CA). The amount of cytokinemRNA was normalized for loading by dividing the pixel value forthe cytokine band by the sum of the pixel values for the twohousekeeping gene mRNAs for L32 and GAPDH. CP55940elicited a dose-related decrease in mRNA levels of IL-1a, IL-6,and TNF-a. (A) Autoradiogram of multiprobe RNase protectionassay. (B) Bar graphic representation of multiprobe RNaseprotection assay depicted in (A). The error bars representstandard error of the mean; P < 0.05 for all treatment groups

56

bonds. Cathepsin L, a cysteine protease, destroys the

T-cell epitope for cytochrome c. HEL is not glycosy-

lated, has a net positive charge but, in contrast to cy-

tochrome c, has four disulfide bonds. A cathepsin B has

been implicated in the degradation of lysozyme. Oval-

bumin has a simple mannose at one site and one

disulfide bond and has a requirement for a cathepsin D

(Asp protease) for its processing. The differential effects

of THC on processing occurred in a drug-dose-depen-

dent fashion, at concentrations that exceeded 10j8 M,

and were stereoselective based on the use of the

enantiomeric cannabinoid pairs, CP55940/CP56667,

suggesting linkage to a cannabinoid receptor. Finally,

using CB1 and CB2 cannabinoid receptor type-specific

antagonists (Fig. 5), it was demonstrated that the

inhibition in the processing of HEL was linked func-

tionally to the CB2 (McCoy et al. 1999). These obser-

vations related to antigen processing were confirmed

using CB2 receptor knockout mice (Buckley et al. 2000;

Chuchawankul et al. 2004).

Anti-inflammatory therapeutic potential

There are many historical reports of the use of mar-

ijuana in the treatment of acute and chronic inflam-

matory diseases such as rheumatism, chronic pain,

gastric distress, and protracted cough (Tomida et al.

2004). The usefulness of marijuana appeared to be

palliative rather than curative. However, because of

the many reports indicating that cannabinoid-based

drugs suppress mechanisms of innate and adaptive im-

munity, these agents are being intensely reevaluated

worldwide for their use in the treatment of inflamma-

tory diseases as well as other conditions such as obesity

and cancer (Baker et al. 2003; Di Marzo et al. 2004;

Fowler et al. 2005). Some promising results have been

obtained in animal models as well as in clinical trials

with human subjects. Interestingly, some of the effec-

tive compounds have low affinity for CB1 and CB2

suggesting that both receptor and nonreceptor mech-

anisms are involved in the mode of action.

Acute and chronic neuroinflammatory diseases

Inflammation of the brain and nervous tissue occurs

frequently in clinical medicine and often with devas-

tating disability and frequently death. Inflammatory

cells and immune mediators migrate from the blood

VEH 10 -10 10-9 10-8 10-7 10-6 10-5

THC (M)

180

0

30

60

90

120

150N

et C

PM

X 1

0-3

*

**

*

Lysozyme

Fig. 4. THC inhibits processing of lysozyme. Clone 63 cells(Ek

� : Ek� and Ad

� : Ad� ) as the antigen-presenting cells were

preincubated with 0.01% ethanol or various concentrations of THC

for 24 h. Hen egg lysozyme (HEL)-specific helper T-cell hybridoma

9.30.B2 (Ad� : Ad

�) served as the processed peptide responder T cells.

T cells and antigen (200 mM) were added to the cultures, and the

secretion of IL-2 by the T cells was measured after 24 h. Assay for

IL-2 was performed by incubating the IL-2-dependent cell line

CTLL-2 with 25% culture supernatants at 37-C for 18 h. The wells

were pulsed with 1 mCi [3H]thymidine (6.7 Ci/mMol, Amersham

Corp., Arlington Heights, IL) and harvested by a PHD cell harvester

after another 6 h. Radiolabel incorporation was measured by liquid

scintillation counting. A standard IL-2 preparation was incubated as

a positive control and consisted of supernatants from MLA-144 cells.

Values are the mean cpm�10j3 in experimental cultures minus the

mean cpm in medium control T SD. Asterisk (*) denotes significantly

different from vehicle control. Each experiment shown is repre-

sentative of three. The medium control was 2839 cpm. Vehicle

control vs. 10j8 and 10j7 M THC: P < 0.05. Vehicle control vs. 10j6

M THC: P < 0.01. Vehicle control vs. 10j5 M THC: P < 0.001. From

McCoy et al. (1995) with permissionTHC + SR144528

THC + SR141716A

THC

SR144528

SR141716A

Control

0 50 100 150 200

IL-2 (pg/ml)

*

*

Fig. 5. The CB2 antagonist SR144528 abolishes THC inhibitionof antigen processing of HEL. Clone 63 cells were incubatedwith vehicle, the CB2 antagonist SR144528 (1 mM), or the CB1

antagonist SR141716A (1 mM) for 4 h followed by treatmentwith vehicle of THC (10 nM) for an additional 24 h. T-cellstimulation assays were performed as described for Fig. 4. Val-ues are the mean secreted IL-2 T SD from triplicate cultures.The experiment is representative of three. Asterisk (*) denotessignificantly different from vehicle control; P < 0.02. The CB2

antagonist, but not the CB1 antagonist, reversed the THC-mediated inhibition of HEL processing. From McCoy et al.(1999) with permission

57

into the brain either acutely following head trauma or

infection or in a more chronic fashion in cases of

multiple sclerosis, Alzheimer’s disease, chronic infec-

tions, and other CNS insults. Animal models initially

suggested beneficial effects of cannabinoids in these

diseases (Fig. 6). For example, THC injection was

reported to attenuate the signs and symptoms of

EAE in rats and guinea pigs (Lyman et al. 1989)

and this effect was attributed to a drug-induced in-

crease in corticosterone (Wirguin et al. 1994). Howev-

er, cannabinoids also can attenuate neuroinflammatory

symptoms by decreasing Th1 activity (Croxford and

Miller 2003) and the activity of brain microglia cells

(Arevalo-Martin et al. 2003) possibly through the

action of both CB1 and CB2 receptors (Klein et al.

2000; Molina-Holgado et al. 2003). Animals with EAE

as well as patients with multiple sclerosis (MS) expe-

rience severe tremor and spasticity as a result of

neuroinflammation. These symptoms in animals were

shown to be attenuated by cannabinoids and mediated

through both CB1 and CB2 receptors (Baker et al.

2000, 2001; Pertwee 2002). That CB2 receptors are in-

volved suggests a role of immune inflammatory pro-

cesses in the etiology of these symptoms, possibly

through drug action on immune cells and attenuation

of the production of immune mediators that might

affect nerve conduction (see pain response in the next

paragraph). In total, the animal studies suggest that

cannabinoids can attenuate the EAE disease process

through both central effects on the brain and periph-

eral effects on immune function. Encouraged by these

animal studies, several clinical trials with MS patients

using the drug Sativex\ (GW Pharmaceuticals, UK), a

mixture of THC and CBD, have shown a beneficial

effect in reducing spasticity (Vaney et al. 2004; Wade

et al. 2004) and bladder dysfunction (Brady et al.

2004).

Cannabinoids from the beginning have been recog-

nized as analgesics able to control pain. This action

occurs at least partly through effects on neurotrans-

mission mediated by CB1 receptors in the CNS

(Howlett et al. 2004) and CB1 and CB2 receptors in

the periphery (Calignano et al. 1998). Chronic or

neuropathic pain can be quite severe and refractory

to treatment and, as the name implies, stems from

some type of injury to nerve tracks. This injury may

result from trauma, infection, ischemia disorders, or

neuroinflammation of unknown etiology such as in the

case of MS. Animal models of neuropathic pain have

shown an attenuating effect of cannabinoids. For

example, experimental pain induced in rats by ligation

of spinal nerves was reversed by the highly potent CB2

agonist, AM1241 (Ibrahim et al. 2003); furthermore,

the effect was reversed by a CB2 antagonist and was

unaltered in CB1 knockout mice. This study showed

the high potency of cannabinoids in suppressing this

type of pain and showed that the effect was totally

mediated by CB2 and not CB1. The mechanism was

speculated to involve the desensitization of afferent

neurons by suppressing immune mediators capable of

sensitizing these neurons. In a similar animal study,

both neuropathic and inflammatory pain were attenu-

ated by the high-affinity ligand, HU-210, and the lower

affinity cannabinoid, ajulemic acid (Mitchell et al.

2005). The effect of the latter was not accompanied

by the psychoactive side effects of HU-210, and the

mechanism of action was speculated to include weak

CB2 effects, effects on vanilloid receptors or on

FAAH, or other anti-inflammatory modes of action

such as suppression of cyclooxygenase and IL-1. These

animal studies have led to clinical studies examining

the attenuating effect of cannabinoids in neuropathic

pain associated with MS. Orally administered THC

(Svendsen et al. 2004) and oromucosal spray delivery

of Sativex\ (Rog et al. 2005) led to significant re-

duction in pain in placebo-controlled studies involving

90 MS patients, and the drugs, although psychoactive,

were reported to be well tolerated.

Acute inflammation of the brain following traumatic

injury appears to be associated with a rise in 2-AG in

the brain (Panikashvili et al. 2001) and to be attenu-

ated by endocannabinoids and the nonpsychoactive

synthetic cannabinoid, HU-211 (Shohami et al. 1997;

Panikashvili et al. 2001; Mechoulam et al. 2002). This

would suggest that both cannabinoid receptor and

nonreceptor mechanisms are involved in the mecha-

nism of action. 2-AG given intravenously to mice after

closed-head injury was observed to cause a significant

CANNABINOIDDRUGS

MultipleSclerosis

TraumaticBrain Injury

NeuropathicPain

Alzheimer’s Disease

Arthritis Septic ShockFig. 6. The efficacy of cannabinoid-based drugs has beenexamined in a variety of preclinical (animal studies) and clinicalmodels and diseases including neuroinflammatory diseases.These include neuropathic pain, multiple sclerosis, traumaticbrain injury, and Alzheimer’s disease, as well as other chronicinflammatory diseases such as arthritic and septic shock

58

reduction in brain edema and better clinical improve-

ment; the effects were CB1 mediated (Panikashvili

et al. 2001) but the mechanism was not described.

Neuroprotection has also been described following

treatment with HU-211 and the mechanism here ap-

pears to involve antagonism of the NMDA receptor

and suppression of TNF-a release in the brain. The

mechanism of the latter effect is not known but may

involve the antioxidant effect of HU-211 (Mechoulam

et al. 2002). These animal results have led to clinical

studies with HU-211 with mixed results. In a phase II

trial, the drug was well tolerated and resulted in some

improvement in intracranial pressure and neurological

outcome (Knoller et al. 2002). However, a recent pre-

liminary report of a phase III study failed to show

efficacy in clinical outcome in a large placebo-con-

trolled study (see Pharmos Corporation, http://www.

pharmoscorp.com/news/pr/pr122004.html). Thus, it ap-

pears that cannabinoid-based drugs can inhibit a num-

ber of proinflammatory mechanisms induced by brain

injury, some involving the glutamate excitotoxicity

pathway, others involving the proinflammatory prod-

ucts from microglia cells, and still others involving re-

active oxygen pathways.

Finally, a recent report suggests that cannabinoids

and receptors mediate disease progression in Alzheim-

er’s disease (Ramirez et al. 2005). Senile plaques from

Alzheimer’s brains expressed both CB1 and CB2; how-

ever, CB1 positive neurons were decreased in these

areas relative to areas containing resting microglia

cells in age-matched controls. Furthermore, intracere-

broventricular injection of high-affinity cannabinoid

ligands into bA25–35-injected rats attenuated microglial

activation, cognitive impairment, and loss of neuronal

markers, and cannabinoids also suppressed the reac-

tivity of microglia cells in culture to treatment with

bA25–35. These results suggested that the endocanna-

binoid system of the brain may be disrupted in

Alzheimer’s disease and that cannabinoids can damp-

en the inflammatory responses and neurodegeneration

associated with this disease.

Arthritis

Cannabinoid-based drugs have been studied in animal

models of arthritis with two cannabinoids being most

extensively studied. The first of these is ajulemic acid,

which is a dimethylheptyl derivative of the cannabi-

noid metabolite, THC-11-oic acid (Burstein et al.

2004). Several years ago, ajulemic acid was shown to

be anti-inflammatory in the paw edema model in mice

and to inhibit leukocyte adhesion when given orally

(Burstein et al. 1992). The compound was also shown

to suppress pain (see above) and to have moderate

affinity for CB1 receptors (Rhee et al. 1997) and

display some psychoactive effects (Dajani et al. 1999).

Additional studies have shown this compound to

suppress leukocyte infiltration and lessen histopathol-

ogy in mouse models of acute inflammation and

chronic adjuvant arthritis (Zurier et al. 1998). The

gastrointestinal symptoms of ajulemic acid, in another

study, were less than that of indomethacin when

administered to rats (Dajani et al. 1999). The mecha-

nisms of the anti-inflammatory effects are not clear at

this time. Because ajulemic acid has some CB1 binding

affinity, cannabinoid receptor mechanisms could be

involved in immune suppression. However, this com-

pound also suppresses prostaglandin production

(Zurier et al. 1998), inhibits the production of IL-1

(Zurier et al. 2003), and activates peroxisome prolif-

erator-activated receptor g while suppressing the IL-8

gene promoter (Liu et al. 2003).

Drug effects on arthritis have also been examined

using another compound, the natural cannabinoid,

CBD, and a dimethylheptyl derivative of CBD, HU-

320 (Bisogno et al. 2001). CBD was shown to block the

progression of collagen-induced arthritis in mice and

to suppress the activation of specific T cells and pro-

duction of TNF-a by joint synovial cells (Malfait et al.

2000). The drug was shown to suppress a number of

immune lymphocyte and macrophage functions

in vitro, suggesting that it attenuated arthritis by sup-

pressing immune activation. The dimethylheptyl de-

rivative, HU-320, more potently suppressed arthritis

along with suppressing immune function These com-

pounds have very low affinity for cannabinoid recep-

tors but do have some effects on vanilloid receptors

and the uptake of AEA (Bisogno et al. 2001); how-

ever, the mechanism of immune suppression is not

clear at this time.

Septic shock

In the pathophysiology of septic shock, pro-inflamma-

tory mediators are released in excess leading to throm-

bosis, vasodilation, capillary leakage, and organ-system

failure. Recent evidence suggests that cannabinoids can

influence vasodilation and that shock symptoms are

attenuated by cannabinoid-based drugs. Early findings

demonstrated that endocannabinoids such as AEA and

2-AG are generated in the blood during LPS-induced

hypotension (Varga et al. 1998). Furthermore, the hypo-

tensive response to either LPS (Wagner et al. 1998) or

endocannabinoids (Varga et al. 1998) was shown to be

attenuated by the CB1 antagonist, SR141716A. As dis-

cussed above, the mechanism of this effect might

59

involve a receptor other than CB1 that is sensitive to

endocannabinoids but not high-affinity agonists such as

CP55940 (see receptors above). Support for a role of

the endocannabinoid system in septic shock comes from

a report showing a 4-fold increase in AEA and 2-AG in

the blood of patients with this condition (Wang et al.

2001). Vascular inflammation subsequent to infarct also

appears to involve cannabinoid-mediated mechanisms.

An animal model of myocardial ischemia/reperfusion

documented an attenuation of infarct size following

treatment with WIN55-212-2 (a nonselective, amino-

alkylindole cannabimimetic agent) and the effect was

inhibited by the CB2 antagonist, AM630 (Di Filippo

et al. 2004). The protective effect of the WIN com-

pound was accompanied by a lowering of leukocyte

influx and decrease in cytokine and chemokine levels in

inflamed tissues. The authors speculated that the drug

was attenuating arteritis by affecting inflammatory cells

through CB2 receptors inhibiting the inflammatory

cascade (Di Filippo et al. 2004). Finally, there is

evidence that septic shock can be attenuated by a

cannabinoid with little cannabimimetic activity. HU-211

has very low affinity for either CB1 or CB2 and, in ad-

dition to suppressing brain edema (see above), was

shown to rescue mice and rats from endotoxic shock

following LPS injection (Gallily et al. 1997). The mech-

anism of action of this drug at least partially involved

the suppression of TNF-a production, a primary

mediator of septic shock. How HU-211 suppresses

TNF-a is not known and will require further study on

the mode of action of these lipid mediators.

Summary

It is clear that cannabinoids modulate the immune

system leading to extensive suppression of cellular

function and cytokine production. The immune system

also contains and is most probably regulated by an

endocannabinoid system of receptors and endogenous

ligands. The function of antigen-presenting cells ap-

pears to be a significant target of cannabinoid action in

that these cells are readily modulated by cannabinoid-

based drugs; they express cannabinoid receptors and

readily produce endocannabinoids and other lipid

mediators. The suppressive action of cannabinoids on

antigen-presenting cell function is intimately involved

in the anti-inflammatory effect of these drugs in

attenuating the symptoms of chronic inflammatory

diseases. Suppression of the many effector mechanisms

of these cells has been demonstrated in the treatment

course of both preclinical and clinical models of

disease such as neuroinflammatory diseases, arthritis,

and septic shock. Continued studies designed to ma-

nipulate the endocannabinoid system operating in the

immune system in general, and antigen-presenting

cells in particular, should be of value in terms of the

development of new therapeutic strategies for the

treatment of chronic inflammatory diseases.

Acknowledgments This study was supported in part by DA03646(TWK) and DA05832 and DA15608 (GAC) from NIDA.

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