Int. J. Immunopharmac, Vol. 15, No. 4, pp. 523-532, 1993. Printed in Great Britain.

0192-0561/93 $6.00 + .00 Pergamon Press Ltd.

International Society for lmmunopharmacology.

S U P P R E S S I O N OF L Y M P H O C Y T E A D E N O S I N E 3' : 5 ' - C Y C L I C MONOPHOSPHATE (cAMP) BY DELTA-9-TETRAHYDROCANNABINOL

SHARON DIAZ,* STEVEN SPECTER *t and RONALD G. COFFEY**

*Department of Medical Microbiology and Immunology and *Department of Pharmacology and Experimental Therapeutics, University of South Florida College of Medicine, Tampa, FL 33612, U.S.A.

(Received 27 October 1992 and in final form 9 February 1993)

Abstract - - Delta-9-tetrahydrocannabinol (THC) is the major psychoactive component of marijuana. Suppression of mitogen-stimulated blastogenesis of human lymphocytes in vitro by THC was previously demonstrated. This effect was shown to be concentration dependent with the non-toxic concentrations 5, 7.5, and 10 Mg THC/ml showing the greatest suppression. However, the mechanism(s) by which THC induces suppression are still unclear. The current study examines the effect of THC on the adenosine 3 ' : 5'-cyclic monophosphate (cAMP) pathway second messenger system, which is involved in activation of human peripheral blood lymphocytes. Lymphocyte cAMP levels were stimulated using three hormone receptor stimulators, isoproterenol, histamine, or 5 ' -N-ethylcarboxamide adenosine (NECA), each of which utilizes a different receptor to enhance cAMP production. THC suppressed cAMP levels independently of the hormone and receptor utilized. Levels of cAMP in non-mitogen-stimulated peripheral blood mononuclear cells and plastic non-adherent lymphocytes, as well as cells stimulated with phytohemmagglutinin, were suppressed by THC. Suppression of cAMP production by THC was further examined to determine whether inhibition involved a GTP-binding protein (G~), which is known to down-regulate cAMP production. Cells were pre-treated with pertussis toxin to inhibit G~ activity; this blocked the THC-induced suppression of cAMP production. These results suggest that THC can exert its effects on second messenger systems at the lymphocyte membrane level, and that a pertussis toxin-sensitive G~ protein may be involved. Thus, second messenger regulated pathways may be involved in THC-induced immune suppression. However, the relationship between alteration of cAMP production and suppression of lymphocyte function due to the presence of THC in the medium remains to be established.

Delta-9-tetrahydrocannabinol (THC), the major psychoactive component o f mari juana, has been shown to be immunosuppressive. T H C suppresses a variety of immune functions including lymphocyte blastogenic t ransformat ion in response to mitogens (Specter, Lancz & Hazelden, 1990), lymphokine- activated killer cell activity (Kawakami, Klein, Newton, Djeu, Specter & Friedman, 1988), natural killer (NK)-cell activity (Kawakami et al., 1988), and leukocyte adhesion (Audette & Burstein, 1990). T H C is also capable of altering the relative numbers of T-helper and T-suppressor lymphocyte subpopulations (Pross, Klein, Newton, Smith, Widen & Friedman, 1990). These effects o f T H C on immune function appear to result in the host

displaying diminished resistance to microorganisms such as Listeria monocytogenes (Morahan, Klykken, Smith, Harris & Munson, 1979), and viral infections (Morahan et al., 1979; Fr iedman et al., 1988; Fischer-Stenger, Updegrove & Cabral , 1992). In addition, there are reports that mari juana use may increase the likelihood of developing certain types of cancer, most notably head and neck cancers (Donald, 1991; Taylor, 1988). Despite the numerous reports on the effect o f T H C on cells o f the immune system, the mechanism(s) by which this compound depresses immune function remain undetermined.

A significant number of reports have suggested that the actions of T H C are modulated through second messenger cell activation systems (Heindel &

~Author to whom correspondence should be addressed at: Department of Medical Microbiology and Immunology (MDC Box 10), University of South Florida College of Medicine, 12901 N. Bruce B. Downs Blvd, Tampa, FL 33612, U.S.A.

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Keith, 1989; Howlett & Fleming, 1984; Howlett, Qualy & Khachatrian, 1986; Little & Martin, 1991; Matsuda, Lolait, Brownstein, Young & Bonner, 1990; Rowley & Rowley, 1990). Recently a cannabinoid receptor was demonstrated to be expressed on neural cell lines (Matsuda el al., 1990), human testis (Gerard, Mollereau, Vassart & Parmentier, 1991) and mouse spleen cells (Kaminski, Abood, Kessler, Martin & Schatz, 1992). This receptor was shown to be coupled to a GTP-binding (G) protein and binding of THC to the receptor resulted in a decrease in adenylate cyclase activity (Matsuda et al., 1990). Although such a receptor has not been directly demonstrated on human lymphocytes, THC may still act to alter lymphoid cell function through similar mechanisms. THC has been shown to inhibit adenylate cyclase activity in a variety of cell types including: neuronal cell lines (HowleR, 1984), human leukemia cells (Rowley & Rowley, 1990), synaptosomes (Little & Martin, 1991), Sertoli cells (Heindel & Keith, 1989), and mouse spleen cells (Schatz, Kessler & Kaminski, 1992). This inhibition is thought to be due to the actions of an inhibitory G protein (G~) (Howlett et al., 1986; Matsuda et al., 1990).

Clearly, it is now established that the interactions of THC with a variety of cell types involve second messenger molecules (Evans, Formukong & Evans, 1987; Chaudry, Thompson, Rubin & Laycock, 1988; Yebra, Klein, Coffey & Friedman, 1991). Only after each step of the molecular pathways of lymphocyte activation are dissected will a comprehensive understanding of the involvement of second messenger systems in THC-induced immunosuppres- sion be elucidated. Thus, the effects of THC on the various signalling pathways must be considered before there is a clear picture of the impact of this drug on regulation of the immune system.

As indicated above, previous studies using other cell types have indicated that THC mediates its actions through a Gi protein (Howlett et al., 1986; Matsuda et al., 1990); therefore, extensive studies were performed to determine if a pertussis toxin- sensitive G~ protein was involved in the observed immunosuppression. Pertussis toxin ADP-ribo- sylates a Gi protein which causes it to become inactivated (Hewlett, Cronin, Moss, Anderson, Myers & Pearson, 1984). Therefore, if THC is acting solely through a pertussis toxin-sensitive G protein, THC should have no effect on cyclic 3 ' : 5 ' adenosine monophosphate (cAMP) concentrations of cells pre-incubated with pertussis toxin.

In this paper we describe the effects of THC on the cAMP second messenger pathway of human

peripheral blood mononuclear cells (PBMC). Our studies show that THC suppresses cAMP levels, which appears to be due to the interaction with a pertussis toxin-sensitive G protein, G~.

EXPERIMENTAL PROCEDURES

Human subjects

Human subjects were healthy, adult, volunteer blood donors. Blood was obtained in the form of buffy coats from the Southwest Florida Blood Bank (Tampa, FL).

Reagents

Tissue culture medium consisted of RPMI-1640 medium purchased from GIBCO (Grand Island, NY), supplemented with 20 mM HEPES (GIBCO), 2 mM L-glutamine purchased from Sigma Chemical Co. (St Louis, MO), 100 U/ml penicillin plus 100 ~g/ml streptomycin (GIBCO) and 10% control- led processed serum replacement-1 (CPSR-1) purchased from Sigma. HBSS was purchased from GIBCO and supplemented with 25 mM HEPES (GIBCO). Phytohemagglutinin, pertussis toxin, histamine, isoproterenol, NECA, and forskolin were purchased from Sigma Chemical Co. (St Louis, MO). Rolipram was generously supplied by Berlex Laboratories (Cedar Knolls, N J).

Lymphocy te preparations

Buffy coats were separated using the Ficoll - Hypaque method (Ficoll - Hypaque, Pharmacia Inc., Piscataway, N J) as described by Boyum (1968). Briefly, whole blood diluted in phosphate buffered saline (PBS) pH 7.4, 1 : 1 (v/v), was layered over Ficoll - Hypaque and centrifuged at 1600 revs/min (400g) for 30 min at room temperature. Flowing centrifugation the band containing the peripheral blood mononuclear cells (PBMC) was removed and washed free of F i c o l l - H y p a q u e using 10 ml PBS. After 3 washes, cells were resuspended in RPMI-1640 supplemented with 10% CPSR-1, 20 mM HEPES, 2 r aM L- glutamine, 100 U penicillin/ml and 100 ~g strepto- mycin/ml. CPSR-1 was used since it contains a reduced amount of lipids and other serum compo- nents as compared to serum. Serum is known to reduce the effective concentration of THC due to binding of the drug to serum constituents, such as albumin (Wahlqvist, Nilsson, Sandberg & Agurell, 1970). This inactivation is diminished with the use of the serum substitute.

cAMP Suppression by THC

Marijuana components

THC was obtained from the National Institute on Drug Abuse (NIDA, Bethesda, MD) as a solution in ethanol at 200 mg/ml. Stock THC in alcohol was stored at - 2 0 ° C . One hundred microliters of THC solution was placed in a tube, and the alcohol evaporated using a stream of nitrogen gas. The dried THC was dissolved in 1 ml reagent grade dimethyl sulfoxide (DMSO) to yield a stock concentration of 20 mg/ml according to a protocol supplied by NIDA. THC in DMSO was prepared fresh on the day of its use. Dilutions of the DMSO diluent equivalent to the amount present in THC dilutions were used as a control, e.g. 10/ag (3.2 × 10 5 M) THC/ml contains 0.05°7o DMSO.

THC treatment

THC was prepared fresh daily in DMSO and diluted in medium immediately prior to addition to cells as previously described (Specter et al., 1990). Cells were incubated for various times, ranging from 10 min to 18 h at 37°C, in a humidified atmosphere of 5070 CO2, 95°70 air. THC ranging from 1 to 10/ag/ ml were used based upon previous studies in this laboratory indicating that these concentrations were immunosuppressive and not cytotoxic (Specter et al., 1990; Specter, Klein, Newton, Mondragon, Widen & Friedman, 1986; Specter, Rivenbark, Newton, Kawakami & Lancz, 1989). THC pre-incubation times were chosen based on the demonstrated effects of the drug in biological assays (Specter et al., 1986, 1989). Ten minute THC incubations were chosen for some assays in order to correlate with published effects of THC on cAMP production in immune cells (Schatz et al., 1992). Three hour THC incubations were chosen to correlate with previous studies in this laboratory demonstrating THC-induced suppression of NK-cell activity (Specter et al., 1989).

cAMP stimulation

Peripheral blood mononuclear cells (2 × 107/ tube) were incubated in RPMI medium for varied time periods detailed for each experiment with the indicated concentration of THC for each experi- ment, DMSO (as a control), or 2.5/ag phytohemag- glutinin (PHA) / ml - - a mitogen used to stimulate T-lymphocytes. At the end of the incubation period cells were centrifuged and resuspended (2 x 10 6

cells/ml) in Hanks' balanced salt solution (HBSS) with 25 mM HEPES. To prevent the breakdown of cAMP by phosphodiesterases, rolipram (50/aM), a specific cAMP phosphodiesterase inhibitor, was added for 10 min prior to cAMP stimulation.

525

Lymphocyte cAMP was stimulated for 10 rain using three different receptor-mediated stimulators: iso- proterenol (Iso, 10 -6 M), which binds to the /3- adrenergic receptor; histamine (Hist, 10-5 M), which uses the H2 receptor, or 5 '-N-ethylcarboxamide ( N E C A , 10 -4 M), an adenosine analog that binds to the A2 receptor, or forskolin (10 -5 M), which activates adenylate cyclase directly, bypassing recep- tor mechanisms. Concentrations of cAMP stimula- tors were chosen based on studies by Box, Portenier & Staehelin (1987) and Coffey & Hadden (1985). The accumulation of cAMP was terminated and cells lysed by the addition of 1.0 M perchloric acid (PCA) as described below.

Purification o f cAMP from lymphocytes

Purification of cAMP was performed as described by Coffey, Davis & Djeu (1988). Briefly, upon termination of cAMP accumulation by the addition of an equal volume of cold 1 M perchloric acid (PCA), cells were pelleted by centrifugation at 3000 revs/min (1000g) for 15 min at 4°C. Super- natant fluids containing released cAMP were puri- fied by column chromatography using neutral alu- mina columns (0.22 g). Columns were washed to remove impurites using 0.5 M PCA and water. Cyclic AMP was eluted from the column using 0.2 M sodium acetate, pH 6.2. Cyclic AMP was measured after acetylation using a competitive radioummuno- assay using rabbit polyclonal antibodies and ~25I-tyrosine-succinyl methyl esters of cAMP pre- pared in our laboratories by the method of Brooker, Harper, Terasaki & Moylan (1979).

Statistics

Data in Fig. 1 were evaluated using a two-tailed Student's t-test. Data in all other figures were evaluated using a Student's t-test for unequal variances.

RESULTS

Suppression by THC o f induction o f peripheral blood leukocyte cAMP

Following a 3 h incubation of lymphocytes with THC, DMSO and/or PHA, cells were incubated for 10 min with the 3 cAMP stimulators. PBMC (2 × 106 cells/ml) were incubated for 10 min with rolipram to prevent cAMP breakdown. Figure 1 shows basal and isoproterenol stimulated cAMP

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Dose-dependent suppression of PBMC cAMP by THC

Cells were incubated with 0.050°7o DMSO, 1, 5, or 10/ag THC/ml and/or 2.5/ag/ml PHA for 10 min followed by a 10 min stimulation with forskolin. THC was not washed out prior to stimulation with forskolin. The reaction was terminated and cAMP levels were determined as described in Experimental Procedures. THC significantly reduced cAMP levels of non-PHA-activated cells at all doses tested (Fig. 3). When PHA was used to stimulate the PBMC, significant decreases were observed at 5 and 10/~g THC/ml as compared to the DMSO controls. THC concentrations of 5 - 10/ag THC/ml were used in all other experiments.

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Fig. 1. Suppression of peripheral blood mononuclear cell cAMP by THC. PBMCs were incubated for 3 h with 0.025°7o DMSO, or 5/ag THC/ml. Cells were then centri- fuged~ resuspended in HBSS, and 50/aM rolipram was added for 10 min followed by the addition of isoproterenol (Iso, 10 -6 M) for 10 min as described in Experimental Procedures. The reaction was terminated with PCA and cAMP levels determined, cAMP levels are expressed as fmole/106 cells. The data represent the mean _+ S. D. for a single experiment performed in triplicate. *Statistical analysis was performed using a two-tailed Student's t-test at

P<0.05.

values expressed as fmole/106 cells from 3 different blood donors. Because of the variability of basal cAMP levels between different blood donors, when these values were averaged, high standard deviation values were attained. In order to compare results from several experiments, data were expressed as percent of control in the subsequent figures, cAMP levels of hormone stimulated cells were consistently elevated as compared to basal levels in Fig. 1 and all other experiments presented in this paper. THC was shown to suppress cAMP levels of peripheral blood mononuclear cells (PBMC) as compared to medium or DMSO controls, when isoproterenol and histamine, but not NECA, were used to enhance cAMP production (Fig. 2). The results with NECA may represent an anomaly, since in other experiments NECA-stimulated cAMP was inhibited by THC. THC also significantly reduced cAMP levels in PHA-activated PBMC, regardless of the cAMP inducer used (PHA data are not shown). Apparent changes in cAMP levels due to the presence of DMSO in the medium were not significant.

Suppression of non-adherent leukocyte cAMP by THC

Having established that THC significantly reduced cAMP levels in PBMC, studies were performed to determine which populations of cells were affected by THC. PBMC were allowed to adhere to plastic tissue culture flasks for 1 h at 37°C, 5°7o CO2 (2 × 108 cells/75 cm 2) in complete medium. The non-adherent cells were collected, washed and used in this assay as a monocyte/macrophage depleted population of lymphocytes. Cells were incubated with THC, DMSO, or medium and analyzed as described above for both PHA-stimulated and non- mitogen-stimulated cells. Results were similar to the data using non-separated PBMC, demonstrating that THC significantly reduced cAMP levels in non- adherent lymphocytes (Fig. 4). In contrast to PBMC, however, THC significantly reduced cAMP levels in lymphocytes stimulated by NECA as well as other hormones. This occurred in the absence or presence (data not shown) of PHA.

Time course studies o f THC-induced suppression of cAMP levels

Studies were performed to determine both the short-term and long-term effects of THC on PBMC. Preliminary studies indicated that a 10 min incubation with THC decreased cAMP concentra- tions in PBMC (Figs 6 and 7). We extended the incubation times to 1, 3, 8, or 18 h with THC, DMSO, or medium, in the presence or absence of PHA, prior to induction of cAMP to determine the effect of THC on these longer incubation times. The results of the time course studies using isoproterenol as the cAMP inducer are shown in Fig. 5. Although all 3 hormones increased cAMP in all cells, the effect of PHA in decreasing cAMP was noted only with

cAMP Suppression by THC 527

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Fig. 2. Effect of THC on peripheral blood mononuclear cell cAMP levels. Cells were incubated for 3 h with medium, 0.025% DMSO or 5/ag THC/ml. Cells were then centrifuged, resuspended in HBSS and 50/am rolipram was added for 10 min followed by the addition of the cAMP stimulators for 10 min. The reaction was terminated with PCA and cAMP levels determined. The data are expressed as percent of medium controls for each cAMP stimulator. The data represent the means _+ S.E.M. for 3 individual experiments performed in triplicate. Control cAMP values (fmole/106 cells) were as follows: Basal, 639; Iso, 3337; Hist, 1475; and NECA, 2347. *Statistical significance compared to the DMSO control at

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Fig. 3. Dose-dependent suppression of PBMC cAMP by THC. Cells were incubated for 10 min with 50/aM rolipram followed by a l0 min incubation with 0.05O7o DMSO, 1, 5, or 10/ag THC/ml. Cells were then stimulated for 10 min with 10-5M forskolin, and the reaction was terminated with PCA. cAMP levels were determined and the data are expressed as percent DMSO control. The data represent the mean _+ S.E. for 3 individual experiments performed in triplicate. Control (DMSO + forskolin) cAMP values were 14,169 fmole/106 cells. *Statistical significance compared

to the DMSO control at P<0.05.

isoproterenol . T H C decreased c A M P levels in the absence of P H A at every t ime point examined except for the 1 h t ime point . The 3 and 18 h t ime points showed the greatest decreases. W h e n P B M C were s t imulated with P H A , no significant differences between the DMSO- and THC- t rea t ed cells were observed, c A M P data using P H A as a lymphocyte ac t ivator were highly var iable between b lood donors for reasons which are unclear at this t ime. Similar results demons t r a t ing T H C reduct ion of c A M P levels were also observed when h is tamine or N E C A was used to induce c A M P produc t ion in these t imed studies (data no t shown). F r o m these results ei ther a 10 rain or a 3 h incuba t ion with T H C were used to demons t ra t e short- and long- te rm effects of THC.

Pertussis toxin alleviates THC-induced suppression of cAMP levels

Previous studies using o ther cell types have indicated tha t T H C mediates its act ions t h r o u g h a G~ prote in; therefore , studies were pe r fo rmed to de termine if a pertussis- toxin-sensi t ive G~ prote in was involved in the observed immunosuppress ion . Pertussis toxin ADP-r ibosy la tes a G~ pro te in which causes it to become inact ivated. Therefore , i f T H C is acting t h rough a pertussis- toxin-sensi t ive G prote in , p re- incubat ing the cells with pertussis toxin should preclude any effect T H C would have on c A M P levels. Cells were pre- incubated with varying concen t ra t ions of pertussis toxin for 24 h fol lowed

528 S. DIAZ et al.

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Fig. 4. Suppression of non-adherent leukocyte cAMP by THC. PBMC were adhered for 1 h to plastic and the non- adherent cells were collected and used in the same experimental design as stated in Fig. 1. Control cAMP values (fmole/l& cells) were as follows: Basal, 918; Iso, 4067; Hist, 1956; and NECA, 2797. *Statistical significance compared to the DMSO

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Fig. 5. Time course studies of THC-induced suppression of isoproterenol-stimulated cAMP levels. PBMC were incubated for 1, 3, 8, or 18 h with 5/ag/ml DMSO and/or 2.5/ag PHA/ml. Cells were then centrifuged, resuspended in HBSS and rolipram added for 10 min followed by the addition of isoproterenol (10 -6 M) for 10 rain to stimulate cAMP production. The reaction was terminated with PCA and cAMP levels determined. The data represent the means _+ S.E.M. for 3 individual experiments preformed in triplicate. Control cAMP values (fmole/106 cells) were as follows: 1 h, 4716; 3 h,

4358; 8 h, 3583; and 18 h, 1869. *Statistical significance compared to the DMSO control at P<0.05.

by a 10 min (Fig. 6) or a 3 h (Fig. 7) incuba t ion with 10/~g T H C / m l . T H C was not washed out pr ior to s t imula t ion with forskol in or isoproterenol . Pre l iminary studies indicated tha t a m i n i m u m of a 24 h incuba t ion of P B M C with pertussis toxin was needed (data no t shown). The results demons t ra t e tha t at concen t ra t ions of pertussis toxin as low as 1 n g / m l , T H C was unab le to decrease c A M P levels s t imulated by forskol in (Fig. 6). W h e n cells were

incuba ted with T H C for 3 h fol lowed by s t imula t ion with isoproterenol , reversal of T H C suppress ion by pertussis toxin was observed at concent ra t ions of 100 and 1000 n g / m l (Fig. 7). As the concen t ra t ions of pertussis toxin increased, the c A M P levels increased in bo th D M S O - and THC- t rea t ed cells (Figs 6 and 7). Pertussis toxin blocked the THC- induced suppress ion of c A M P levels in bo th forskol in (Fig. 6) and isoproterenol-s t imulated

cAMP Suppression by THC

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Fig. 6. Pertussis toxin blocks THC-induced suppression of forskolin stimulated PBMC cAMP levels. PBMC were pre-incubated with various concentrations of pertussis toxin for 24 h. Cells were then treated for 10 rain with 10/ag THC/ml or 0.05% DMSO, followed by a 10 min stimulation with forskolin. Data represent the means -+ S.E.M. for 3 individual experiments performed in tripli- cate. Control (DMSO + forskolin) cAMP values were 3528 fmole/106 ceils. *Statistical significance compared to the

DMSO control at P<0.05.

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Fig. 7. Pertussis toxin alleviates THC-induced suppression of isoproterenol stimulated PBMC cAMP levels. PBMC were pre-incubated with various doses of pertussis toxin ranging from 1 ng/ml to 1 gg/ml for 24 h followed by 3 h incubation with 10 gg THC/ml. Isoproterenol was used to stimulate cAMP levels. Data represent results from 3 experiments performed in triplicate, expressed as percent of DMSO control. Control (DMSO + isoproterenol) cAMP values were 1227 fmole/106 ceils. *Statistical significance compared to the DMSO control at P<0.05.

(Fig. 7) PBMC. Cell viability was determined to be >90% by trypan blue exclusion in all experiments using pertussis toxin.

DISCUSSION

These data indicate that THC suppresses cAMP levels in both resting and PHA-stimulated human peripheral blood mononuclear cells, as well as non- adherent lymphocytes. Results were achieved using forskolin or three different receptor-mediated stimulators of cAMP, histamine, isoproterenol and NECA, each of which acts through a different hormone receptor. THC suppressed cAMP levels independently of the hormone receptor used to stimulate cAMP. Data using NECA to stimulate cAMP production were inconsistent. The observed decrease in cAMP levels mediated by THC was shown to be concentration-dependent.

Others (Howlett et al., 1986; Matsuda et al., 1990) have shown that THC lowers adenylate cyclase activity in other tissues via a pertussis-toxin-sensitive G~ protein. Pertussis toxin was used to determine

whether the observed suppression in cAMP levels was a result of THC exerting its effects on a GTP- binding protein that inhibits adenylate cyclase activity. Bordetella pertussis secretes an exotoxin that causes ADP-ribosylation of G~ which renders G~ inactive. When ceils were incubated with pertussis toxin prior to incubation with THC the toxin should have abrogated suppression of cAMP accumulation, provided that THC acts through G~. The concentration of pertussis toxin that caused the reversal of THC suppression of cAMP levels was dependent on the length of incubation with THC and the choice of cAMP stimulator. The results indicated that pertussis toxin blocked the THC-mediated suppression of cAMP production. Thus, depression of cAMP induction appears to be mediated via a pertussin-toxin-sensitive G~ protein. This is consistent with the findings of other cell types and suggests that the cannabinoid receptor may be present on human PBMC.

Cyclic AMP is a key regulatory molecule for leukocyte function (Coffey, 1988) and is known to inhibit several lymphocyte functions (Hadden & Coffey, 1990; Kammer, 1988). Therefore, the

530

observed decreases in cAMP levels following exposure of lymphocytes to THC is in contrast to what might be expected, since immunosuppression caused by THC might be expected to correlate with increased, not decreased, cAMP levels. One possibility is that THC-induced immunosuppression is not mediated via alterations in cAMP levels. Preliminary studies indicate that THC is also altering leukotriene production in PBMC. However, a clearer understanding of the mechanism(s) of THC effects on the cAMP pathway and its relationship to THC-induced immune suppression will only be attained after further examination of drug effects on metabolic pathways in lymphoid cells. Studies are currently in progress to determine how THC is altering cAMP levels. Each aspect of the pathway will be examined including alterations in adenylate cyclase (the catalytic subunit responsible for converting ATP to cAMP) levels and further examination of the involvement of GTP binding proteins in THC effects on cAMP levels.

The importance of understanding the molecular interactions of THC and host cell metabolic pathways is critical for a number of reasons. First, is the determination of the relative risk/benefit ratios of the use of THC for medicinal purposes. THC has been used clinically as an antiemetic for patients undergoing cancer chemotherapy (Lane, Smith, Sullivan & Plasse, 1990) and symptomatic HIV patients (Plasse, Gorter, Krasnow, Lane, Shepard & Wadleigh, 1991), for treatment of immune-mediated disease such as experimental autoimmune encephalomyelitis (Lyman, Sonett, Brosnan, Elkin & Bornstein, 1989), treatment of spasticity and ataxia in multiple sclerosis (Meinck, Schonle & Conrad, 1989), and has the potential to be used in certain inflammatory conditions, such as asthma (Evans et

S. DIAZ et al.

al., 1987). In order to fully understand the impact of administration of a drug such as THC, the effect of THC on all cell types and systems must be taken into account. Secondly, individuals who abuse psychoactive drugs are a known high risk group for contracting human immunodeficiency virus (HIV). The possible role that THC may play in exacerbating the progression of HIV positive, asymptomatic patients to full blown acquired immunodeficiency syndrome (AIDS) is an important consideration. A number of reports suggest that psychoactive drugs, including marijuana, which have demonstrated immunosuppressive activity, may serve as co-factors for the development of AIDS (Pillai, Nair & Watson, 1991; Watson, 1989). Finally, marijuana remains the most commonly used illicit drug (NIDA, 1991), thus increasing our need to understand its detrimental effects on the host. In a 1990 NIDA household survey it was found that almost 10 million Americans use marijuana on a frequent basis (NIDA, 1991). Furthermore, the potency of marijuana has dramatically increased in the last decade (Pitts, O'Neil & Leggo, 1990). Therefore, it is important to determine how THC may be impacting the health of those individuals who are chronic smokers of marijuana.

These results clearly demonstrate that THC can diminish activity of a second messenger system in lymphoctye plasma membranes and that a pertussis toxin sensitive G~ protein is probably involved. However, it is still necessary to determine the relationship between THC altered cAMP levels and suppression of immunity in the presence of this drug.

Acknowledgements - - This study was supported by grants DA 04141 and DA 07245 from the National Institute on Drug Abuse, Public Health Service.

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