INFLUENCE OF MARUUANA COMPONENTS (THC AND CBD) ON HUMAN MONONUCLEAR CELL CYTOKINE SECRETION IN VITRO Bernhard Watzl!, Philip Scuderi2, Ronald R. Watsonl lDepartment of Family and Community Medicine, Arizona Health Sciences Center and 2Department of Microbiology and Immunology Arizona Cancer Center, University of Arizona, Tucson, AZ 85724 INTRODUCTION Cytokines are a class of polypeptides produced by cells of the immune system. They coordinate the immune response to an antigenic challenge and play key roles in immunomodulation and host defense (1,2), and exert also metabolic effects (3,4). Therefore, a change in cytokine secretion caused by drugs of abuse could have an impact on immunological systems and metabolism. Delta-9-tetrahydrocannabinol (THe), the major psychoactive component of marijuana, has been shown to modulate virus and mitogen induced cytokine secretion in mice. Intraperitoneal administered THC (5-100 mg/kg) decreased the plasma concentration of alpha-/betainterferon (IFN) significantly (5). Chronic in vivo exposure of mice to THC (50 mg/kg for 56 days) also reduced the secretion of alpha-/beta-IFN in cultured, mitogen-stimulated spleen cells (6). In vitro culture of murine spleen cells together with THC (2.5-10 Ilg/ml) showed dose-dependent responses in the alpha-/beta-IFN secretion. Low concentrations had no effect on alpha-/beta-IFN secretion, while 5 to 10 Ilg/ml were significantly suppressive (6). However, all these immunomodulatory concentrations were high and well above the pharmacological range of I-toO ng/ml of THC found in the plasma of human marijuana smokers (7). Friedman et aI., also investigated the effect of THC on the secretion of different cytokines by cultured murine spleen cells using bioassays (8). Only concentrations above the pharmacological range were used (5-10 Ilg/ml). All suppressed in a dose-dependent manner the secretion of cytokines. Lipopolysaccharide (LPS)-induced secretion of IFN (alpha-/beta) in splenocytes, adherent and nonadherent cells was reduced to 85%, a reduction was also observed by chronic administration of THC to these cells. The secretion of interleukin-l alpha (IL-l) and interleukin-2 (lL-2) by splenocytes cultured with mitogens and THe, and of peritoneal macrophages from mice chronically injected with THC, decreased significantly. Beside the cytokine production or release from cells, THC affects multiple responses of the immune system (9,10,11), including the activity of natural killer (NK) cells (12-14). NK cell function modification could be mediated by the decrease in IFN or IL-2 secretion. In one study, IL-2 was able to prevent and reverse the THC-induced depression of NK cell activity. It is not clear, if there is an agonistic

Drugs of Abuse, Immllllil)l, aNi ImmlllJOtkrlCiency Edited by H. Friedman e/ al., P1enwn Press, New York, 1991

63

interaction between IL-2 and THC, or if IL-2, in contrast to THC's suppressive effect, stimulates NK cell activity without interfering with THC. THC is only one of more than 60 cannabinoids in the marijuana plant (15). Cannabidiol (CBD) is also one of the major constituents, but has no psychoactivity. After smoking marijuana the systemic availability of CBD is 31 % higher than THC, which is in the range of 10-25%. They both have comparable biological half-life times (7). Therefore, the immune system is more exposed to CBD than to THC, but only very few animal (16) and no human studies have been published about the effects of CBD on the immune response. Thus, we have examined the effects of THC and CBD on the mitogen-induced secretion of human IL-1, IL-2, tumor necrosis factor alpha (lNF) and gamma-IFN. MATERIALS AND METHODS Cells and Culture Conditions Human peripheral blood was collected in EDTA tubes from healthy adult donors (age 25-69), with no history of drug use. The mononuclear cells (PBMC) were separated by Ficoll-Hypaque (Organon Teknika, Durham, NC) and suspended at a density of 1 x 106 cells/ml in complete tissue culture medium consisting of RPMI-1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin and streptomycin.

Delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) were graciously provided by Dr. Paul Consroe (College of Pharmacy), who obtained the drugs from the National Institute on Drug Abuse, Research Technology Branch, Rockville, MD. THC and CBD were provided as ethyl alcohol solutions. The vehicle for both cannabinoids was dimethyl sulfoxide (DMSO; Sigma Chemical, St. Louis, MO), except for incubations using the mitogen phytohemagglutinin (PHA; Sigma). In this case, THC and CBD ethyl alcohol solutions were directly suspended in complete culture medium at a concentration of 2 mg THC or CBD per ml medium and stored at -20°C. In all the other cases, the ethyl alcohol was evaporated from the cannabinoid stock with a stream of nitrogen gas and the cannabinoid residue was suspended at 20 mg per ml in DMSO. THC/DMSO and CBD/DMSO were diluted in warm RPMI-1640 medium at a concentration of 2 mg/ml and stored at -20°C. The concentration of the vehicle control was equivalent to the highest concentration used in the THC/CBD dilutions (0.1%). c;ytokine Induction Mononuclear cells (1 x lOS cells) were added to individual wells of flat-bottom 96-well tissue culture plates (Falcon, Oxnard, CA). To induce cytokine secretion cells were incubated with the following mitogens (all final concentrations):1L-1, 24 hrs with pokeweed mitogen (PWM, O.l·p.g/ml; Sigma); IL-2, 72 hrs with concanavalin A (Con A, 5 p.g/ml; Sigma); lNF, 24 hrs with lipopolysaccharide (LPS, 10 p.g/ml; Difco, Detroit, MI); gamma-IFN, 72 hrs with PHA (5 p.g/ml). For the control, 100 p.l of cell suspension were incubated with 100 p.l of the individual mitogens. The vehicle controls were prepared with the amount of vehicle in the THC/CBD preparation at 10 p.g/ml. THC or CBD were added to the wells to give a final concentration of 0.01, 0.1, 1, 2.5, 5 and 10 p.g/ml. Cells were incubated for 64

various times in a humidified atmosphere of 5% CO2, 95% air. Mter incubation, the culture plates were centrifuged (180 x g, 5 min) and the supernatants were collected for cytokine assays. Cytokine Assays To detect the cytokines, monoclonal antibodies specific for IL-l (clone C42, Olympus, Lake Success, NY), IL-2 (Genzyme, Boston, MA), TNF (clone F12, Olympus) and IFN (clone A07, Olympus) and a set of standards made with human recombinant IL-l, IL-2, TNF and IFN (generously provided by Genentech Inc., San Francisco, CA) were used. The enzyme-linked immunosorbent assay (ELISA) was performed by coating 96-well plates (Immunol II, Dynatech Inc., McLean, VA) with 0.2 ~g/well (IL-l), 0.83 Ilg/well (IL-2), 0.2 Ilg/well (TNF) or 0.12 ~g/well (IFN) of the murine monoclonal antibody with specificity for these human cytokines. Between subsequent steps in the assay, coated plates were washed twice with phosphate-buffered saline containing 0.05% Tween-20. All reagents used in these assays were incubated for 1 hr at room temperature with coated wells. Fifty III of supernatants were added to the wells. For the standard curve recombinant cytokines were added to the culture medium ranging from 0.07-10 ng/ml. Mter exposure to the supernatants, assay plates were washed and then exposed to rabbit polyclonal antibodies anti-IL-l, anti-IL-2, anti-TNF and anti-IFN. The production of rabbit polyclonal antiserum specific for these cytokines was accomplished by immunizing New Zealand rabbits with the recombinant cytokines. Mter a similar incubation and wash procedure a peroxidase-conjugated goat anti-rabbit IgG specific antiserum, which had been absorbed against human IgG (American Qualex Inc., La Mirada, CA) was added. The peroxidase substrate used was 2,2'-Azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (Sigma). Optical density readings were made on a Titertek multiscan (Flow Labs, McLean, VA) with a 405 nm filter after 30 min of substrate addition. The quantity of cytokines in culture supernatants was determined by comparison with a set of standards made with recombinant human cytokines. All data are reported as mean of duplicates. Statistics Data were analyzed using the oneway analysis of variance. RESULTS Interleukin-l Figure 1 shows the effect of THC and CBD on the secretion of IL-l. For THe', cells from 11 donors were examined and for CBD cells from 7 other donors. Cells and mitogen alone as controls showed high differences in their IL-l secretion, which demonstrates the high intersubject variability in IL-l secretion observed also by others (23). DMSO as the vehicle for both cannabinoids did not affect the IL-l secretion. Only high concentrations of THC (10 Ilg/ml) decreased the IL-l significantly, CBD suppressed the secretion significantly at lower concentrations, which were slightly above the pharmacological range. With 20 Ilg/ml CBD the IL-l secretion was totally inhibited. Interleukin-2 For each component, THC and CBD, 5 different donors participated in this study. 65

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Fig. 1. *p ~ 0.05 vs. control. PBMC were stimulated with PWM (0.1 ~g/ml) for 24 hours. The vehicle for TIlC/CBD was DMSO. (TIlC n= 11; CBD n=7).

The controls (cells and mitogen) showed a high intersubject variability in the IL-2 secretion, which confirmed data obtained by others (23) (Figure 2). IL-2 secretion was suppressed by the vehicle control. This means, that DMSO had a stronger influence on PBMC than both cannabinoids and it is therefore difficult to assess the effect of CBD. Compared to the control, CBD with DMSO suppressed the IL-2 secretion. In comparison to the vehicle control, the release of IL-2 was increased by CBD. No concentration of TIlC had any effect on IL-2 secretion except 1 ~g/ml, which induced a 100% increase in cytokine release.

Effect of THC or CBO on IL -2 Secretion of Cultured Human PBMC 0.7

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Control Control Vehicle

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*p ~ 0.05 vs. control. PBMC were stimulated with Con A (5 ~g/ml) for 72 hours. The vehicle for TIlC/CBD was DMSO. (TIlC n = 5, CBD n = 5).

Table 1. Effect of THC or CBO on 1NF Secretion of Cultured Human PBMC Group

Control Vehicle

O.Ola

THC

1.07b ±0.60

0.94 ±0.46

CBO

0.31 ±0.09

0.29 ±0.15

0.1

1.0

2.5

n.d.

1.15 ±0.74

1.05 ±0.60

0.95 ±0.56

1.19 ±0.70

0.94 ±1.06

0.30 ±0.18

0.24 ±0.18

0.09 ±0.09

0.02 ±0.05

0.01 ±0.03

0.01 ±0.03

5

10

aTHC or CBO final concentration (Jlgjml). bTNF concentration (ngjml).

Tumor necrosis factor (TNF) Eleven donors participated in the THC experiments and 15 more donors in the CBO experiments. TNF secretion is known to have a high intersubject variability (23), which was also seen in this study for the controls of both components (Table 1). THC had no effect on leukocyte TNF release. CBO in the physiological range did not modulate the TNF secretion, concentrations above 0.1 Jlgjml blocked nearly totally the cytokine secretion (Table 1). The vehicle had no effect on the TNF release.

Table 2. Effect of the,Yehicles OMSO or Ethanol on Gamma-IFN Secretion of Cultured Human PBMC Change in gamma-IFN secretion OMSO (Ill/ml) 0 .00001 .0001 .001 .01 .1 1 10

%a

Ethanol (Ill/ml)

%

100 191 225 197 156 188 247 147

0 0.0005 0.005 0.05 0.125 0.25 0.5 1.0

100 105 132 124 98 109 135 127

8Expressed as percent of control. 67

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Fig. 3.

*p ~ 0.05 vs. control. **p ~ 0.01 vs. control. PBMC were stimulated with PHA (5 Ilg/ml) for 72 hrs. The vehicle for TIlC/CBD was ethanol. (TIlC n = 12).

Gamma-interferon (IFN) The commonly used vehicle, DMSO in combination with PHA stimulated intensively the gamma-IFN secretion (Table 2). Therefore, ethanol was used as a vehicle for the drugs, which did not increase significantly the gamma-IFN secretion. Peripheral blood leukocytes from 10 donors were used for the TIlC experiment and 15 different donors for the CBD experiment. In contrast to the cytokines IL-1, IL-2 and TNF, gamma-IFN showed a much lower intersubject variability for the controls (Figure 3). TIlC and CBD at low concentration (0.1 Ilg/ml) augmented the gammaIFN secretion by 74 and 80%. Increasing concentrations depressed the cytokine secretion and concentrations higher than 5 Ilg/ml suppressed the release totally. This was not due to toxic effects of TIlC or CBD, because the cell viability after 72 hr incubation was 80% for TIlC and 70% with CBD. DISCUSSION Our data show that psychoactive and nonpsychoactive components of marijuana modulate cytokine secretion by human PBMC in vitro. CBD had a stronger suppressive impact on the secretion of these cytokines than TIlC. CBD suppressed the IL-1, TNF and gamma-IFN secretion, while TIlC only suppressed the secretion of IL-1 and gamma-IFN. Both cell types, monocytes and lymphocytes, seemed to be sensitive to the effects of these cannabinoids at physiological concentrations. So far, no other studies were published about the effects of TIlC or CBD on human cytokine secretion. The suppression of the IL-1 secretion with high concentrations of TIlC has also been observed with cultured murine spleen cells (8), but we could not see a suppressive effect on the secretion of IL-2. We found a significant increase of the gammaIFN release with physiological concentrations of TIlC or CBD, which were not reported in any other study. At the lowest concentration used by others (6), TIlC (2.5 Ilg/rnl) showed no effect compared with control. At concentrations above 5 Ilg/rnl TIlC suppressed the release of gamma-IFN in our study and the release of 68

alpha-/beta-IFN in others (6,8). The observed bell-shaped dose-response curve of the gamma-IFN secretion was also seen with the human lymphocyte transformation assay (17). Very low concentrations of THC stimulated the lymphocyte transformation, while increasing concentrations of THC inhibited transformation. Physiological concentrations of THC in vitro did not affect the activity of murine NK cells (19), which is in agreement with the normal secretion of IL-2 and the stimulated gammaIFN secretion observed in our study. The activity of human NK cells was not decreased by 1 ~g/rnl (12) or 3 ~g/rnl (13) of THC. Only very high concentrations of THC (5 ~g/rnl) decreased the NK cell activity in vitro 24 (12-14). The inhibition of the NK cell activity by high concentrations of THC was not associated with a product of the cyclooxygenase pathway of arachidonic acid, because indomethacin did not prevent the inhibition of NK cell activity (14). THC elevates in vitro the arachidonic acid levels of murine peritoneal macrophages (19). In vivo, THC decreased the activity of murine NK cells (13,20). THC further reduced the plasma levels of norepinephrine, epinephrine and corticosterone, and increased the plasma beta-endorphin levels (20). This demonstrates clearly that in vivo THC acts not only directly on lymphoid cells, but also modulates immune responses via its impact on the neuroendocrine-immune network. The mechanism of the in vitro effects of THC or CBD on lymphoid cells are not known. A recent study has shown that a cannabinoid receptor exists on rat brain membranes, which appears to be associated with certain of the typical cannabinoid responses (21). It is not known, however, if the cannabinoid effects on lymphoid cells are also mediated by binding to this receptor. THC and CBD are very lipophilic and may alter the lipid membranes of lymphoid cells. The functional activity of lymphoid cells depends on the integrity of the lipid membranes. Therefore, cannabinoids could modify the membrane fluidity and permeability, the localization of receptors on the surface of the membranes, the activity of membrane-located phospholipases or the signal inductions for second messengers (22). ACKNOWLEDGMENT This study was supported by NIDA grants AA 08037 and DA 04827. REFERENCES 1. 2. 3. 4. 5. 6.

7.

S. B. Mizel, The interleukins, F ASEB L 3:2379 (1989). F. R. Balkwill, and R. Burke, The cytokine network, Immun. Today 10:299 (1989). R. F. Grimble, Cytokines: Their relevance to nutrition, Europ. 1.. Clin. Nutr. 43:217 (1989). K. C. Klasing, Nutritional aspects of leukocytic cytokines, Amer. L Clin. Nutr. 1436 (1988). G. A Cabral, J. C. Lockrnuller, and E. M. Mishkin, Delta-9-tetrahydrocannabinol decreases alpha-beta interferon response to herpes simplex virus type 2 in the B6C3Fl mouse, Proc. Soc. ~ BioI. Med. 181:305 (1986). D. K. Blanchard, C. Newton, T. W. Klein, W. E. Stewart II, and H. Friedman, In vitro and in vivo suppressive effects of delta-9-tetrahydrocannabinol in interferon production by murine spleen cells, Int. 1.. Immunopharmac. 8:819 (1986). S. Agurell, M. Halldin, J. E. Lindgren, A Ohlsson, M. Widman, H. Gillespie, and L. Hollister, Pharmacokinetics and metabolism of delta-I-tetrahydrocannabinol and other cannabinoids with emphasis on man, Pharmacol. Reviews 38:21 (1986). 69

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pharm. 44:125 (1988). A. E. Munson, and K. O. Fehr, in: "Adverse Health and Behavioral Consequences of Cannabis Use," K. O. Fehr and H. Kalant, ed., Addiction Research Foundation, Toronto (1983). M. D. Yahya, R. and R. Watson, Immunomodulation by morphine and marijuana, ~ Sciences 41:2503 (1987). L. E. Hollister, Marijuana and immunity, L Psychoactive DrugS 20:3 (1988). S. C. Specter, T. W. Klein, C. Newton, M. Mondragon, R. Widen, and H. Friedman, Marijuana effects on immunity: Suppression of human natural killer cell activity by delta-9-tetrahydrocannabinol, Int. J... Immunopharmac. 8:741 (1986). T. W. Klein, C. Newton, and H. Friedman, Inhibition of natural killer cell function by marijuana components, J... Toxicol. Environ. Health 20:321 (1987). S. Specter, M. Rivenbark, C. Newton, Y. Kawakami, and G. Lancz, Prevention and reversal of delta-9-tetrahydrocannabinol induced depression of natural killer cell activity by interleukin-2, Int. J... Immunopharmac. 11:63 (1989). C. E. Turner, Marijuana and cannabis: Research why the conflict? in: "Marijuana '84," D. Y. Harvey, ed., IRL Press, Oxford (1985). S. Zimmerman, A. M. Zimmerman, I. L. Cameron, and H. L. Laurence, DeltaI-tetrahydrocannabinol, cannabidiol and cannabinol effects on the immune response of mice, Pharmacology 15:10 (1977). Y. D. Luo, M. L. Shen, and D. E. Ou, Does delta-9-tetrahydrocannabinol suppress human immune functions? FASEB ,l. 3:Abstr. 291 (1989). F. Lu and D. W. Ou, Cocaine or delta-9-tetrahydrocannabinol does not affect cellular cytotoxicity in vitro, Int. J.. Immunopharmac. 11:849 (1989). S. Burstein, S. A. Hunter, K. Ozman, and L. Renzulli, Cannabinoid-induced elevation of Jipoxygenase products in mouse peritoneal macrophages, Biochem. Pharmacol. 33:2653 (1984). V. Patel, M. Borysenko, M. S. A. Kumar, and W. J. Millard, Effects of acute and subchronic delta-9-tetrahydrocannabinol administration on the plasma catecholamine, beta-endorphin, and corticosterone levels and splenic natural killer cell activity in rats, Proc. ~ ~ BioI. Med. 180:400 (1985). W. A. Devane, F. A. Dysarz III, M. R. Johnson, L. S. Melvin, and A. C. Howelett, Determination and characterization of a cannabinoid receptor in rat brain, Mol. Pharmacol. 34:605 (1988). B. R. Martin, Cellular effects of cannabinoids, Pharmacol. Reviews 38:45 (1986). S. Endres, J. G. Cannon, R. Ghorbain, R. A. Dempsey, S. D. Sisson, G. Lonnemann, J. W. D. Van der Meer, S. M. Wolff, and D. A. Dinarello, In vitro production of IL-1 beta, IL-1 alpha, TNF and IL-2 in healthy subjects: distribution, effect of cyclooxygenase inhibition and evidence of independent gene regulation, Eur. L Immunol. 19:2327 (1989).

Influence of marijuana components (THC and CBD) on human mononuclear cell cytokine secretion in vitro.

INFLUENCE OF MARUUANA COMPONENTS (THC AND CBD) ON HUMAN MONONUCLEAR CELL CYTOKINE SECRETION IN VITRO Bernhard Watzl!, Philip Scuderi2, Ronald R. Watso...
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