0 1 9 2 - 0 5 6 1 / 9 1 $3.00 + .00 Pergamon Press plc. International Society for l m m u n o p h a r m a c o l o g y .
lnt. J. lmmunopharmac., Vol. 13, No. 8, pp. 1091 - 1097, 1991. Printed in Great Britain.
MARIJUANA COMPONENTS STIMULATE HUMAN PERIPHERAL B L O O D M O N O N U C L E A R CELL SECRETION OF I N T E R F E R O N G A M M A A N D S U P P R E S S INTERLEUKIN-1 A L P H A I N VITRO BERNHARD WATZL,* PHILIP SCUDERIt and RONALD R. WATSON* *Department of Family and Community Medicine, and tDepartment of Microbiology and Immunology, Arizona Health Sciences Center, and tArizona Cancer Center, The University of Arizona, Tucson, AZ 85724, U.S.A. (Received 11 January 1991 and in final form 31 May 1991)
Abslract - - We investigated the in vitro effects of both psychoactive and nonpsychoactive marijuana components on leukocyte secretion of the immunoregulatory cytokines interleukin-1 alpha (IL-1), tumor necrosis factor alpha (TNF), interferon-gamma (IFN) and interleukin-2 (IL-2). Psychoactive delta-9tetrahydrocannabinol (THC) and nonpsychoactive cannabidiol (CBD) were added to cultures of mitogenactivated human peripheral blood mononuclear cells (PBMC) and the concentrations of IL-1, TNF, IFN and IL-2 in culture supernatants were measured by ELISA systems. Concentrations of THC and CBD, comparable to plasma levels found after smoking marijuana (10 - 100 ng/ml), increased the concentration of measurable IFN (139 and 68%), while high concentrations of both cannabinoids (5 -20/ag/ml) completely blocked synthesis and/or release of this cytokine. CBD was also shown to decrease the measurable quantity of both IL-1 and TNF. In contrast to the effects on IFN, IL-I and TNF, both cannabinoids, had no effect on IL-2 secretion. This report suggests that both psychoactive and nonpsychoactive components of marijuana are immunomodulating and can potentially alter cytokine secretion of human PBMC.
Delta-9-tetrahydrocannabinol (THC) is one of more than 60 cannabinoids in the marijuana plant (Turner, 1985) and is the major psychoactive component in plant tissue. Cannabidiol (CBD), also a major component of marijuana, has no demonstratable psychoactivity. In marijuana users, cells of the immune system are exposed to THC, CBD and other protein-bound cannabinoids. Several studies have demonstrated a suppressive effect of TH C on the immune response (Munson & Fehr, 1983; Yahya & Watson, 1987; Hollister, 1988) including the suppression of natural killer (NK) cell activity (Specter, Klein, Newton, Mondragon, Widen & Friedman, 1986; Klein, Newton & Friedman, 1987; Specter, Rivenbark, Newton, Kawakami & Lancz, 1989), inhibition of macrophage phagocytosis (Friedman, LopezCepero, Klein & Friedman, 1986) and lymphocyte proliferation (Pross, Klein, Newton & Friedman, 1987; Specter, Lancz & Haseiden, 1990). In contrast to these observations, lymphocytes from individuals who smoked marijuana or ingested T H C did not demonstrate any changes in their ability to respond to T-lymphocyte mitogens (White, Brin & Jamichi,
1975; Lau, Tubergen, Barr, Domino, Benowitz & Jones, 1976; Dax, Pilotte, Adler, Nagel & Lange, 1989). Delta-9-tetrahydrocannabinol has been shown to modulate virus and mitogen induced cytokine secretion in mice. Intraperitoneally administered T H C ( 5 - 1 0 0 mg/kg) significantly decreased the plasma concentration of interferon-alpha and -beta (Cabral, Lockmuller & Mishkin, 1986). Chronic in vivo exposure of mice to THC (50 mg/kg for 56 days) also reduced the concentrations of interferon-alpha and -beta in supernatants of cultured, mitogenstimulated spleen cells (Blanchard, Newton, Klein, Stewart & Friedman, 1986). In vitro, the incubation of murine spleen cells together with TH C ( 2 . 5 - 10 tag/ml) produced dose-dependent responses on measurable IFN. In these studies lower concentrations of THC had no effect on IFN, while 5 - 10 tag/ml decreased the concentration (Blanchard et al., 1986). Friedman, Klein, Specter, Pross, Newton, Blanchard & Widen (1988) also have shown a suppressive effect of TH C on the secretion of different cytokines by cultured murine spleen cells using bioassays at concentrations of 5 - 10 tag/ml. In
1091
B. WATZLet al.
1092
most studies to date, however, the immunomodulatory concentrations of THC used were well above the physiologically achievable range of 1 - 100 ng/ml of THC which has been found in the plasma of human marijuana smokers (Agurell, Halldin, Lindgren, Ohlsson, Widman, Gillespie & Hollister, 1986). None of the previous studies has investigated the effects of cannabinoids on the secretion of tumor necrosis factor (TNF) and little information is available about the modulation of human cytokine secretion by physiologically achievable levels of the cannabinoids, including nonpsychoactive compounds such as CBD. In the present study, we have examined the effects of THC and CBD on the concentration of secreted human IL-1, IL-2, TNF and IFN at drug concentrations found in the blood of marijuana smokers. EXPERIMENTAL
PROCEDURES
Cells and culture conditions Human peripheral blood was collected in EDTA tubes from healthy adult donors (age 2 5 - 6 0 ) . The mononuclear cells (PBMC) from each donor were separated by F i c o l l - H y p a q u e (Organon Teknika, Durham, NC) and suspended at a density of 1 × 106 cells/ml in complete tissue culture medium consisting of RPMI-1640 supplemented with 1007o fetal bovine serum (FBS), 2 mM L-glutamine, penicillin and streptomycin. Isolation o f adherent and nonadherent mononuclear cells Adherent and nonadherent cells were isolated from human PBMC as described by Kumagai, Hoh, Hinuma & Tada (1979). Briefly, F i c o l l - H y p a q u e purified PBMC at 4 x 106 cells/ml were incubated in 175 cm 2 flasks previously coated overnight at 4°C with heat-inactivated FBS for 1.5 h at 37°C in a CO: incubator. Nonadherent cells were removed by two rounds of adherence to plastic. Adherent cells were removed from FBS-coated flasks after incubation with Ca 2+, Mg2+-free PBS, pH 7.4, containing EDTA (0.2%) and FBS (5%) for 30 min at 37°C. Both adherent and nonadherent cells were washed with complete RPMI-1640 media and adjusted to 1 x 1 0 6 cells/ml. Materials Delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) were gifts from Dr Paul Consroe (College of Pharmacy, University of Arizona), 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 either dimethyl sulfoxide (DMSO; Sigma Chemical, St. Louis, MO) or ethanol. When ethanol was used as a vehicle, THC and CBD ethyl alcohol solutions were directly suspended in complete culture medium at a concentration of 2 mg THC or CBD per ml of medium and stored at - 2 0 ° C . When DMSO was used as a vehicle, 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 in DMSO and CBD in DMSO were diluted in warm (37°C) RPMI-1640 medium at a concentration of 2 mg/ml and stored at - 2 0 ° C . Cytokine induction Peripheral blood mononuclear cells from each donor, adherent or nonadherent cells (1 x 105 cells), were added to individual wells of flat-bottomed 96-well tissue culture plates (Falcon, Oxnard, CA). To induce cytokine secretion cells were exposed to the followi,g mitogens (all final concentrations): for IL-1, the cells were incubated for 24 h with pokeweed mitogen (PWM, 0.1 tag/ml; Sigma); for IL-2, 72 h with concanavalin A (Con A, 5/ag/ml; Sigma); for TNF, 24 h with lipopolysaccharide (LPS extracted from Escherichia coli 0111 :B4, 10 tag/ml; Difco, Detroit, MI); and for IFN, 72 h with phytohemagglutinin (PHA, 5/ag/ml, Sigma). As a control, 100 ~1 of cell suspension was incubated with 100/al of the individual mitogens. The vehicle controls were prepared with the same amount of vehicle used in each of the THC/CBD preparations at 10/ag/ml (0.0507o DMSO or ethanol). THC or CBD were added to the wells to give final concentrations of 0.01, 0.1, 1, 2.5, 5, 10 and 20/ag/ml. Cells were incubated for various lengths of time in a humidified atmosphere of 507o CO2, 9507o air. After incubation, the culture plates were centrifuged (180 g, 5 min), the supernatants were collected and assayed for the individual cytokine. Cytokine assays To detect the secreted cytokines, monoclonal antibodies specific for IL-1 (clone C42, Olympus, Lake Success, NY), IL-2 (Genzyme, Boston, MA), TNF (clone Fl2, Olympus) and IFN (clone A07, Olympus) and a set of standards made with human recombinant IL-I, IL-2, TNF and IFN (generously provided by Genentech Inc., San Francisco, CA)
In Vitro Secretion of 1FN-y and IL-1
were used. Individual enzyme-linked immunosorbent assays (ELISA) were produced by coating 96-well plates (Immulon II, Dynatech Inc., McLean, VA) with one of the following monoclonal antibodies: 0.2~g/well (anti-IL-1), 0.83/~g/well (anti-IL-2), 0.2/~g/well (anti-TNF) and 0.12/~g/well (anti-IFN). Plates were incubated at 4°C overnight. Between subsequent steps in the assay, coated plates were washed twice with phosphate-buffered saline containing 0.05°7o Tween-20. All reagents used in these assays were incubated on the assay plates for 1 h at room temperature. After washing the antibody coated plates, either 50/A of culture supernatants or 50 ~1 of cytokine standard was added to the wells. After exposure to supernatants, assay plates were washed and then exposed to rabbit polyclonal antibodies specific for anti-IL-1, 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. After a similar incubation and wash procedure a peroxidaseconjugated goat anti-rabbit IgG specific antiserum, which had been absorbed against human IgG (American Qualex Inc., La Miranda, CA) was added. The peroxidase substrate used was 2,2'-Azino-bis-3-ethylbenzthiazoline-6-sulfonicacid (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 one-way analysis of variance. Where significant overall differences were noted, a least significant difference range test was performed in order to isolate individual group differences.
RESULTS
1093
0.50 0.45 0.40 \
0.35
¢= 0.30 0.25 _~ 0.20 0.15 0.10 0.05 0.00 Vehicle 0.01
0.1
1
2.5
5
10
20
~g/ml
Fig. 1. Effect of THC and CBD on |L-1 secretion of cultured human PBMC. *P
lnt. J. lmmunopharmac., Vol. 13, No. 8, pp. 1091 - 1097, 1991. Printed in Great Britain.
MARIJUANA COMPONENTS STIMULATE HUMAN PERIPHERAL B L O O D M O N O N U C L E A R CELL SECRETION OF I N T E R F E R O N G A M M A A N D S U P P R E S S INTERLEUKIN-1 A L P H A I N VITRO BERNHARD WATZL,* PHILIP SCUDERIt and RONALD R. WATSON* *Department of Family and Community Medicine, and tDepartment of Microbiology and Immunology, Arizona Health Sciences Center, and tArizona Cancer Center, The University of Arizona, Tucson, AZ 85724, U.S.A. (Received 11 January 1991 and in final form 31 May 1991)
Abslract - - We investigated the in vitro effects of both psychoactive and nonpsychoactive marijuana components on leukocyte secretion of the immunoregulatory cytokines interleukin-1 alpha (IL-1), tumor necrosis factor alpha (TNF), interferon-gamma (IFN) and interleukin-2 (IL-2). Psychoactive delta-9tetrahydrocannabinol (THC) and nonpsychoactive cannabidiol (CBD) were added to cultures of mitogenactivated human peripheral blood mononuclear cells (PBMC) and the concentrations of IL-1, TNF, IFN and IL-2 in culture supernatants were measured by ELISA systems. Concentrations of THC and CBD, comparable to plasma levels found after smoking marijuana (10 - 100 ng/ml), increased the concentration of measurable IFN (139 and 68%), while high concentrations of both cannabinoids (5 -20/ag/ml) completely blocked synthesis and/or release of this cytokine. CBD was also shown to decrease the measurable quantity of both IL-1 and TNF. In contrast to the effects on IFN, IL-I and TNF, both cannabinoids, had no effect on IL-2 secretion. This report suggests that both psychoactive and nonpsychoactive components of marijuana are immunomodulating and can potentially alter cytokine secretion of human PBMC.
Delta-9-tetrahydrocannabinol (THC) is one of more than 60 cannabinoids in the marijuana plant (Turner, 1985) and is the major psychoactive component in plant tissue. Cannabidiol (CBD), also a major component of marijuana, has no demonstratable psychoactivity. In marijuana users, cells of the immune system are exposed to THC, CBD and other protein-bound cannabinoids. Several studies have demonstrated a suppressive effect of TH C on the immune response (Munson & Fehr, 1983; Yahya & Watson, 1987; Hollister, 1988) including the suppression of natural killer (NK) cell activity (Specter, Klein, Newton, Mondragon, Widen & Friedman, 1986; Klein, Newton & Friedman, 1987; Specter, Rivenbark, Newton, Kawakami & Lancz, 1989), inhibition of macrophage phagocytosis (Friedman, LopezCepero, Klein & Friedman, 1986) and lymphocyte proliferation (Pross, Klein, Newton & Friedman, 1987; Specter, Lancz & Haseiden, 1990). In contrast to these observations, lymphocytes from individuals who smoked marijuana or ingested T H C did not demonstrate any changes in their ability to respond to T-lymphocyte mitogens (White, Brin & Jamichi,
1975; Lau, Tubergen, Barr, Domino, Benowitz & Jones, 1976; Dax, Pilotte, Adler, Nagel & Lange, 1989). Delta-9-tetrahydrocannabinol has been shown to modulate virus and mitogen induced cytokine secretion in mice. Intraperitoneally administered T H C ( 5 - 1 0 0 mg/kg) significantly decreased the plasma concentration of interferon-alpha and -beta (Cabral, Lockmuller & Mishkin, 1986). Chronic in vivo exposure of mice to THC (50 mg/kg for 56 days) also reduced the concentrations of interferon-alpha and -beta in supernatants of cultured, mitogenstimulated spleen cells (Blanchard, Newton, Klein, Stewart & Friedman, 1986). In vitro, the incubation of murine spleen cells together with TH C ( 2 . 5 - 10 tag/ml) produced dose-dependent responses on measurable IFN. In these studies lower concentrations of THC had no effect on IFN, while 5 - 10 tag/ml decreased the concentration (Blanchard et al., 1986). Friedman, Klein, Specter, Pross, Newton, Blanchard & Widen (1988) also have shown a suppressive effect of TH C on the secretion of different cytokines by cultured murine spleen cells using bioassays at concentrations of 5 - 10 tag/ml. In
1091
B. WATZLet al.
1092
most studies to date, however, the immunomodulatory concentrations of THC used were well above the physiologically achievable range of 1 - 100 ng/ml of THC which has been found in the plasma of human marijuana smokers (Agurell, Halldin, Lindgren, Ohlsson, Widman, Gillespie & Hollister, 1986). None of the previous studies has investigated the effects of cannabinoids on the secretion of tumor necrosis factor (TNF) and little information is available about the modulation of human cytokine secretion by physiologically achievable levels of the cannabinoids, including nonpsychoactive compounds such as CBD. In the present study, we have examined the effects of THC and CBD on the concentration of secreted human IL-1, IL-2, TNF and IFN at drug concentrations found in the blood of marijuana smokers. EXPERIMENTAL
PROCEDURES
Cells and culture conditions Human peripheral blood was collected in EDTA tubes from healthy adult donors (age 2 5 - 6 0 ) . The mononuclear cells (PBMC) from each donor were separated by F i c o l l - H y p a q u e (Organon Teknika, Durham, NC) and suspended at a density of 1 × 106 cells/ml in complete tissue culture medium consisting of RPMI-1640 supplemented with 1007o fetal bovine serum (FBS), 2 mM L-glutamine, penicillin and streptomycin. Isolation o f adherent and nonadherent mononuclear cells Adherent and nonadherent cells were isolated from human PBMC as described by Kumagai, Hoh, Hinuma & Tada (1979). Briefly, F i c o l l - H y p a q u e purified PBMC at 4 x 106 cells/ml were incubated in 175 cm 2 flasks previously coated overnight at 4°C with heat-inactivated FBS for 1.5 h at 37°C in a CO: incubator. Nonadherent cells were removed by two rounds of adherence to plastic. Adherent cells were removed from FBS-coated flasks after incubation with Ca 2+, Mg2+-free PBS, pH 7.4, containing EDTA (0.2%) and FBS (5%) for 30 min at 37°C. Both adherent and nonadherent cells were washed with complete RPMI-1640 media and adjusted to 1 x 1 0 6 cells/ml. Materials Delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) were gifts from Dr Paul Consroe (College of Pharmacy, University of Arizona), 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 either dimethyl sulfoxide (DMSO; Sigma Chemical, St. Louis, MO) or ethanol. When ethanol was used as a vehicle, THC and CBD ethyl alcohol solutions were directly suspended in complete culture medium at a concentration of 2 mg THC or CBD per ml of medium and stored at - 2 0 ° C . When DMSO was used as a vehicle, 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 in DMSO and CBD in DMSO were diluted in warm (37°C) RPMI-1640 medium at a concentration of 2 mg/ml and stored at - 2 0 ° C . Cytokine induction Peripheral blood mononuclear cells from each donor, adherent or nonadherent cells (1 x 105 cells), were added to individual wells of flat-bottomed 96-well tissue culture plates (Falcon, Oxnard, CA). To induce cytokine secretion cells were exposed to the followi,g mitogens (all final concentrations): for IL-1, the cells were incubated for 24 h with pokeweed mitogen (PWM, 0.1 tag/ml; Sigma); for IL-2, 72 h with concanavalin A (Con A, 5/ag/ml; Sigma); for TNF, 24 h with lipopolysaccharide (LPS extracted from Escherichia coli 0111 :B4, 10 tag/ml; Difco, Detroit, MI); and for IFN, 72 h with phytohemagglutinin (PHA, 5/ag/ml, Sigma). As a control, 100 ~1 of cell suspension was incubated with 100/al of the individual mitogens. The vehicle controls were prepared with the same amount of vehicle used in each of the THC/CBD preparations at 10/ag/ml (0.0507o DMSO or ethanol). THC or CBD were added to the wells to give final concentrations of 0.01, 0.1, 1, 2.5, 5, 10 and 20/ag/ml. Cells were incubated for various lengths of time in a humidified atmosphere of 507o CO2, 9507o air. After incubation, the culture plates were centrifuged (180 g, 5 min), the supernatants were collected and assayed for the individual cytokine. Cytokine assays To detect the secreted cytokines, monoclonal antibodies specific for IL-1 (clone C42, Olympus, Lake Success, NY), IL-2 (Genzyme, Boston, MA), TNF (clone Fl2, Olympus) and IFN (clone A07, Olympus) and a set of standards made with human recombinant IL-I, IL-2, TNF and IFN (generously provided by Genentech Inc., San Francisco, CA)
In Vitro Secretion of 1FN-y and IL-1
were used. Individual enzyme-linked immunosorbent assays (ELISA) were produced by coating 96-well plates (Immulon II, Dynatech Inc., McLean, VA) with one of the following monoclonal antibodies: 0.2~g/well (anti-IL-1), 0.83/~g/well (anti-IL-2), 0.2/~g/well (anti-TNF) and 0.12/~g/well (anti-IFN). Plates were incubated at 4°C overnight. Between subsequent steps in the assay, coated plates were washed twice with phosphate-buffered saline containing 0.05°7o Tween-20. All reagents used in these assays were incubated on the assay plates for 1 h at room temperature. After washing the antibody coated plates, either 50/A of culture supernatants or 50 ~1 of cytokine standard was added to the wells. After exposure to supernatants, assay plates were washed and then exposed to rabbit polyclonal antibodies specific for anti-IL-1, 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. After a similar incubation and wash procedure a peroxidaseconjugated goat anti-rabbit IgG specific antiserum, which had been absorbed against human IgG (American Qualex Inc., La Miranda, CA) was added. The peroxidase substrate used was 2,2'-Azino-bis-3-ethylbenzthiazoline-6-sulfonicacid (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 one-way analysis of variance. Where significant overall differences were noted, a least significant difference range test was performed in order to isolate individual group differences.
RESULTS
1093
0.50 0.45 0.40 \
0.35
¢= 0.30 0.25 _~ 0.20 0.15 0.10 0.05 0.00 Vehicle 0.01
0.1
1
2.5
5
10
20
~g/ml
Fig. 1. Effect of THC and CBD on |L-1 secretion of cultured human PBMC. *P
