Int. J. lmmunopharmac., Vol. 12, No. 3, pp. 261-267, 1990. Printed in Great Britain.
0192-0561/90 $3.00 + .00 International Society for lmmunopharmaeology.
M A R I J U A N A A N D IMMUNITY: T E T R A H Y D R O C A N N A B I N O L M E D I A T E D INHIBITION OF L Y M P H O C Y T E BLASTOGENESIS STEVEN SPECTER,* GERALD LANCZ and JUDITH HAZELDEN Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, 12901 N. Bruce B. Downs Blvd, Tampa, FL 33612, U.S.A. (Received 8 May 1989 and in final form 27 October 1989)
Abstract - - The effects of delta-9-tetrahydrocannabinol (THC) and its major metabolite, l 1-OH delta9-tetrahydrocannabinol (ll-OH THC) on mitogen driven lymphocyte blastogenic transformation (LBT) were studied. THC inhibited LBT responses to the T lymphocyte mitogens phytohemagglutinin and concanavalin A but not the B lymphocyte stimulant pokeweed mitogen. The metabolite ll-OH THC caused a comparable, but less significant, inhibition of LBT responses to the T cell mitogens. These inhibitions were dependent upon the drug doses and the time of incubation with the lymphocytes. There was no significant inhibitory activity of THC to the LBT when it was added 24 or 48 h after mitogen. In addition, exposure of lymphocytes to THC for 3 h, followed by removal of the drug prior to addition of mitogen had no effect on the cells' ability to respond to the mitogen. Thus, there appears to be a specified temporal period during which exposure of lymphocytes to THC results in an inhibition of blastogenesis. Interleukin 2 (30 units/ml) could not preclude the THC induced inhibition of lymphocyte blastogenesis. We conclude that THC and ll-OH THC inhibit T lymphocyte blastogenesis. However, unlike the THC mediated inhibition of natural killer cell activity (as shown previously), the process is not responsive to the cytokine interleukin 2.
The role that drugs of abuse may play in the pathogenesis of infectious diseases is disputed. Potentially these drugs may affect regulation of the immune system, potentiate primary infections, or promote re-activation of latent, quiescent infection. Any of these effects may contribute to exacerbation of human immunodeficiency virus (HIV) infection leading to the development of frank acquired immunodeficiency syndrome (AIDS). Several previous studies on the effect of marijuana or its major psychoactive component, delta-9-tetrahydrocannabinol (THC), on cells of the immune system have yielded contradictory results (reviewed in Munson & O'Brien-Fehr, 1983). White, Brin & Janicki (1975) and Lau, Tubergen, Barr, Domino, Benowitz & Jones (1976) both report that 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. In the former case, lymphocytes were taken from individuals who were chronic marijuana users. In the latter study, the individuals ingested
THC daily, up to 210mg/day for 18 days. By contrast, Nahas and coworkers reported a marked depression of LBT responses in individuals who smoked marijuana (Nahas, Suciu-Foca, Armand & Morishima, 1974). Petersen, Graham & Lemberger (1976) also reported alteration in LBT responses as well as T-cell rosette formation in individuals receiving marijuana while under hospital supervision. However, the results in the study by Petersen et al. were inconsistent, leading the authors to speculate that decreased immunocyte function may be related to other factors. In-vitro studies by Nahas, Morishima & Desoize (1977) indicated that THC could directly depress LBT responses to PHA. However, the concentrations of TH C (62.3 tag/ml or 2 × 10 4 M) which were required to inhibit lymphocyte blastogenesis in the study by Nahas and coworkers were toxic to human peripheral blood lymphocytes in our studies (Specter, Klein, Newton, Mondragon & Friedman, 1986). This study also indicated that 10/ag T H C / m l was not toxic to lymphocytes over an 18 h incubation period. Subsequent studies in our
*Author to whom correspondence should be addressed. 261
S. SPECTER et al.
262
laboratory have indicated that 10/Jg T H C / m l was not toxic to lymphocytes during a 48 h incubation (unpublished data). More recent studies using murine spleen cells indicate that THC inhibits lymphocyte blastogenic transformation (LBT) following stimulation with phytomitogens or bacterial lipopolysaccharide (Klein, Newton, Widen & Friedman, 1985; Pross, Klein, Newton & Friedman, 1987). In the studies by Klein et al. (1985) the 10/Jg T H C / m l concentration was not toxic to mouse lymphocytes after 48 h of culture. This indicated that the inhibition of lymphocyte function was not due to a cytotoxic effect of THC on lymphocytes but rather the result of some metabolic or structural alteration of the cells. Thus, we studied the response of human peripheral blood lymphocytes to phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) in the presence of micromolar, subtoxic concentrations of THC. The results indicate that THC inhibits T lymphocyte blastogenesis induced by PHA or Con A but not by PWM.
EXPERIMENTAL
PROCEDURES
Human subjects Human subjects were volunteers recruited from the community around the University of South Florida. A unit of blood was collected at the Southwest Florida Blood Bank and the buffy coat was separated. Studies were performed with blood from individuals who denied using marijuana or other controlled substances. Lymphocyte preparations Peripheral blood lymphocytes were separated from the buffy coat by centrifugation through F i c o l l - H y p a q u e as previously described (Specter, Rivenbark, Newton, Kawakami & Lancz, 1989). Marijuana components Delta-9-THC, the major psychoactive ingredient in marijuana, was obtained from the National Institute on Drug Abuse (NIDA), Bethesda, MD. The THC was received as a solution in ethanol at 200 mg/ml. Stock THC in ethanol was stored at - 20°C. Reconstitution and dilution were performed using dimethyl sulfoxide (DMSO) as diluent as described previously (Specter et al., 1989). THC testing in vitro was performed over a range of 1 tJg/ml to 10/Jg/ml (3.2 × 10 6_ 3.2 × 10 5M).
Controls consisted of cells exposed to medium only or cells exposed to dilutions of the DMSO diluent which were equivalent to the amount present in THC dilutions (0.5% DMSO or less). ll-OH-delta-9-THC (ll-OH-THC), is a major psychoactive metabolite of the naturally occurring delta-9-THC found in marijuana. This metabolite was obtained from the NIDA in an ethanol solution at a concentration of 56 tag/ml. Because of the lower concentration of l l-OH-THC it was diluted in RPMI-1640 medium directly from the ethanol stock. Medium supplemented with equivalent concentrations of ethanol were used in controls.
Interleukin 2 Interleukin 2 (IL-2) was a generous gilt from Dr W. R. Benjamin, H o f f m a n - L a R o c h e , Nutley, NJ.
THC treatment THC was freshly prepared on the day of experimentation and added to lymphocyte suspensions 3 h before culture with mitogen (pre-incubation), at the time of culture in the presence of mitogens, or at times specified thereafter. THC was either allowed to remain in culture for the duration of the experiment or cells were washed free of excess drug after a 3 h incubation, as specified in each experiment.
Lymphocyte blastogenesis assay Peripheral blood lymphocytes were added to 96-well flat bottom microtiter plates (Costar, Cambridge, MA) at a concentration of 2 × 105 cells per well in 200 tJl. Cells were incubated at 37°C in an atmosphere of 5% CO2, 95°70 air for a period of 66 to 72 h. 3H-thymidine was added to the cultures for the final 18 h of incubation to measure blastogenesis. Cells were collected on fiber filter paper, using a cell harvester. The filters were placed in scintillation fluid and counts per minute determined using liquid scintillation spectrometry (Zhang, Friedman, Hsu, Specter, Friedman & Nonoyama, 1984).
Interleukin 2 treatment Based on data from previous experiments with natural killer (NK) cells (Specter et al., 1989) and preliminary experiments testing lymphocyte blastogenesis, IL-2 was used at a concentration of 30 units/ml. IL-2 was added to cultures for the LBT simultaneously with THC.
Marijuana and Immunity Table
1.
Inhibition
of
mitogen induced lymphocyte delta-9-tetrahydroeannabinol
263 blastogenesis
by
Counts per minute*
Medium DMSO* THC (~g/ml) 1 2.5 5 7.5 10
PHA
Con A
PWM
45,977 + 3439 46,411 _+ 1886
38,762 + 645 40,084 _+ 1662
13,639 _+ 1779 12,778 _+ 1191
47,919 _+ 4163 (103.3) 44,045 + 4935 (94.9) 39,175 +_ 5887 (84.4) 27,933 _+ 7918 (60.1) 9070 _+ 3349 (19.5)
39,507 _+ 2055 (98.6) 36,613 + 1874 (91.3) 33,635 _+ 1451 (83.9) 24,797 _+ 2212 (61.9) 9427 _+ 1909 (23.5)
13,948 _+ 1293 (109.2) 14,234 _+ 1022 (111.4) 14,874 _+ 942 (116.4) 16,140 _+ 510 (126.3) 12,316 _+ 1471 (96.4)
*Human peripheral blood mononuclear cells were cultured with the indicated mitogen _+ THC for 72 h. +Mean of four experiments + standard error of the mean, using triplicate cultures per experiment. *Dimethyl sulfoxide is the diluent for THC. Concentration listed is equivalent to the amount of DMSO in 10 t~g THC/ml. Lower concentrations of DMSO gave results similar to medium alone. ~Per cent versus DMSO control. ~Statistically significant at P
0192-0561/90 $3.00 + .00 International Society for lmmunopharmaeology.
M A R I J U A N A A N D IMMUNITY: T E T R A H Y D R O C A N N A B I N O L M E D I A T E D INHIBITION OF L Y M P H O C Y T E BLASTOGENESIS STEVEN SPECTER,* GERALD LANCZ and JUDITH HAZELDEN Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, 12901 N. Bruce B. Downs Blvd, Tampa, FL 33612, U.S.A. (Received 8 May 1989 and in final form 27 October 1989)
Abstract - - The effects of delta-9-tetrahydrocannabinol (THC) and its major metabolite, l 1-OH delta9-tetrahydrocannabinol (ll-OH THC) on mitogen driven lymphocyte blastogenic transformation (LBT) were studied. THC inhibited LBT responses to the T lymphocyte mitogens phytohemagglutinin and concanavalin A but not the B lymphocyte stimulant pokeweed mitogen. The metabolite ll-OH THC caused a comparable, but less significant, inhibition of LBT responses to the T cell mitogens. These inhibitions were dependent upon the drug doses and the time of incubation with the lymphocytes. There was no significant inhibitory activity of THC to the LBT when it was added 24 or 48 h after mitogen. In addition, exposure of lymphocytes to THC for 3 h, followed by removal of the drug prior to addition of mitogen had no effect on the cells' ability to respond to the mitogen. Thus, there appears to be a specified temporal period during which exposure of lymphocytes to THC results in an inhibition of blastogenesis. Interleukin 2 (30 units/ml) could not preclude the THC induced inhibition of lymphocyte blastogenesis. We conclude that THC and ll-OH THC inhibit T lymphocyte blastogenesis. However, unlike the THC mediated inhibition of natural killer cell activity (as shown previously), the process is not responsive to the cytokine interleukin 2.
The role that drugs of abuse may play in the pathogenesis of infectious diseases is disputed. Potentially these drugs may affect regulation of the immune system, potentiate primary infections, or promote re-activation of latent, quiescent infection. Any of these effects may contribute to exacerbation of human immunodeficiency virus (HIV) infection leading to the development of frank acquired immunodeficiency syndrome (AIDS). Several previous studies on the effect of marijuana or its major psychoactive component, delta-9-tetrahydrocannabinol (THC), on cells of the immune system have yielded contradictory results (reviewed in Munson & O'Brien-Fehr, 1983). White, Brin & Janicki (1975) and Lau, Tubergen, Barr, Domino, Benowitz & Jones (1976) both report that 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. In the former case, lymphocytes were taken from individuals who were chronic marijuana users. In the latter study, the individuals ingested
THC daily, up to 210mg/day for 18 days. By contrast, Nahas and coworkers reported a marked depression of LBT responses in individuals who smoked marijuana (Nahas, Suciu-Foca, Armand & Morishima, 1974). Petersen, Graham & Lemberger (1976) also reported alteration in LBT responses as well as T-cell rosette formation in individuals receiving marijuana while under hospital supervision. However, the results in the study by Petersen et al. were inconsistent, leading the authors to speculate that decreased immunocyte function may be related to other factors. In-vitro studies by Nahas, Morishima & Desoize (1977) indicated that THC could directly depress LBT responses to PHA. However, the concentrations of TH C (62.3 tag/ml or 2 × 10 4 M) which were required to inhibit lymphocyte blastogenesis in the study by Nahas and coworkers were toxic to human peripheral blood lymphocytes in our studies (Specter, Klein, Newton, Mondragon & Friedman, 1986). This study also indicated that 10/ag T H C / m l was not toxic to lymphocytes over an 18 h incubation period. Subsequent studies in our
*Author to whom correspondence should be addressed. 261
S. SPECTER et al.
262
laboratory have indicated that 10/Jg T H C / m l was not toxic to lymphocytes during a 48 h incubation (unpublished data). More recent studies using murine spleen cells indicate that THC inhibits lymphocyte blastogenic transformation (LBT) following stimulation with phytomitogens or bacterial lipopolysaccharide (Klein, Newton, Widen & Friedman, 1985; Pross, Klein, Newton & Friedman, 1987). In the studies by Klein et al. (1985) the 10/Jg T H C / m l concentration was not toxic to mouse lymphocytes after 48 h of culture. This indicated that the inhibition of lymphocyte function was not due to a cytotoxic effect of THC on lymphocytes but rather the result of some metabolic or structural alteration of the cells. Thus, we studied the response of human peripheral blood lymphocytes to phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) in the presence of micromolar, subtoxic concentrations of THC. The results indicate that THC inhibits T lymphocyte blastogenesis induced by PHA or Con A but not by PWM.
EXPERIMENTAL
PROCEDURES
Human subjects Human subjects were volunteers recruited from the community around the University of South Florida. A unit of blood was collected at the Southwest Florida Blood Bank and the buffy coat was separated. Studies were performed with blood from individuals who denied using marijuana or other controlled substances. Lymphocyte preparations Peripheral blood lymphocytes were separated from the buffy coat by centrifugation through F i c o l l - H y p a q u e as previously described (Specter, Rivenbark, Newton, Kawakami & Lancz, 1989). Marijuana components Delta-9-THC, the major psychoactive ingredient in marijuana, was obtained from the National Institute on Drug Abuse (NIDA), Bethesda, MD. The THC was received as a solution in ethanol at 200 mg/ml. Stock THC in ethanol was stored at - 20°C. Reconstitution and dilution were performed using dimethyl sulfoxide (DMSO) as diluent as described previously (Specter et al., 1989). THC testing in vitro was performed over a range of 1 tJg/ml to 10/Jg/ml (3.2 × 10 6_ 3.2 × 10 5M).
Controls consisted of cells exposed to medium only or cells exposed to dilutions of the DMSO diluent which were equivalent to the amount present in THC dilutions (0.5% DMSO or less). ll-OH-delta-9-THC (ll-OH-THC), is a major psychoactive metabolite of the naturally occurring delta-9-THC found in marijuana. This metabolite was obtained from the NIDA in an ethanol solution at a concentration of 56 tag/ml. Because of the lower concentration of l l-OH-THC it was diluted in RPMI-1640 medium directly from the ethanol stock. Medium supplemented with equivalent concentrations of ethanol were used in controls.
Interleukin 2 Interleukin 2 (IL-2) was a generous gilt from Dr W. R. Benjamin, H o f f m a n - L a R o c h e , Nutley, NJ.
THC treatment THC was freshly prepared on the day of experimentation and added to lymphocyte suspensions 3 h before culture with mitogen (pre-incubation), at the time of culture in the presence of mitogens, or at times specified thereafter. THC was either allowed to remain in culture for the duration of the experiment or cells were washed free of excess drug after a 3 h incubation, as specified in each experiment.
Lymphocyte blastogenesis assay Peripheral blood lymphocytes were added to 96-well flat bottom microtiter plates (Costar, Cambridge, MA) at a concentration of 2 × 105 cells per well in 200 tJl. Cells were incubated at 37°C in an atmosphere of 5% CO2, 95°70 air for a period of 66 to 72 h. 3H-thymidine was added to the cultures for the final 18 h of incubation to measure blastogenesis. Cells were collected on fiber filter paper, using a cell harvester. The filters were placed in scintillation fluid and counts per minute determined using liquid scintillation spectrometry (Zhang, Friedman, Hsu, Specter, Friedman & Nonoyama, 1984).
Interleukin 2 treatment Based on data from previous experiments with natural killer (NK) cells (Specter et al., 1989) and preliminary experiments testing lymphocyte blastogenesis, IL-2 was used at a concentration of 30 units/ml. IL-2 was added to cultures for the LBT simultaneously with THC.
Marijuana and Immunity Table
1.
Inhibition
of
mitogen induced lymphocyte delta-9-tetrahydroeannabinol
263 blastogenesis
by
Counts per minute*
Medium DMSO* THC (~g/ml) 1 2.5 5 7.5 10
PHA
Con A
PWM
45,977 + 3439 46,411 _+ 1886
38,762 + 645 40,084 _+ 1662
13,639 _+ 1779 12,778 _+ 1191
47,919 _+ 4163 (103.3) 44,045 + 4935 (94.9) 39,175 +_ 5887 (84.4) 27,933 _+ 7918 (60.1) 9070 _+ 3349 (19.5)
39,507 _+ 2055 (98.6) 36,613 + 1874 (91.3) 33,635 _+ 1451 (83.9) 24,797 _+ 2212 (61.9) 9427 _+ 1909 (23.5)
13,948 _+ 1293 (109.2) 14,234 _+ 1022 (111.4) 14,874 _+ 942 (116.4) 16,140 _+ 510 (126.3) 12,316 _+ 1471 (96.4)
*Human peripheral blood mononuclear cells were cultured with the indicated mitogen _+ THC for 72 h. +Mean of four experiments + standard error of the mean, using triplicate cultures per experiment. *Dimethyl sulfoxide is the diluent for THC. Concentration listed is equivalent to the amount of DMSO in 10 t~g THC/ml. Lower concentrations of DMSO gave results similar to medium alone. ~Per cent versus DMSO control. ~Statistically significant at P
