0192-0561/91 $3.00 + .00 Pergamon Press plc. International Society for Immunopharmacology.
Int. J. Immunopharmac., Vol. 13, No. 4, pp. 4 1 l - 4 1 7 , 1991. Printed in Great Britain.
DELTA-9-TETRAHYDROCANNABINOL AUGMENTS MURINE RETROVIRAL INDUCED IMMUNOSUPPRESSION A N D INFECTION STEVEN SPECTER,* GERALD LANCZ, GINA WESTRICH and HERMAN FRIEDMAN 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 20 August 1990 and in final form 26 October 1990)
Abstract -- Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, and the murine retrovirus, Friend leukemia virus (FLV), have been demonstrated to depress cellular immune function, including lymphocyte hlastogenic transformation and natural killer cell activity. The present study demonstrates that the two agents can work in concert to depress these immune activities more severely than either agent administered by itself. When 7.5 - 10 ~g/ml THC was added in vitro to spleen cells from mice infected 2 - 4 weeks earlier with FLV there was a noticeable decrease, beyond that seen with the drug or virus alone, for both lymphocyte hlastogenesis and natural killer cell cytotoxicity. In addition, when both FLV and THC were administered to mice concurrently with infection by herpes simplex virus (HSV), mortality attributed to the retrovirus infection occurred significantly more rapidly than in the absence of the drug and HSV. The data indicate that THC acted in the presence of a HSV infection to enhance the FLV induced mortality. By extrapolation to the human condition, these results suggest that marijuana could serve as a cofactor, possibly in conjunction with opportunistic pathogens, in the progression of infection due to the human immunodeficiency virus from latency to overt acquired immunodeficiency syndrome.
Epidemiological studies examining the time of human immunodeficiency virus (HIV) infection relative to the onset of the acquired immunodeficiency syndrome (AIDS) reveal an extended incubation period with, a mean of 10 years, lapses between infection and overt disease. This observation has led to speculation that, in addition to the HIV, co-factors are involved in the development of overt disease. Most of the studies that have been reported to date have focused on DNA viruses as co-factors in AIDS development, since several DNA viruses have been shown to transactivate HIV (Davis, Kenney, Kamine, Pagano & Huang, 1987; Gendelman, Phelps, Feigenbaum, Ostrove, Adachi, Khoury, Ginsburg & Martin, 1986; Mosca, Bednarik, Raj, Rosen, Sodroski, Hazeltine & Pitha, 1987). However, recent epidemiologic studies that have examined the incidence of AIDS in high risk populations, i.e. intravenous drug abusers and promiscuous male homosexuals, discovered a high incidence of narcotics use (Selik, 1990; Watson, 1990a). The concept that narcotics and other addictive drugs might serve as co-factors fostering
the progression of events associated with the development of overt AIDS has been examined in two recent publications (Friedman, Klein & Specter, in press; Watson, 1990a). Much of the recent interest in drugs of abuse as cofactors in augmenting the development of AIDS comes from the observations that marijuana, cocaine, opiates and alcohol each are capable of depressing immune functions (Friedman et al., in press; Watson, 1990b). In this regard, our laboratories have demonstrated that a broad spectrum of immune functions are depressed by the major psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) (Klein & Friedman, 1990; Specter, Lancz & Friedman, 1990a). Exposure of human or mouse lymphocytes to TH C in vitro results in a decrease in mitogen driven lymphocyte blastogenic transformation (LBT) (Klein, Newton, Widen & Friedman, 1985; Specter, Lancz & Hazelden, 1990b) and decreased natural killer (NK) cell activity (Klein, Newton & Friedman, 1987; Specter, Klein, Newton, Mondragon, Widen & Friedman, 1986). Furthermore, administration of
*Author to whom correspondence should he addressed. 411
S. SPECTER et al.
THC to mice also results in depressed immune function (Klein & Friedman, 1990). Delta-9tetrahydrocannabinol also has been demonstrated to affect the replicative capability of herpes simplex virus (HSV) (Lancz, Specter & Brown, submitted). Thus, THC may influence genetic expression of HSV, a virus known to transactivate HIV (Mosca et al., 1987), thereby possibly influencing the progression of HIV induced disease. These observations led us to investigate whether THC might exacerbate the immunosuppressive effects of the murine retrovirus, Friend leukemia virus (FLV). The immunosuppressive effects of FLV infection have been described (Friedman, 1975; Specter, Basolo & Bendinelli, 1987) and this retrovirus infection has been considered as a murine model for AIDS (Soldaini, Matteucci, LopezCepero, Specter, Friedman & Bendinelli, 1989). While the immunosuppressive aspects or characteristics of FLV and HIV have many similarities, especially the depression of immune function, there are some differences between these viral infections, the most notable being that FLV infection results in a neoplasia, while HIV causes a cytolytic infection of lymphocytes (Soldaini et al., 1989). The current study indicates that THC added in vitro enhances the immunosuppression seen in spleen cells from FLV infected mice. Suppression of NK cell activity and lymphocyte blastogenic responsiveness to mitogens was observed. Furthermore, THC administered to FLV infected mice enhanced the rate of mortality when these animals were concurrently infected with HSV.
Mice. Six to eight week old BALB/c female mice were purchased from Jackson Laboratories (Bar Harbor, ME), housed in plastic cages and fed water and mouse pellets ad libitum.
Viruses. Friend leukemia virus: a polycythemia inducing strain of FLV containing both lymphatic leukemia virus and spleen focus forming virus components was used throughout the study. Stock virus was prepared as a 10°70 (w/v) suspension of spleens collected 21 days post infection and homogenized in phosphate buffered saline, pH 7.4. Crude homogenates were clarified by centrifugation at 2000 g for 30 min at 4°C, and the supernatant fluids were collected and stored at - 7 0 ° C . Stock
preparations of virus contained approximately 5 x 103 infectious doses per ml. Herpes simplex virus: HSV type 1 strain F obtained from B. Roizman (University of Chicago) was used. Virus has grown in rabbit skin cells and then purified by centrifugation through a sucrose gradient as previously described (Lancz, 1980). Stock preparations of virus contained approximately 2 x 108 plaque forming units (PFU) and were stored at - 70°C. THC. Delta-9-tetrahydrocannabinol was obtained from the National Institute on Drug Abuse (Bethesda, MD). The THC was received as a stock solution in ethanol at 200 mg/ml, which was stored at - 2 0 ° C . Reconstitution and dilution were performed using dimethyl sulfoxide (DMSO) as the diluent as described previously (Specter et al., 1989). The activity of THC was tested in vitro over a range of 1 - 10/ag/ml (3.2 x 10 6 3.2 x 10-5 M), concentrations previously shown to be non-cytotoxic to lymphocytes (Specter et al., 1986). Controls consisted of spleen cells exposed to medium only or cells exposed to medium containing the DMSO diluent at a concentration that was equivalent to the amount of DMSO present in the sample receiving the highest concentration of THC (0.1°70 DMSO). For in vivo studies THC was suspended in mouse serum at a concentration of 2 mg/ml and 1 ml was administered by intraperitoneal injection. Mitogens. Phytohemagglutinin (PHA) (Sigma, St. Louis, MO) was used at a concentration of 2.5/ag/ml. Escherichia coli lipopolysaccharide (LPS) (Difco Laboratories, Kalamazoo, MI) was used at a concentration of 10 tag/ml. T H C treatment f o r in vitro studies. Delta-9tetrahydrocannabinol was added to 10 6 spleen cells at the concentrations listed in each experiment. Cells were either pre-treated with THC for 3 h or drug was added at the time of culture initiation, as indicated in each experiment. L y m p h o c y t e blastogenesis. Lymphocyte blastogenic transformation was performed as previously described (Klein et al., 1985). Spleens were aseptically removed from mice and minced to prepare a suspension of single cells. One hundred microliters containing 2 x 105 cells were added to wells of a flat bottomed 96 well microtiter plate along with 50 ~l of drug diluted in medium or medium alone. Fifty microliters of medium
THC Augmentation Table 1. Effect of delta-9-tetrahydrocannabinol and Friend leukemia virus infection on lipopolysaccharide stimulation of lymphocyte blastogenesis*
0* Group DMSO (control) THC (/~g/ml) 1
2.5 5 7.5 10
123 99 56 27 11
ND 121 35 5 2
Days post infection 7 14
Percent of control response 100 26
<1 10 1 <1 <1
4 18 12 4 <1
67 110 93 67 43
10 23 20 10 2
*Spleen cell suspensions were exposed to E. coli lipopolysaccharide (2.5 ~g/ml) for 66 h. DMSO (0.1%) or THC (at the indicated concentration) was added simultaneously with LPS. Cells were pulsed with 3H-thymidine for the last 18 h of the assay. *Non-infected mice. *Days post FLV infection. ~All groups were compared with this control. ND: not done.
containing mitogen was then added to yield a final volume of 200/A per well. Cells and drug were incubated for 6 6 h at 37°C in a humidified atmosphere of 5% CO2, 95% air. Ten microliters of medium containing 0.5/aCi 3H-thymidine was then added to each well for the last 18 h o f incubation. Samples, collected on glass fiber filter paper using a multiple automated sample harvester, were air dried and placed into vials containing liquid scintillation cocktail and assayed for c o u n t s / m i n using a beta spectrometer. All samples were run in triplicate and experiments repeated three times.
Natural killer cell activity. The NK cell assay was performed exactly as described (Moody, Specter, Bendinelli & Friedman, 1984). A n 18 h NK assay was used to allow for sufficient cytotoxicity, since B A L B / c mice normally express relatively low levels of NK cell activity in a 4 h assay. Infection o f mice. Mice were infected with FLV by an intraperitoneal (i.p.) injection of 0.2 ml o f a 10-fold dilution of stock virus. This dose causes a lethal infection in all animals within 3 0 - 4 5 days. Mice infected with HSV received an i.p. injection of 0.1 ml virus diluted in PBS to a concentration of 2 × 10 4 P F U / m l , a dose previously determined to be lethal for 20% o f these mice. RESULTS
Lymphocyte blastogenesis was inhibited by either FLV or T H C regardless of whether LPS or P H A was used to stimulate the cells (Tables 1 and 2).
Inhibition due to FLV infection alone was seen at 2 weeks post infection (p.i.), with an inhibition of the LBT response to LPS being approximately 80% from 2-4 weeks p.i. (Table 1). Delta-9tetrahydrocannabinol depressed the LBT response to LPS in a dose related manner with strong suppression (73%) noted at 7.5 tag/ml and almost total ablation (89%) of the response at 10/ag/ml. A comparison was made of the LPS induced LBT response of spleen cells from non-infected mice with those obtained at 3 days post FLV infection. Delta-9tetrahydrocannabinol added to these cells showed a dose related decrease in the LBT which was more severe in virus infected mice. Interestingly, it appeared that FLV infection at 7 days p.i. interfered with the T H C - m e d i a t e d (5 - 10 tag/ml) inhibition of LPS induced LBT. At 2 - 4 weeks p.i. there was a notable combined effect by FLV and >--5/ag T H C / m l in suppressing the LPS induced LBT (Table 1). The LBT responses to P H A were similarly inhibited by FLV infection at 2 - 4 weeks p.i. (>95%) as well as by T H C at concentrations at 7 . 5 / a g / m l (50%) or 10 tag/ml (98%) in non-infected animals (Table 2). Phytohemagglutinin blastogenesis was not affected by the F L V / T H C combination at 3 days p.i. beyond what was observed with virus or drug alone. As with LPS, FLV enhanced the P H A induced LBT response in the presence of T H C at 7 days p.i. Suppression of the PHA-stimulated LBT due to the combination of FLV and T H C was greater than either used alone at 21 and 28 days p.i. when cells were exposed to the drug at 5 - 10/ag T H C / m l (Table 2).
S. SPECTER e t al.
Table 2. Effect of delta-9-tetrahydrocannabinol and Friend leukemia virus infection on phytohemagglutinin stimulation of lymphocyte blastogenesis*
0t Group DMSO (control) THC (gg/ml)
152 128 86 50 2
87 101 71 17 2
2.5 5 7.5 10
Days post infection 7 14
Percent of control response 116 5
4 3 <1
3 3 2
131 132 117 96 78
10 7 5 2
*Spleen cell suspensions were exposed to phytohemagglutinin (2.5 t~g/ml) for 66 h. DMSO (0.1%) or THC (at the indicated concentration) was added simultaneously with PHA. Cells were pulsed with ~H-thymidine for the last 18 h of the assay. tNon-infected mice. *Days post FLV infection. ~AI1 groups were compared with this control. Table 3. Suppression of natural killer cell activity by delta9-tetrahydrocannabinol added in vitro to spleen cells from Friend leukemia virus infected mice
Medium (control) DMSO (Diluent control) THC (~g/ml) 1 2.5 5 7.5
Percentspecificlysis Non-infected FLVin~cted* 41.5±6.0 5.4±1.6 40.8±3.5 7.1±4.8 42.4±3.9 43.3±3.3 42.7±2.0 30.2±4.2
4.5±2.6 6.3±3.3 5.7±2.9 0.0
*Mice were infected with approximately 500 IDs0 F L V , spleens were r e m o v e d 14 days later, a n d single cell suspensions exposed to T H C for 3 h before p e r f o r m ing a n 18 h N K assay. E a c h p o i n t represents the m e a n f r o m two mice tested independently. E f f e c t o r to target ratio 100 : 1.
N a t u r a l killer cell activity was suppressed i n d e p e n d e n t l y by b o t h F L V a n d T H C (Table 3). By 14 days p.i. the N K response was depressed by a b o u t 85-90°70, a l t h o u g h there was n o depression o f NK activity n o t e d at 7 days p.i. (data not shown). Cells f r o m n o n - i n f e c t e d mice t h a t were exposed to ~<5.0/~g T H C / m l in vitro were u n a f f e c t e d , while 7.5 ~ g / m l only showed a b o u t 75°7o as m u c h activity as controls, 10/~g T H C / m l totally a b l a t e d NK cell activity (not shown). W h e n spleen cells f r o m F L V infected mice were exposed to T H C in vitro there was no additive effect between virus a n d drug at ~<5.0 jag T H C / m l but at 7.5 /~g/ml the c o m b i n a t i o n completely inhibited cytotoxicity. A t earlier time points there was n o evidence of a n additive effect between drug
a n d virus a n d at later time points suppression was t o o great due to virus alone to see any additional effect by T H C (data not shown). Dual virus infection with F L V a n d H S V resulted in a slight b u t insignificant increase in the m o r t a l i t y rate due to either virus, w h e n F L V infection was established 7 days before H S V infection (Fig. 1). The m e a n time to d e a t h for F L V i n f e c t i o n alone was 33.0 _+ 2.0 days, for F L V a n d T H C was 33.4 _+ 4.3, while F L V a n d H S V was 40.5 + 7.6 days. However, w h e n 1 m g T H C was a d m i n i s t e r e d b o t h 2 days before a n d after H S V infection there was a significant reduction (as assessed using the Wilcoxon r a n k test P < . 0 5 w h e n g r o u p 4 was c o m p a r e d with groups 1, 2 a n d 3) in the m e a n time to d e a t h (23.8 ___ 3.0 days). A single injection o f T H C given 2 days before H S V did not alter the mortality rate. W h e n H S V only was used to infect mice, mortality occurred within 14 days (2/10 mice) or mice survived w i t h o u t sign of illness for >3 m o n t h s , w h e n the experiment was t e r m i n a t e d . Mice t h a t died d u r i n g the first 14 days were not used to assess m o r t a l i t y related to FLV, but n o g r o u p h a d m o r e t h a n 2 deaths t h a t could be a t t r i b u t e d to HSV.
The data presented in this study s u p p o r t the hypothesis t h a t T H C m a y act as a co-factor in F L V infection leading to a f u r t h e r i n h i b i t i o n o f i m m u n e f u n c t i o n a n d increased progression of infection. This conclusion is t e m p e r e d by the n a r r o w range of suppressive activity due to T H C a n d the rapidly
THC Augmentation Group 1
--'~-'- Group 2
. . . . . . .
.~ . . . . . . . .
~ . . . .
"_ . . . . . . .
2 6 2 8 SO 3 2 8 4 : 1 6 : 1 8 40 D a y s post FLV infection
Fig. 1. Increased mortality rate in mice infected with FLV and HSV that received THC. Mice were infected with approximately 500 IDa0FLV and 7 days later infected with herpes simplex virus (2 × 103 P F U - 1 LDz0),some mice also received THC (1 mg intraperitoneally/mouse/injection). Group 1, FLV only; Group 2, FLV and THC (9 days p.i.) only; Group 3, FLV and HSV only; and Group 4, FLV, HSV and THC (5 and 9 days post FLV); n equals the number of mice in each group. Using the Wilcoxon rank test Group 4 was statistically different from groups 1, 2 and 3; P<0.05. progressive nature of the acquired immunodeficiency due to FLV, which together limit the opportunity to observe a combined effect of drug and virus. These limitations notwithstanding, the data support the notion that the suppressive effects of THC and FLV on the immune system are additive, with some suggestion of a synergistic inhibition by these two agents, especially at and after 2 weeks post FLV infection. The early combined effect noted at 3 days post FLV infection is likely attributed to virus and drug whereas the effects seen at 14 days p.i. or later can be attributed to the virus and tumor acting in concert with THC. This does not explain the failure to see suppression due to T H C in spleen cells from mice infected 7 days earlier with FLV. Continued study of this retrovirus model is required to uncover the reason(s) for the protective effect of T H C on immunosuppression observed in FLV infected mice at 7 days p.i. Furthermore, these studies should determine whether or not T H C can promote replication of FLV or whether the drug and virus combination will promote replication of an opportunistic infection such as HSV. It is interesting to note that T HC administration to mice both 2 days before and after HSV resulted in an increase in mortality due to this virus in previous studies performed in our laboratories (unpublished observations). However, since all deaths occur
before 14 days p.i. with HSV it is unlikely that this contributed to the mortality noted here. Several groups had one or two mice which died within this time frame in this experiment but were discounted in determining FLV related deaths. Definitive evidence that drugs of abuse act as cofactors in the development of AIDS remains to be established. In vivo effects of THC on immunity are less amenable to experimentation. In immune function studies employing volunteers who smoked marijuana or a purified THC preparation in a controlled environment, or using chronic marijuana smokers, a significant alteration of immune status was not observed (reviewed in Specter et al., 1990a). It should be noted that relatively high concentrations of TH C are needed to generate suppressive effects in vitro (5 - 10/~g T H C / m l ) in the presence of medium containing 10% serum. Levels of THC in the blood of chronic smokers of marijuana generally do not exceed 0.5 tag/ml. However, it is important to recognize that THC accumulates to considerably higher concentration in the throat and lungs of smokers and many THC effects may occur in this region. There are several reports of pathology in the throat of marijuana smokers including inflammatory changes (Guarisco, Cheney, LeJeune & Reed, 1988) and evidence of increased incidence of cancer in anatomical areas in which TH C would be expected to
S. SPECTERet al.
accumulate (Donald, 1986; Taylor, 1988; J. Endicott, personal communication). These changes may be a reflection of the irritant effect of marijuana in the head and neck region, recognizing that both irritant and immunosuppressive effects of this drug might contribute to the oncogenic process. Data on the TH C levels in draining lymph nodes would permit a more definitive assessment of microenvironmental effects of the drug in the head and neck region. At present, no such data have been collected. The evidence at this time is suggestive, but not compelling, that marijuana may act as a co-factor in the exacerbation of AIDS in HIV infected individuals, based on the present findings and reports investigating marijuana and disease cited above. There is presently no evidence that marjuana use is associated with increased incidence of acute infection in man. Nevertheless, our data show that mortality associated with FLV infection occurred sooner in the presence of a concurrent infection (HSV) and after administration of THC, while either agent alone had no significant effect. Juel-Jensen (1972) reported that smoking marijuana caused exacerbation of HSV latent infection. Others have demonstrated that T HC will increase susceptibility to HSV or bacterial infection in experimental animal models (Holsapple, McNerney, Mishkin & Cabral, 1985; Morahan, Klykken, Smith, Harris & Munson, 1979). In subsequent studies Cabral and co-workers
reported that decreased levels of interferon were associated with increased susceptibility to HSV in mice given TH C (Cabral, Lockmuller & Mishkin, 1986). In this regard, Blanchard and co-workers demonstrated that TH C can directly inhibit the induction of interferon in vivo and in vitro (Blanchard, Newton, Klein, Stewart & Friedman, 1986). Thus, there is evidence to suggest that chronic types of infections may be exacerbated by marijuana. From the extended incubation period and the myriad of unusual opportunistic infections seen in AIDS patients, substances, chemicals or infectious agents that can depress immune function may serve as co-factor(s) contributing to the development of overt disease in HIV infected persons. The high percentage of AIDS patients who use narcotic drugs in general and marijuana in particular propels these substances to the forefront of a list of agents that may contribute to the pathogenesis of the HIV and opportunistic organisms which cause fatal disease in AIDS patients. Smoking marijuana should, therefore, be considered as a potential hazard in immunocompromised individuals beyond any other detrimental effects documented in otherwise healthy persons. Acknowledgements - - This work was supported in part by
grant Nos DA04141, DA05363, DA05794 from the Public Health Service, National Institute on Drug Abuse.
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