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TOXICOLOGY

Arch. Toxicol. 40, 97--101 (1978)

9 Springer-Verlag 1978

Hashish Smoking and T-Lymphoeytes E. Kaklamani 1, D. Trichopoulos 1, A. Koutselinis 2, M. Drouga t, and D. Karalis 1 1 Department of Hygiene and Epidemiology, 2 Department of Forensic Medicine and Toxicology,School of Medicine, University of Athens, Goudi 609, Athens, Greece

Abstract. To investigate the effect of long term hashish use on T-lymphocytes we have measured the incorporation of 14C-thymidine by peripheral blood lymphocytes, unstimulated and stimulated by phytohemagglutinin, as well as the proportion and number of E-rosettes in 12 healthy male chronic users of the drug before and after a smoking session, and in 15 control subjects. The results show that hashish smoking does not impair the response of lymphocytes to PHA, at least at the concentration of PHA used. Furthermore there is a possibility that among chronic users a hashish smoking session may have a slight stimulatory effect. Key words: Hashisch -- Cannabinoids -- T-lymphocytes -- Immunology. Zusammenfassung. Zur Untersuchung des Langzeiteffekts yon Haschischgebrauch auf T-Lymphozyten haben wir den Einbau yon 14C-Thymidin in Lymphozyten des str6menden Blutes ohne und mit Stimulierung dureh Phythfimagglutinine (PHA) gemessen. Zugleich wurde das Zahlenverh~iltnis yon E-Rosetten bei zwSlf gesunden m~innlichen chronischen Haschischkonsumenten vor und nach einer Rauchperiode bestimmt sowie mit 15 Kontrollpersonen verglichen. Haschischrauchen ergab keine Schiidigung der Lymphozytenrekation auf PHA, wenigstens bei der verwendeten PHA-Konzentration. Es ist nieht ausgeschlossen, dab nach chronischem Haschischrauchen eine leichte Stimulierung eintritt.

Introduction The effect of marijuana on the immune system has been the subject of several studies in humans and animals (Nahas et al., 1974, 1976; Gupta et aL, 1974; Silverstein and Lessin, 1974; White et al., 1975; Rosenkrantz et al., 1976; Rosenkrantz, Send offprint requests to D. Trichopoulos at the above address

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1976; L a u et al., 1976; R a c h e l e f s k y et al., 1976). A l t h o u g h the results are conflicting there is s o m e e v i d e n c e that c a n n a b i n o i d s suppress the i m m u n e r e s p o n s e ( N a h a s et al., 1974, 1976; G u p t a et al., 1974; R o s e n k r a n t z et al., 1976; R o s e n k r a n t z , 1976). H o w e v e r , there is n o e p i d e m i o l o g i c a l or clinical observation, thus far, to suggest t h a t c h r o n i c users o f m a r i j u a n a are m o r e susceptible to neoplastic or infectious diseases ( M u n s o n , 1975) as it m i g h t be e x p e c t e d in c h r o n i c i m m u n o s u p p r e s s e d individuals. O n the o t h e r hand, it has been r e p o r t e d that m a r i j u a n a intoxication is a c c o m p a n i e d by a m a r k e d p h a r m a c o l o g i c a l t o l e r a n c e in several animal species expressed as a necessity to increase d o s a g e to o b t a i n the effect o f initial d o s a g e (Williams et al., 1946; M c M i l l a n et al., 1970). This t o l e r a n c e b e c o m e s evident after a time period similar to t h a t r e q u i r e d for the d e v e l o p m e n t o f a cellular i m m u n e response. This o b s e r v a t i o n and o t h e r e x p e r i m e n t a l d a t a ( N a h a s et al., 1973) f a v o r the h y p o t h e s i s t h a t t o l e r a n c e is due to the d e v e l o p m e n t o f an i m m u n e response to cannabinoids. T o investigate the effect o f l o n g - t e r m hashish use on T - l y m p h o c y t e s we h a v e m e a s u r e d the i n c o r p o r a t i o n o f ~4C-thymidine by peripheral b l o o d l y m p h o c y t e s unstimulated and stimulated by p h y t o h e m a g g l u t i n i n e ( P H A ) as well as the p r o p o r t i o n and n u m b e r o f E-rosettes in 12 h e a l t h y m a l e c h r o n i c users o f the drug and c o m p a r e d the results to t h o s e f r o m a c o n t r o l g r o u p o f 15 h e a l t h y subjects.

Materials and Methods Subjects. Twelve volunteers chronic hashish smokers 40--45 years of age were included in the study. They had been using hashish for more than 20 years but took no other psychotropic drugs. Tobacco smoking and alcohol use did not disqualify the subjects from the study. Some of them had signs of chronic cannabinismus. According to information given by the subjects they had smoked every available kind of hashish product (pure resin, marijuana etc.), using in most instances a "nargileh pipe". Nargileh is a pipe used chiefly in the Near East that cools the tobacco (or hashish) smoke by passing it through a reservoir of water. Some of them reported to have smoked, in certain occasions, up to 100 g of crude hashish at a time. The subjects were not receiving medications at the time of study and were in good health. The control group consisted of 15 healthy adults of low socioeconomic class who consumed no cannabinoids or any other drug. Five of the controls were moderate users of tobacco. Hashish. In the experiments we have used pure hashish resin which was extracted from the flowering tops of female cannabis plants. The resin used was analysed by chromatographic methods. The concentration of A9-TI--IC was 1.6%. To evaluate the chronic effect of hashish, blood specimens were taken at least 20 h after the last abuse of the drug. Then, the subjects were allowed to smoke 20 g of pure resin by a "nargileh pipe" for a time period not exceeding 15 min and a new blood specimen was taken 30-60 min later. During this time interval the highest concentration of the drug in the blood stream is also noted (Galanter et al., 1972). For each hashish user at least one control subject was tested at the same time. Lymphocyte Preparation. 20 ml heparinized (500 IU Leo) venous blood were mixed with an equal volume of saline layered on 30 ml of Lymphoprep (Nyegaard, Oslo) (9.6 w/v sodium metrizoate + 5.6% Ficol) and centrifuged at 400 g for 30 min. Cells at the interface were removed and washed three times with RPMI 1640 medium (GIBCO). The cells were suspended in the same medium and the lymphocytes were counted. Lymphoeytes Transformation by Phytohemagglutinin (PHA); 1 x 106. Lymphocytes were cultured in 1 ml RPMI 1640 supplemented with 20% fetal calf serum (B-D Merieux No. 8 937 1) and antibiotics

Hashish Smoking and T-Lymphocytes

99

(100 units/ml penicillin and 100 vg/mi streptomycin). Triplicate cultures were stimulated with 0.04 ml PHA-M (Difco) and incubated in an atmosphere of 5% CO2 at 37~ C. Unstimulated cells were also cultured in triplicate. 24 h before harvesting cultures were labelled by adding 0.5 ~xCi 14C-thymidine (CIS, specific activity 55.3 mCi/mM). The rate of synthesis of DNA was determined by measuring the incorporation of 14C-thymidine into the trichloroacetic acid (TCA) precipitable material. After 72 h the setting for the stimulated cultures and 24 h after the setting for the unstimulated cultures cells were recovered by centrifugation, washed in saline, and fixed in cold TCA. The precipitate after vigorous agitation with vortex mixer were transfered on Watman discs and dried. The filters were placed into scintillation vials with 10 ml of toluene-based scintillation fluid. Radioactivity was measured in a liquid scientillator counter and expressed as net counts per minute (cpm). 14C-thymidine uptake values are not normally distributed. Therefore comparisons of thymidine uptake before and after hashish smoking were done by the Wilcoxon test for pair differences and comparisons between smokers and healthy controls were done by the Wilcoxon test for two samples (Siegel 1956). Furthermore median rather than mean values were used as measures of location of the distributions in Table 1.

Spontanous Rosettes (E.R.). The technique suggested by Hudson (1973) was followed with minor modifications. 0.1 mi of a suspension containing 1 x 107 washed lymphocytes per ml of RPMI was mixed with 0.2 ml of a 2% suspension of washed sheep red blood cells (SRBC), and 0.1 ml of fetal calf serum (absorbed with SRBC). The mixture was spun at 150 g for 10 rain and incubated at room temperature for 1 h and then at 4~ C for 18 h. The cells were resuspended gently, mixed with one drop of 0.4% brillant cresyl blue and counted. Rosettes developed under these conditions were found to be stable. Lymphocytes covered with at least four SRBC were considered as E-rosettes. The absolute number of T-lymphocytes was estimated from the percentage of E-rosettes and the total number of lymphocytes per cmm.

Results The results concerning 14C-thymidine uptake are summarized in Table 1. It was found that am o n g hashish smokers the sponaneous uptake o f 14C-thymidine was significantly higher after the smoking session (P < 0.05). Before smoking session spontaneous uptake a m o n g hashish smokers was higher than am o n g normal controis but not significantly so (P > 0.10). A m o n g hashish smokers thymidine uptake after P H A stimulation was also higher after a smoking session (P < 0.10). N o differences were observed after P H A stimulation between hashish smokers before a smoking session and controls. N o significant differences were found between the hashish smokers (either before or after smoking session), and normal controls with respect to the proportion and the absolute number o f E-rosette forming lymphocytes (Table 2).

Discussion The results o f the present study indicate that long-term hashish smoking has not a per m an en t depressing effect on lymphocytes response to P H A . Other investigators have noted that cannabinoids when present in the culture medium inhibit thymidine incorporation. Th e degree of inhibition however, is a function o f the tetrahydrocannabinol ( T H C ) and serum concentration in the culture medium and it is reversed by washing (Nahas et al., 1976). Serum has a protective effect which m a y be due to the binding o f T H C to serum lipoproteins (Wahlqvist et al., 1970). Therefore, some of

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Table 1. ~4C-thymidineincorporation by peripheral blood lymphocytes in chronic hashish users before and after smoking session and in healthy controls; thymidine incorporation was measured as counts per minute (cpm) of radioactivity in cultures unstimulated or stimulated in vitro with PHA Chronic hashish users

Controls

Before smoking sessions

After smoking sessions

..............................

Spontaneous uptake-cpm

PHA uptake-cpm

Spontaneous uptake-cpm

PHA uptake-cpm

Spontaneous uptake-cpm

PHA uptake-cpm

5,152 6,748 791 1,152 1,281 1,833 1,084 1,459 N.D. a N.D? N.D. a 681

10,766 19,844 58,745 24,495 25,388 33,564 49,628 56,798 8 753 28,658 24,773 33,790

6,824 7,446 1,392 1,123 1,169 N.D. a 1,694 2,019 N.D. a N.D. a 562 1,030

21,594 23,007 32,530 29,456 26,432 43,124 47,389 63,621 20,979 38,219 26,217 32,846

6,398 1,742 2,652 1,889 N.D. a 744 801 1,003 724 436 790 863 1,034 1,010 577

18,817 24,332 39,789 62,966 39,739 22,797 28,590 34,544 21,499 30,284 24,794 22,099 52,850 47,992 24,059

12

9

12

14

15

27,023

1,392

30,993

933

28,590

NO.

9 Median 1,281 a = not done

Table 2. Proportion and absolute number of E-rosette forming lymphocytes in chronic hashish users before and after smoking session and in healthy controls; mean (+ Standard Error of mean) Chronic hashish users Before smoking sessions E.R. (%) E.R. (No./mm 3)

57.5 (+ 1.1) 1,479

(+ 158)

Controls After smoking sessions 57.6 (+ 0.9) 1,478

(+_ 145)

59.8 (_+ 1.2) 1,525

(+ 97)

the discrepancies reported by several investigators m ay be due to technical differences. With respect to the effect o f hashish on the values of T-lymphocytes the results presented in this paper should not be c o m p a r e d with those o f G u p t a et al. (1974) who have applied the "active rosette test" (Wybran and Fudenberg, 1973). The slightly higher uptake of 14C-thymidine after the smoking session, observed in this investigation, suggest that hashish smoking m ay have a stimulatory effect on

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101

T-lymphocytes in chronic users of the drug. However there is a substantial overlapping in the values of the observed distributions and further studies are required before firm conclusions could be drawn. A higher uptake of 3H-thymidine by lymphocytes of chronic users receiving T H C has also been reported by Lau et al. (1976), and N a h a s et al. (1973) have shown that T H C can be immunogenic in experimental animals. Confirmation of these findings would indicate that lymphocytes from heavy chronic hashish smokers m a y transform in the presence of optimal a m o u n t s of hashish products.

References Galanter, M., Wyatt, R. J., Lemberger, L., Weingartner, H., Vaughan, T. B., Roth, W. T.: Effects on humans of A9-tetrahydrocannabinol administered by smoking. Science 176, 934-936 (1972) Gupta, S., Grieco, M. H., Cushman, P., Jr.: Impairment of rosett-forming T-lymphocytes in chronic marihuana smokers. New Engl. J. Med. 291, 874--877 (1974) Hudson, L.: Differentiation and function of lymphoid cells. Current titles in Immunol. Transplant. and Allergy 1, 394--395 (1973) Lau, A. J., Tubergen, D. G., Barr, M., Jr., Domino, E. F., Benowitz,N., Jones, R. T.: Phytohemagglutinin-induced lymphocyte transformation in humans receiving Ag-tetrahydrocannabinol. Science 192, 805-807 (1976) McMillan, D. E., Harris, I. S., Frankenheim, J. M., Kennedy, J. S.: L-A9-trans-tetrahydrocannabinolin pigeons: Tolerance to the behavioral effects. Science 169, 501-503 (1970) Munson, A. E.: Marihuana and immunity. In: Marihuana and health hazards (J. R. Trinkleberg, ed.), pp. 39-45. New York: Academic Press 1975 Nahas, G. G., Zagury, D., Schwartz, I. W., Nagei, M. D.: Evidence for the possibleimmunogenecityof Ag-tetrahydrocannabinol (THC) in rodents. Nature (Lond.) 243, 407--408 (1973) Nahas, G. G., Suciu-Foka, N., Armand, J. P., Morishima, A.: Inhibition of cellular mediated immunity in marihuana smokers. Science 183, 419--420 (1974) Nahas, G. G., Desoize, B., Hsu, J., Morishima, A.: Inhibitory effect of A9-tetrahydrocannabinolon nucleic acid synthesis and proteins in cultured lymphocytes. In: Marihuana: chemistry, biochemistry and cellular effects. (G. G. Nahas, ed.), pp. 299--312. Berlin-Heidelberg-NewYork: Springer 1976 Rachelefsky, G. S., Opelz, G., Mickey, M. R., Lessin, P., Kiuchi, M., Silverstein,M. J., Stiehm, E. R.: Intact humoral and cell-mediatedimmunity in chronic marijuana smoking. J. Allergy clin. Immunol. 58, 483--490 (1976) Rosenkrantz, H.: The immune response and marihuana. In: Marihuana: chemistry, biochemistry and cellular effects (G. G. Nahas, ed.), pp. 441--456. Berlin-Heidelberg-NewYork: Springer 1976 Rosenkrantz, H., Miller, A. J., Esber, H. J.: Ag-tetrahydrocannabinol suppression of the primary immune response in rats. J. Toxicol. Environ. Health 1, 119--125 (1976) Siegel, S.: Nonparametric statistics for the behavioral sciences, p. 116. New York: McGraw-Hill 1956 Silverstein, M. J., Lessin, P. J.: Normal skin test responses in chronic marijuana users. Science 186, 740--741 (1974) Wahlqvist, M., Nilsson, I. M., Sandberg, F., Agurell, S.: Binding of delta-9-THC to human plasma proteins. Biochem. Pharmacol. 19, 2579--2584 (1970) White, S. C., Brin, S. C., Janicki, B. W.: Mitogen-inducedblastogenic responses of lymphocytes from marihuana smokers. Science 188, 71-72 (1975) Williams, E. G., Himmelsbach, C. K., Wikler, A., Ruble, D. C.: Sudies on marihuana and pyrahexyl compound. Publ. Hlth Rep. (Wash.) 61, 1059--1083 (1946) Wybran, J., Fudenberg, H. H.: Thymus-derived rosette-forming cells in various human disease state: cancer, lymphoma, bacterial and viral infections and other diseases. J. clin. Invest. 52, 1026--1032 (1973) Received October 28, 1977