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Inhibition of cytokine release by cocaine
Int. J. lmmunopharmac., Vol. 16, No. 4, pp. 295-300, 1994 Elsevier Science Ltd International Society for lmmunopharmacology Printed in Great Britain. All rights reserved 0192-0561/94 $6.00 + .00
Pergamon 0192
- 0561(93)E0020-A
INHIBITION OF CYTOKINE RELEASE BY COCAINE HONG-MING SHEN, JOHN L. KENNEDY and DAVID W. OU* Department of Pathology, College of Medicine, The University of Illinois at Chicago and VA West Side Medical Center, Chicago, IL 60612, U.S.A. (Received 7 September 1993 and in final form 1 December 1993)
- - The influence of cocaine on cytokine release by mouse peritoneal macrophages has been investigated in vitro. LPS-stimulated TNF and IL-1 secretion were suppressed by cocaine in higher dosages. This suppression was shown to be dose-related. The synthesis of cAMP, however, was stimulated by cocaine. Though cAMP is generally considered a mediator for cytokine release, further studies are required to demonstrate whether cocaine directly affects cytokine release by macrophages or indirectly influences cytokine release through stimulating cAMP synthesis. Abstract
Besides the previously described cocaine effects on the central nerve system (CNS), the influences of cocaine on the immune system in vivo and in vitro have been identified. These include: (1) lymphocyte proliferation (Luo, Patel, Wiederhold & Ou, 1992; Klein, Newton & Friedman, 1988); (2) natural killer (NK) cell activity (Di-Francesco, Pica, Croce, Favalli, Tubaro & Garaci, 1990; Dyke, Stesin, Jones, Chuntharapai & ~eaman, 1986; Lu & Ou, 1989); (3) lymphocyte subset alteration (Lopez, Huang, Watzl, C h e n & Watson, 1991; Bagasra & Forman, 1989); (4)phagocyte activity (Ou, Shen & Luo, 1989); (5)antibody formation (Ou, Shen & Luo, 1989); etc. The actions of cocaine on cytokine and prostaglandin E (PGE) release were reported recently. The results from the analysis of drug users' hair samples show that cocaine increases the deposition of interleukin-2 (IL-2) in the hair, but not interleukin-1 (IL-1) and gamma-interferon (gammaIFN) (Chen, Pillai, Erickson, Martinez, Estrada & Watson, 1991). Furthermore, PGEs are significantly increased in drug users' amniotic fluid (Ahluwalia, Clark, Wetney, Smith, James & Rajguru, 1992). It is well known that the tumor necrosis factor (TNF) and interleukin-1 (IL-I) as cytokines are endogenous pyrogens and can activate not only monocytes/macrophages, but also lymphocytes, endothelial cells, neutrophils, chondrocytes, fibroblasts, etc. (Delustro, Mulkins & Allison, 1987;
Hamilton & Slywka, 1981; Chirgwin, Przybyla, MacDonald & Rutter, 1979). These two cytokines share a wide spectrum of biological activities, but the regulation of their production shows some dissimilarities (Lachman, Dinarello, Liansa & Fidler, 1986; Mannel, Moore & Mergenhagen, 1980; Onozaki, Matsushima, Aggarwal & Oppenheim, 1985). However, TNF and IL-1 production are both inhibited by increasing intracellular cyclic adenosine monophosphate (cAMP) levels (Renz, Gong, Schmidt, Nain & Gemsa, 1988; Hart, Whitty, Piccoli & Hamilton, 1989). In this study we further characterized the influences of cocaine on cytokine and cAMP release by mouse peritoneal macrophages in vitro, using several well-defined methods, described elsewhere (Coligan, Kruisbeek, Margulies, Shevach & Strober, 1991; Rosenwasser & Dinarello, 1981; Yamamoto & Suzuki, 1987). The release of IL-1 and TNF in vitro was inhibited by higher concentrations of cocaine. However, the activity of adenylate cyclase in the macrophages was stimulated by cocaine in a dosedependent fashion. , EXPERIMENTAL
PROCEDURES
Reagents
Cocaine, lipopolysaccharide, concanavalin A, actinomycin D, arachidonic acid, 3 ' - 5 ' cyclic
*Author to whom correspondence should be addressed at: Department of Pathology, College of Medicine, 1853 West Polk Street, Chicago, IL 60612, U.S.A. 295
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adenosine monophosphate, anti-cAMP serum were obtained from SIGMA. 3 ' - 5 ' cyclic adenosine monophosphate [125-I] tracer was from Dupont. [Methyl-~H] thymidine (2.0 curies/mmol) was from Amersham.
1.00 0.90 0.80 0.70 0.60
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Cells The thymus from Balb/c mice, 4 - 6 weeks old, was removed, rinsed and washed with phosphatebuffered saline. Thymocytes were prepared manually by pressing each intact thymus through a sterile wire mesh. Peritoneal macrophages were isolated from C57BL mice 6 - 8 weeks after the i.p. injection of 5 ml of saline. The cells were suspended in RPMI1640 medium supplemented with 10% fetal calf serum (FCS). L929 cells were a gift from Dr J. L. Taylor of the Department of Microbiology, Medical College of Wisconsin.
Assay of TNF-alpha Macrophages (105/well) were incubated with 1 /ag/ml LPS and 5, 20, 80, 200, 400 and 800 gg/ml of cocaine, respectively in RPMI-1640 with 10% FCS for 4 h. The TNF-alpha activity was measured using actinomycin-D-treated L929 targets (Coligan et al., 1991). L929 cells (5.5 × 104/well) were grown in RPMI-1640 with 10% FCS to confluence before replacement of the medium with actinomycin D suspended in 100 ~1 of medium (final concentration 5 ng/mi) together with an equivalent volume of macrophage culture supernatant containing TNF which kills actinomycin-D-treated L929 cells dosedependently. After incubation for 20 h, the cell monolayers were stained with crystal violet (0.05% in 20% ethanol) for 10 min. One hundred tA 100% methanol was added into a dried microplate, and the remaining cells were enumerated by reading the absorbance at 6 1 0 n m on a Bio-Tek EL 310 microtiterplate reader.
Assay of IL-1 IL-1 was assayed by the murine thymocyte comitogenesis assay (Rosenwasser & Dinarello, 1981). Macrophages (105/well) from the C57BL mouse peritoneum were incubated with the same concentrations of cocaine as described above (see assay of TNF) for 3 h. Cells were washed twice with Hanks' balanced salt solution (HBSS), and the viability was determined. The cells were then cultured with LPS (20 ~g/ml) for 18 h. Thymocytes (106/well) from 4 - 6 - w e e k - o l d Balb/C mice were incubated with Con A (the final concentration is
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Fig. I. Suppression of TNF release by cocaine. Mouse peritoneal macrophages (105/well) were incubated with LPS (1 ~g/ml) and different concentrations of cocaine at 37°C, 5% COz for 4 h. One hundred ~1 supernatant and one hundred ~1 actinomycin D (final concentration 5 gg/ml) were added to the well where TNF-sensitive L929 cells were cultured. After 20 h incubation, the cells were stained with crystal violet and the O.D. value was read at 610 nm by a Bio-Tek microtiterplate reader. The higher O.D. represents lower TNF toxicity directed against the target cells and vice versa. **P<0.01; ***P<0.0001. Results refer to three separate experiments.
1 gg/ml) and 50 ~1 macrophage culture supernatant for 72 h at 37°C in 5% COz. [Methyl-3H]-thymidine (2 Ci/mmol, 0.5 uC/well) was added to each well 24 h before harvesting onto glass fiber strips by use of a cell harvester.
Assay of cAMP Cyclic adenosine monophosphate was assayed by radioimmunoassay (Yamamoto & Suzuki, 1987). Macrophages (5 × 105/ml) were cultured with 3/Jg/ml LPS and 10 5M arachidonic acid in RPMI-1640 with 10%0 FCS for 3 h. At the end of the incubation period, the culture medium was removed and replaced with 0.25 ml HBSS. The cells were then disrupted by f r e e z e - t h a w i n g (three times) after the addition of trichloroacetic acid (TCA) to a final concentration of 5%. The f r o z e n - t h a w e d sample was centrifuged at 3000 revs/min at 4°C for 20 rain. The supernatant was acidified by the addition of 2 N HCL (15 gl). Excess TCA was extracted 10 times from the supernatant with 1 ml each of ethyl ether. Ethyl ether was then removed by heating for 45 min at 80°C. Fifty ~1 supernatant was placed into 1.5 ml centrifuge tubes and then succinylated by 10 gl succinic anhydride with triethylamide, and the measurement of cAMP quantity was carried by a radioimmunoassay. The radioactivity was deter° mined in a scintillation counter.
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Fig. 2. Suppression of IL-1 release by cocaine. Mouse peritoneal macrophages (105/well) were cultured with cocaine for 3 h. The cocaine-treated cells were washed twice and reincubated with LPS (20/ag/ml) for another 18 h. Fifty /al supernatant and Con A (final concentration 1 ~g/ml) were added into the wells where thymocytes (106/well) were incubated. [Methyl-3H]-thymidine (2 Ci/mmol, 0.5 uCi/well) was added and the amount of [methyl-3H]-thymidine incorporation was observed on a scintillation counter. */='<0.05; **P<0.01; ***P<0.0001. Results refer to three separate experiments.
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Fig. 3. Stimulation of cAMP synthesis by cocaine. Mouse peritoneal macrophages (105/well) were cultured with 3 t~g/ml LPS and 10 5M arachidonic acid for 3 h. The cells were lysed and radioimmunoassay was performed for quantitating cAMP amount (Experimental Procedures). **P<0.01; ***P<0.0001. Results refer to three separate experiments.
297
cocaine for 4 h, and the supernatant was removed and placed into wells containing actinomycin D (final concentration 5/ag/ml). Figure 1 shows that TNF secretion by C57BL mouse peritoneal macrophages was significantly increased by the stimulation of 1 /ag/ml LPS, as compared to the blank which contains only medium without macrophages. Because L929 cells were stained by crystal violet, lower O.D. values indicate that more L929 cells were killed by macrophage-secreted TNF, therefore, representing more TNF in macrophage-mediated medium. The secretion of TNF by macrophages is decreased to 93070 of the control level in the presence of 80 ~g/ml; 81070 in the presence of 200 ~g/ml; 64070 in the presence of 400 tag/ml; 18070 in the presence of 800/ag/ml. The inhibition of TNF released by macrophages occurs only at higher concentrations (above 200/ag/ml), and at 800 ~g/ml TNF secretion by macrophages was almost totally inhibited by cocaine.
Inhibition of IL-1 release by cocaine (Fig. 2) Macrophages were preincubated with cocaine for 3 h and then with 20 pg/ml LPS for 20 h. Cocaine does not significantly inhibit IL-1 secretion by macrophages below 80/ag/ml of dose concentration. However, it is evident that cocaine has obvious effects on secretion of macrophages above 200 tag/ml of dosage. In the presence of 200, 400 and 800 tag/ml the counts/min is decreased to 72, 70 and 55°7o of the control level, respectively, which means that the secretion of IL-1 by peritoneal macrophages is dose-dependently inhibited by cocaine. We also cocultured macrophages with LPS and cocaine for 4 h, and the results were similar to the preincubation of cocaine (data not shown). To find out whether cocaine is able to inhibit PMA-stimulated IL-1 secretion, we utilized cocaine preincubation and then PMA stimulation instead of LPS. We found that the data were similar to those from LPS stimulated secretion (data not shown).
Statistics
Stimulation of cAMP synthesis (Fig. 3)
Statistical significance of the data was determined by Student's t-test using BMDP program. A P value of less than 0.05 was considered to be significant.
The above results left open the question of whether cocaine directly inhibits TNF and IL-1 secretion or indirectly inhibits cytokine secretion through mediator dependent pathways, cAMP as a mediator for cytokine release was investigated (Hart et al., 1989). We applied the radioimmunoassay to analyze the effect of cocaine on cAMP synthesis to determine whether the intracellular cAMP level was affected. The results show that cocaine obviously stimulates adenylate cyclase activity (Fig. 3), which
RESULTS
Inhibition of TNF release by cocaine (Fig. 1) Macrophages were treated with LPS acting as a cytokine inducer and different concentrations of
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is increased to 198,207,235 and 245% of the control level in the presence of 80, 200, 400 and 800 ~g/ml of cocaine, respectively. This suggests that cAMP might play some role in mediating cytokine release when cocaine is present. However, to answer the question definitively, further investigation is required.
DISCUSSION
Our experiments reported here reveal that cocaine inhibits TNF release by macrophages. At higher concentrations (800 ~g/ml), TNF release was almost totally inhibited (18% of the control level). Suppression of TNF secretion in serum by intraperitoneal injections (i.p.) of cocaine, using the TNF ELISA test, was observed ( C h e n & Watson, 1991). However, by intraperitoneal treatment with increasing cocaine doses for four weeks, the mouse splenocytes were stimulated to increase the secretion of gamma-IFN and TNF-alpha (Lopez et al., 1991). Therefore, the effects of cocaine on the TNF-alpha function are quite complicated, depending on the individual and the experimental situation. Like TNF-alpha, IL-1 is an endogenous pyrogen. It is considered to be important in the development and maintenance of inflammation (Dinarello, 1989; Akira, Ilirano, Taga & Kishimoto, 1990) and tumor growth arrestment (Rangnekar, Waheed & Rangnekar, 1992; Ruggiero & Baglioni, 1987). The present observations indicate that the level of IL-1 released by mouse peritoneal macrophages decreased after the macrophages were treated with high doses of cocaine (above 200 t~g/ml), which are compatible with results that in vitro, high concentration of cocaine is required to suppress concanavalin A (Con A) or phytohemagglutinin (PHA) stimulated T-lymphocyte proliferation (Klein et al., 1988) and hydrogen peroxide, superoxide, and nitrite secretion by mouse peritoneal macrophage stimulated with
PMA (Shen, Sha & Ou, in preparation). A reasonable explanation for the evidence that the drug level is much lower in the blood of cocaine addicts than those of which we used in vitro is that in vivo the effects of cocaine on the immune system are influenced by some other factors. Cocaine is regarded to be able to affect endogenous neuropeptide and neurotransmitter release by blocking or inhibiting the reuptake of them (Gold, Washton & Dackis, 1985; Cunningham & Callahan, 1991; Tyrala, Mathews & Rao, 1992) and induce beta-endorphin (beta-END), adrenocorticotropic hormone (ACTH) and corticosterone secretion (Sweep, Wiegant, De-Vry & Van-Ree, 1989; Torres & Rivier, 1992), which have been shown to play an important regulatory role in the immune responses (Besedovsky, Rey, Sorkin, Prada & Keller, 1979; Blalock, 1984; Payan, Levine & Geotzl, 1984), indicating that in vivo the effects of cocaine on the immune system may be influenced by CNS. The general line of thinking about the effects of cocaine on CNS has led some investigators to speculate that an induction of beta-endorphin in plasma after intraperitoneal administration of cocaine causes PGE2 release by monocytes, which could mediate the suppression of TNF-alpha and IL-1 (Watzl & Watson, 1990). Our results were partly in agreement with this speculation. Using radioimmunoassay we have demonstrated that intracellular levels of cyclic adenosine 3',5'-monophosphate were dose-dependently enhanced by cocaine-stimulation when LPS (3 t~g/ml) and arachidonic acid (10 _5 M) were present. In the immune system, prostaglandins E are mediated by their ability to increase intracellular cAMP, which has been widely attributed to inhibit macrophage functions (Haynes, Whitehouse & Roberts, 1992; Brandwein, 1986; Schultz, Pavlidis, Stoychkov & Chirigos, 1979). However, further investigations are required to know whether cocaine directly inhibits cytokines release or indirectly acts on it through stimulating prostaglandin E which induces cAMP synthesis.
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Isolation of biologically active ribonucteic acid from sources enriched in ribonuclease. Biochemistry, 18, 5294-5299. COLIGAN, J. E., KRUISBEEK,A. M., MARGULIES, D. H., SHEVACH, E. M. & STROBER, W. (1991). Current Protocols in Immunology, 6.10.1 - 6.10.5. John Wiley, New York. CUNNINGHAM,K. A. & CALLAHAN,P. M. (1991). Monoamine reuptake inhibitors enhance the discriminative state induced by cocaine in the rat. Psychopharmac. Berl., 104, 177- 180. DELUSTRO, B. M., MULKINS,M. A. & ALLISON,A. C. (1987). Monoclonal antibodies to human recombinant interleukin 1 (IL-l)-beta: quantitation of IL-l-beta and inhibition of biological activity. J. Immun., 138, 4236-4242. D1-FRANCESCO, P., PICA, F., CROCE, C., FAVALLI, C., TUBARO, E. & GARACI, E. (1990). Effect of acute or daily cocaine administration on cellular immune response and virus infection in mice. Natn. Immun. Cell Growth Regul., 9, 397 - 405. DXNARELLO, C. A. (1989). Interleukin-1 and its biologically related cytokines. Adv. Immun., 44, 153- 205. DYKE, C. V., STESIN, A., JONES, R., CHUNTHARAPAI,A. & SEAMAN,W. (1986). Cocaine increases natural killer cell activity. J. clin. Invest., 77, 1387- 1390. GOLD, M. S., WASHTON, A. M. & DACKIS, C. A. (1985). Cocaine abuse: neurochemistry, phenomenology, and treatment. NIDA Res. Monogr., 61, 130-150. HAMILTON, J. A. & SLYWKA, J. (1981).Stimulation of human synovial fibr0blast plasminogen activator production by mononuclear cell supernatants. J. Immun., 126, 851- 855. HART, P. H., WHITTY, G. A., PICCOLI, D. C. & HAMILTON, J. A. (1989). Control by IFN-gamma and PGE2 and TNF-alpha and IL-I production by human monocytes. Immunology, 66, 376-383. HAYNES, D. R., WHITEHOUSE, M. W. & ROBERTS, B. V. (1992). The prostaglandin El" analogue misoprostol, regulates inflammatory cytokines and immune function in vitro like the natural prostaglandins El, E2 and E3. Immunology, 76, 251-257. KLEIN, T. W., NEWTON, C. A. & FRIEDMAN, H. (1988). Suppression of human and mouse lymphocyte proliferation by cocaine. Adv. Biochem. Psychopharmac., 44, 139- 143. LACHMAN, L. B., DINARELLO, C. A., LIANSA, N. D. & FIDLER, I. J. (1986). Natural and recombinant human interleukinbeta is cytotoxic for human melanoma cells. J. Immun., 136, 3098-3102. LOPEZ, M. C., HUANG, D. S., WATZL, B., CHEN, G. J. & WATSON, B. R. (1991). Splenocytes subsets in normal and protein malnourished mice after long-term exposure to cocaine or morphine. Life Sci., 49, 1253 - 1262. Lu, F. & Ou, D. W. (1989). Cocaine or delta-9-tetrahydrocannabinol does not affect cellular cytotoxicity in vitro. Int. J. Immunopharmac., 11, 849- 852. LUO, Y. D., PATEL,M. K., WIEDERHOLD,M. D. & Ou, D. W. (1992). Effects of cannabinoids and cocaine on the mitogeninduced transformations of lymphocytes of human and mouse origins. Int. J. Immunopharmac., 14, 49-56. MANNEL, D. N., MOORE, R. N. & MERGENHAGEN, S. E. (1980). Macrophages as a source of tumoricidal activity (tumor nectrotizing factor). Infect. Immun., 30, 523- 530. ONAZAKI, K., MATSUSHIMA,K., AGGARWAL,B. B. & OPPENHEIM, J. J. (1985). Human interleukin-1 is a cytocidal factor for several tumor cell lines. J. Immun., 135, 3962-3968. OU, D. W., SHEN, M. L. & LUO, Y. D. (1989). Effects of cocaine on the immune system of Balb/c mice. Clin. Immun. Immunopath., 52, 305 - 312. PAVAN, D. G., LEVINE, J. D. & GEOTZL, E. J. (1984). Modulation of immunity and hypersensitivity by sensory neuropeptides. J. Immun., 132, 1601 - 1604. RANGNEKAR,V. V., WAHEED, S. & RA*IGNEKAR,V. M. (1992). Interleukin-l-inducible tumor growth arrest is characterized by activation of cell type-specific "early" gene expression programs. J. biol. Chem., 267, 6740-6748. RENZ, H., GONG, J. H., SCHMIDT, A., NAIN, M. & GEMSA, D. (1988). Release of tumor necrosis factor-alpha from macrophages enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides. J. Immun., 141, 2388-2393. ROSENWASSER,L. J. & DINARELLO,C. A. (1981). Ability of human leukocytic pyrogen to enhance phytohemagglutinininduced murine thymocyte proliferation. Cell. Immun., 63, 134- 142. RUGGIERO, V. & BAGLIONI, C. (1987). Synergistic anti-proliferative activity of interleukin-1 and tumor necrosis factor. J. Immun., 138, 661-663.
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SCHULTZ, R. M., PAVLIDIS, N. A., STOYCHKOV,J. N. & CH1RIGOS, M. A. (1979). Prevention of macrophage tumoricidal activity by agents known to increase cellular cyclic AMP. Cell. Irnmun., 42, 71 - 7 8 . SWEEP, C. G., WIEGANT, V. M., DE-VRv & VAN-REE, J. M. (1989). Beta-endorphin in brain limbic structures as neurochemical correlate of psychic dependence on drugs. Life Sci., 44, 1133- 1140. TORRES, G. & RIVlER, C. (1992). Cocaine-induced stimulation of the rat hypothalamic-pituitary-adrenal axis is progressively attenuated following hourly-interval regimens of the drug. Life Sci., 51, 1041 - 1048. TYRALA, E. E., MATHEWS,S. V. & RAO, G. S. (1992). Effect of intrauterine exposure to cocaine on acetylcholinesterase in primary cultures of fetal mouse brain cells. Neurotoxic. Terat., 14, 229-233. WATZL, B. & WATSON, R. R. (1990). Immunomodulation by cocaine - - a neuroendocrine mediated response. Life Sci., 46, 1319- 1329. YAMAMOTO, H. & SUZUKI, T. (1987). Prostaglandin E2-induced activation of adenosine 3'-5' cyclic monophosphatedependent protein kinases of a murine macrophage-like cell line (p388Dl). J. Immun., 139, 3416-3421.
Pergamon 0192
- 0561(93)E0020-A
INHIBITION OF CYTOKINE RELEASE BY COCAINE HONG-MING SHEN, JOHN L. KENNEDY and DAVID W. OU* Department of Pathology, College of Medicine, The University of Illinois at Chicago and VA West Side Medical Center, Chicago, IL 60612, U.S.A. (Received 7 September 1993 and in final form 1 December 1993)
- - The influence of cocaine on cytokine release by mouse peritoneal macrophages has been investigated in vitro. LPS-stimulated TNF and IL-1 secretion were suppressed by cocaine in higher dosages. This suppression was shown to be dose-related. The synthesis of cAMP, however, was stimulated by cocaine. Though cAMP is generally considered a mediator for cytokine release, further studies are required to demonstrate whether cocaine directly affects cytokine release by macrophages or indirectly influences cytokine release through stimulating cAMP synthesis. Abstract
Besides the previously described cocaine effects on the central nerve system (CNS), the influences of cocaine on the immune system in vivo and in vitro have been identified. These include: (1) lymphocyte proliferation (Luo, Patel, Wiederhold & Ou, 1992; Klein, Newton & Friedman, 1988); (2) natural killer (NK) cell activity (Di-Francesco, Pica, Croce, Favalli, Tubaro & Garaci, 1990; Dyke, Stesin, Jones, Chuntharapai & ~eaman, 1986; Lu & Ou, 1989); (3) lymphocyte subset alteration (Lopez, Huang, Watzl, C h e n & Watson, 1991; Bagasra & Forman, 1989); (4)phagocyte activity (Ou, Shen & Luo, 1989); (5)antibody formation (Ou, Shen & Luo, 1989); etc. The actions of cocaine on cytokine and prostaglandin E (PGE) release were reported recently. The results from the analysis of drug users' hair samples show that cocaine increases the deposition of interleukin-2 (IL-2) in the hair, but not interleukin-1 (IL-1) and gamma-interferon (gammaIFN) (Chen, Pillai, Erickson, Martinez, Estrada & Watson, 1991). Furthermore, PGEs are significantly increased in drug users' amniotic fluid (Ahluwalia, Clark, Wetney, Smith, James & Rajguru, 1992). It is well known that the tumor necrosis factor (TNF) and interleukin-1 (IL-I) as cytokines are endogenous pyrogens and can activate not only monocytes/macrophages, but also lymphocytes, endothelial cells, neutrophils, chondrocytes, fibroblasts, etc. (Delustro, Mulkins & Allison, 1987;
Hamilton & Slywka, 1981; Chirgwin, Przybyla, MacDonald & Rutter, 1979). These two cytokines share a wide spectrum of biological activities, but the regulation of their production shows some dissimilarities (Lachman, Dinarello, Liansa & Fidler, 1986; Mannel, Moore & Mergenhagen, 1980; Onozaki, Matsushima, Aggarwal & Oppenheim, 1985). However, TNF and IL-1 production are both inhibited by increasing intracellular cyclic adenosine monophosphate (cAMP) levels (Renz, Gong, Schmidt, Nain & Gemsa, 1988; Hart, Whitty, Piccoli & Hamilton, 1989). In this study we further characterized the influences of cocaine on cytokine and cAMP release by mouse peritoneal macrophages in vitro, using several well-defined methods, described elsewhere (Coligan, Kruisbeek, Margulies, Shevach & Strober, 1991; Rosenwasser & Dinarello, 1981; Yamamoto & Suzuki, 1987). The release of IL-1 and TNF in vitro was inhibited by higher concentrations of cocaine. However, the activity of adenylate cyclase in the macrophages was stimulated by cocaine in a dosedependent fashion. , EXPERIMENTAL
PROCEDURES
Reagents
Cocaine, lipopolysaccharide, concanavalin A, actinomycin D, arachidonic acid, 3 ' - 5 ' cyclic
*Author to whom correspondence should be addressed at: Department of Pathology, College of Medicine, 1853 West Polk Street, Chicago, IL 60612, U.S.A. 295
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adenosine monophosphate, anti-cAMP serum were obtained from SIGMA. 3 ' - 5 ' cyclic adenosine monophosphate [125-I] tracer was from Dupont. [Methyl-~H] thymidine (2.0 curies/mmol) was from Amersham.
1.00 0.90 0.80 0.70 0.60
c5 ©
0.50 0.40
Cells The thymus from Balb/c mice, 4 - 6 weeks old, was removed, rinsed and washed with phosphatebuffered saline. Thymocytes were prepared manually by pressing each intact thymus through a sterile wire mesh. Peritoneal macrophages were isolated from C57BL mice 6 - 8 weeks after the i.p. injection of 5 ml of saline. The cells were suspended in RPMI1640 medium supplemented with 10% fetal calf serum (FCS). L929 cells were a gift from Dr J. L. Taylor of the Department of Microbiology, Medical College of Wisconsin.
Assay of TNF-alpha Macrophages (105/well) were incubated with 1 /ag/ml LPS and 5, 20, 80, 200, 400 and 800 gg/ml of cocaine, respectively in RPMI-1640 with 10% FCS for 4 h. The TNF-alpha activity was measured using actinomycin-D-treated L929 targets (Coligan et al., 1991). L929 cells (5.5 × 104/well) were grown in RPMI-1640 with 10% FCS to confluence before replacement of the medium with actinomycin D suspended in 100 ~1 of medium (final concentration 5 ng/mi) together with an equivalent volume of macrophage culture supernatant containing TNF which kills actinomycin-D-treated L929 cells dosedependently. After incubation for 20 h, the cell monolayers were stained with crystal violet (0.05% in 20% ethanol) for 10 min. One hundred tA 100% methanol was added into a dried microplate, and the remaining cells were enumerated by reading the absorbance at 6 1 0 n m on a Bio-Tek EL 310 microtiterplate reader.
Assay of IL-1 IL-1 was assayed by the murine thymocyte comitogenesis assay (Rosenwasser & Dinarello, 1981). Macrophages (105/well) from the C57BL mouse peritoneum were incubated with the same concentrations of cocaine as described above (see assay of TNF) for 3 h. Cells were washed twice with Hanks' balanced salt solution (HBSS), and the viability was determined. The cells were then cultured with LPS (20 ~g/ml) for 18 h. Thymocytes (106/well) from 4 - 6 - w e e k - o l d Balb/C mice were incubated with Con A (the final concentration is
0.30 0.20 0.10 0.00 blank
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Fig. I. Suppression of TNF release by cocaine. Mouse peritoneal macrophages (105/well) were incubated with LPS (1 ~g/ml) and different concentrations of cocaine at 37°C, 5% COz for 4 h. One hundred ~1 supernatant and one hundred ~1 actinomycin D (final concentration 5 gg/ml) were added to the well where TNF-sensitive L929 cells were cultured. After 20 h incubation, the cells were stained with crystal violet and the O.D. value was read at 610 nm by a Bio-Tek microtiterplate reader. The higher O.D. represents lower TNF toxicity directed against the target cells and vice versa. **P<0.01; ***P<0.0001. Results refer to three separate experiments.
1 gg/ml) and 50 ~1 macrophage culture supernatant for 72 h at 37°C in 5% COz. [Methyl-3H]-thymidine (2 Ci/mmol, 0.5 uC/well) was added to each well 24 h before harvesting onto glass fiber strips by use of a cell harvester.
Assay of cAMP Cyclic adenosine monophosphate was assayed by radioimmunoassay (Yamamoto & Suzuki, 1987). Macrophages (5 × 105/ml) were cultured with 3/Jg/ml LPS and 10 5M arachidonic acid in RPMI-1640 with 10%0 FCS for 3 h. At the end of the incubation period, the culture medium was removed and replaced with 0.25 ml HBSS. The cells were then disrupted by f r e e z e - t h a w i n g (three times) after the addition of trichloroacetic acid (TCA) to a final concentration of 5%. The f r o z e n - t h a w e d sample was centrifuged at 3000 revs/min at 4°C for 20 rain. The supernatant was acidified by the addition of 2 N HCL (15 gl). Excess TCA was extracted 10 times from the supernatant with 1 ml each of ethyl ether. Ethyl ether was then removed by heating for 45 min at 80°C. Fifty ~1 supernatant was placed into 1.5 ml centrifuge tubes and then succinylated by 10 gl succinic anhydride with triethylamide, and the measurement of cAMP quantity was carried by a radioimmunoassay. The radioactivity was deter° mined in a scintillation counter.
Inhibition of Cytokine Release by Cocaine 2500 2000 1500 1000 500 0 0
5
20
80
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Cocaine (ug/ml)
Fig. 2. Suppression of IL-1 release by cocaine. Mouse peritoneal macrophages (105/well) were cultured with cocaine for 3 h. The cocaine-treated cells were washed twice and reincubated with LPS (20/ag/ml) for another 18 h. Fifty /al supernatant and Con A (final concentration 1 ~g/ml) were added into the wells where thymocytes (106/well) were incubated. [Methyl-3H]-thymidine (2 Ci/mmol, 0.5 uCi/well) was added and the amount of [methyl-3H]-thymidine incorporation was observed on a scintillation counter. */='<0.05; **P<0.01; ***P<0.0001. Results refer to three separate experiments.
18 ¸ "~
16
1o
,~ <
8 6 4
2 0 0
5
20
80
200
400
800
Cocaine (ug/ml)
Fig. 3. Stimulation of cAMP synthesis by cocaine. Mouse peritoneal macrophages (105/well) were cultured with 3 t~g/ml LPS and 10 5M arachidonic acid for 3 h. The cells were lysed and radioimmunoassay was performed for quantitating cAMP amount (Experimental Procedures). **P<0.01; ***P<0.0001. Results refer to three separate experiments.
297
cocaine for 4 h, and the supernatant was removed and placed into wells containing actinomycin D (final concentration 5/ag/ml). Figure 1 shows that TNF secretion by C57BL mouse peritoneal macrophages was significantly increased by the stimulation of 1 /ag/ml LPS, as compared to the blank which contains only medium without macrophages. Because L929 cells were stained by crystal violet, lower O.D. values indicate that more L929 cells were killed by macrophage-secreted TNF, therefore, representing more TNF in macrophage-mediated medium. The secretion of TNF by macrophages is decreased to 93070 of the control level in the presence of 80 ~g/ml; 81070 in the presence of 200 ~g/ml; 64070 in the presence of 400 tag/ml; 18070 in the presence of 800/ag/ml. The inhibition of TNF released by macrophages occurs only at higher concentrations (above 200/ag/ml), and at 800 ~g/ml TNF secretion by macrophages was almost totally inhibited by cocaine.
Inhibition of IL-1 release by cocaine (Fig. 2) Macrophages were preincubated with cocaine for 3 h and then with 20 pg/ml LPS for 20 h. Cocaine does not significantly inhibit IL-1 secretion by macrophages below 80/ag/ml of dose concentration. However, it is evident that cocaine has obvious effects on secretion of macrophages above 200 tag/ml of dosage. In the presence of 200, 400 and 800 tag/ml the counts/min is decreased to 72, 70 and 55°7o of the control level, respectively, which means that the secretion of IL-1 by peritoneal macrophages is dose-dependently inhibited by cocaine. We also cocultured macrophages with LPS and cocaine for 4 h, and the results were similar to the preincubation of cocaine (data not shown). To find out whether cocaine is able to inhibit PMA-stimulated IL-1 secretion, we utilized cocaine preincubation and then PMA stimulation instead of LPS. We found that the data were similar to those from LPS stimulated secretion (data not shown).
Statistics
Stimulation of cAMP synthesis (Fig. 3)
Statistical significance of the data was determined by Student's t-test using BMDP program. A P value of less than 0.05 was considered to be significant.
The above results left open the question of whether cocaine directly inhibits TNF and IL-1 secretion or indirectly inhibits cytokine secretion through mediator dependent pathways, cAMP as a mediator for cytokine release was investigated (Hart et al., 1989). We applied the radioimmunoassay to analyze the effect of cocaine on cAMP synthesis to determine whether the intracellular cAMP level was affected. The results show that cocaine obviously stimulates adenylate cyclase activity (Fig. 3), which
RESULTS
Inhibition of TNF release by cocaine (Fig. 1) Macrophages were treated with LPS acting as a cytokine inducer and different concentrations of
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is increased to 198,207,235 and 245% of the control level in the presence of 80, 200, 400 and 800 ~g/ml of cocaine, respectively. This suggests that cAMP might play some role in mediating cytokine release when cocaine is present. However, to answer the question definitively, further investigation is required.
DISCUSSION
Our experiments reported here reveal that cocaine inhibits TNF release by macrophages. At higher concentrations (800 ~g/ml), TNF release was almost totally inhibited (18% of the control level). Suppression of TNF secretion in serum by intraperitoneal injections (i.p.) of cocaine, using the TNF ELISA test, was observed ( C h e n & Watson, 1991). However, by intraperitoneal treatment with increasing cocaine doses for four weeks, the mouse splenocytes were stimulated to increase the secretion of gamma-IFN and TNF-alpha (Lopez et al., 1991). Therefore, the effects of cocaine on the TNF-alpha function are quite complicated, depending on the individual and the experimental situation. Like TNF-alpha, IL-1 is an endogenous pyrogen. It is considered to be important in the development and maintenance of inflammation (Dinarello, 1989; Akira, Ilirano, Taga & Kishimoto, 1990) and tumor growth arrestment (Rangnekar, Waheed & Rangnekar, 1992; Ruggiero & Baglioni, 1987). The present observations indicate that the level of IL-1 released by mouse peritoneal macrophages decreased after the macrophages were treated with high doses of cocaine (above 200 t~g/ml), which are compatible with results that in vitro, high concentration of cocaine is required to suppress concanavalin A (Con A) or phytohemagglutinin (PHA) stimulated T-lymphocyte proliferation (Klein et al., 1988) and hydrogen peroxide, superoxide, and nitrite secretion by mouse peritoneal macrophage stimulated with
PMA (Shen, Sha & Ou, in preparation). A reasonable explanation for the evidence that the drug level is much lower in the blood of cocaine addicts than those of which we used in vitro is that in vivo the effects of cocaine on the immune system are influenced by some other factors. Cocaine is regarded to be able to affect endogenous neuropeptide and neurotransmitter release by blocking or inhibiting the reuptake of them (Gold, Washton & Dackis, 1985; Cunningham & Callahan, 1991; Tyrala, Mathews & Rao, 1992) and induce beta-endorphin (beta-END), adrenocorticotropic hormone (ACTH) and corticosterone secretion (Sweep, Wiegant, De-Vry & Van-Ree, 1989; Torres & Rivier, 1992), which have been shown to play an important regulatory role in the immune responses (Besedovsky, Rey, Sorkin, Prada & Keller, 1979; Blalock, 1984; Payan, Levine & Geotzl, 1984), indicating that in vivo the effects of cocaine on the immune system may be influenced by CNS. The general line of thinking about the effects of cocaine on CNS has led some investigators to speculate that an induction of beta-endorphin in plasma after intraperitoneal administration of cocaine causes PGE2 release by monocytes, which could mediate the suppression of TNF-alpha and IL-1 (Watzl & Watson, 1990). Our results were partly in agreement with this speculation. Using radioimmunoassay we have demonstrated that intracellular levels of cyclic adenosine 3',5'-monophosphate were dose-dependently enhanced by cocaine-stimulation when LPS (3 t~g/ml) and arachidonic acid (10 _5 M) were present. In the immune system, prostaglandins E are mediated by their ability to increase intracellular cAMP, which has been widely attributed to inhibit macrophage functions (Haynes, Whitehouse & Roberts, 1992; Brandwein, 1986; Schultz, Pavlidis, Stoychkov & Chirigos, 1979). However, further investigations are required to know whether cocaine directly inhibits cytokines release or indirectly acts on it through stimulating prostaglandin E which induces cAMP synthesis.
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