Toxicology Letters, 59 (1991) 147-152 Q 1991 Elsevier Science Publishers B.V. 0378-4274/91/$ 3.50
Stimulation of natural killer cell activity by murine retroviral infection and cocaine
Torka S. Poet”*, Radhakrishna and Ronald Ross Watson* *Department of Pharmacology University of Arizona,
Pillai*T*, Steven Wood*
and Toxicology and ‘Department
of Family and Community Medicine,
Tucson, AZ (U.S.A.)
(Received 17 April 1991) (Accepted 3 September 1991) Key words: Cocaine; Murine AIDS; LP-BM5 murine leukemia virus; Immunomodulation; cell
SUMMARY The effects ofcocaine and murine AIDS on natural killer (NK) cell activity in C57BL/6 mice was studied. Cocaine may play a major role in the development and progression to AIDS in the human population. Chronic intraperitoneal injection of cocaine was shown to cause an increase in NK cell activity over those of saline-treated animals. Infection with LP-BM5 murine leukemia retrovirus was also shown to increase NK cell activity. NK cell activity was increased in retrovirally infected mice treated with cocaine beyond that of mice treated with cocaine alone. This study indicates an important immunomodulatory effect of cocaine on NK cell activity, especially when combined with the effects caused by retroviral infection.
The use of cocaine in the United States has reached epidemic proportions. The often-quoted 1985 National Institute on Drug Abuse (NIDA) survey reported that 22 million people in the United States had used cocaine. Cocaine is generally thought to be immunosuppressive [l], and thus may enhance a person’s susceptibility to retroviral infection. A recent study found a correlation between intravenous cocaine use and human immunodeficiency virus (HIV) seropositivity . A variety of co-factors *Current address: Regional Cancer Center, Thiruvangently Apuram, India. correspondence: Ronald R. Watson, Department of Family and Community Medicine, Arizona Health Sciences Center, Tucson, AZ 85724, U.S.A.
may be involved in the etiology and pathogenesis of the acquired immunodeficiency syndrome (AIDS), including drugs of abuse [3,4]. The hypothesis that cocaine may be a co-factor in the development of AIDS has been strengthened by a report that cocaine enhances HIV replication in mononuclear cell co-cultures . Natural killer (NK) cells are non-major histocompatibility-dependent cytotoxic cells. They play a major role in the killing of tumor and virally-infected cells. They also have powerful immunoregulatory roles, including effects on bone marrow maturation and regulation of antibody production [6,7]. NK cells produce interferon , which, in turn, can augment NK cell activity, and sensitize tissue cells to lysis by NK and T-cells [8,9]. Retroviruses have been shown to decrease interferon production in vitro [lo]. To study the pathogenesis of AIDS a range of animal models has been suggested [ 111. The LP-MB5 murine leukemia virus causes an immunosuppression syndrome in mice with characteristics similar to the changes caused by the HIV infection in humans, such as an early phase of B-cell hyperactivity and polyclonal activation, loss of T-cell function occurring before reduction in helper T-cell functions and reductions in NK cell functions [ 1l-141. The condition produced through the actions of this virus has been termed ‘murine AIDS’ or ‘MAIDS’. For immunological studies the MAIDS model has many advantages, such as an accurate reflection of the early stages of AIDS, availability of genetically identical inbred animals, defined immunological parameters and the ability to reproduce disease in large numbers of animals within a relatively short period of time 112,131. Therefore we employed this model to investigate the effects of retroviral infection and chronic cocaine injection on NK cell function. MATERIALS AND METHODS
The LP-BM5 murine leukemia virus, a combination of ecotropic, recombinant and defective murine leukemia viruses, is a non-human pathogen and is not transmitted naturally to uninfected mice co-housed in the same room . Animals used for studies were C57BL/6 female mice and were infected with 0.1 ml of the virus inoculum intraperitoneally as described previously . The mice were infected 1 month prior to beginning the injections of cocaine. Cocaine administration
Infected and non-infected mice were injected intraperitoneally with 20 mg/kg of cocaine hydrochloride (kindly provided by the National Institute of Drug Abuse) in 0.1 ml saline or with 0.1 ml saline daily for 5 weeks. Mice were terminated under ether, blood samples collected from an axillary vein and spleens removed aseptically. Single cell suspensions were prepared in RPM1 medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics (complete medium). Cells were centrifuged at 180 x g for 10 min and red blood cells were lysed in 0.144 M NH&l lysing buffer for 2 min and washed with complete medium. Cells were then processed for
NK cell function as described earlier . Serum samples were stored at -70°C until analysis for cocaine levels. Natural killer cell activity NK cell function was measured using a fluorescence concentration and release assay modified from the method of Wierda et al. . Unlike the “Cr release assay, measurement of specific lysis in this assay is based on the direct measurement of dye retained by the remaining viable target cells after the assay using the Pandex Fluorescence Concentrator and Analyzer (FCA) (Baxter Heathcare, Mudelein, IL). The carboxyfluorescein derivative 2’,7’-bis(carboxyethyl)-5’,6’-carboxyfluorescein (BCECF) was used as the target cell label. The assay has been found to be superior to the 51Cr release assay and is easily reproducible  [Pillai et al., unpublished data]. The Moloney murine leukemia-virus induced T-cell lymphoma line, YAC-1, was used as target for the NK cells. Tumor target cells were serially passaged in complete RPM1 1640 medium and used in cytotoxicity assays within 48 h of the last passage. Viability was determined to be more than 98% by Trypan Blue dye exclusion. Target cells were labeled with BCECF (Molecular Probes, Eugene, OR) as explained earlier . The cells were then incubated at different effecter/target (E/T) cell concentrations (50:1,25:1, 12.51) in U-bottom microtiter plates (Falcon 3077, Becton & Dickinson, NJ) at 37°C in a humidified atmosphere of 5.0% carbon dioxide in air for 3 h. Spontaneous and maximum release was determined as described earlier 1171.After incubation, 100~~1aliquots from each well of the assay plate were transferred to wells in a Pandex harvesting plate (Baxter Healthcare Corp, Mudelein, IL). Automated washing and processing for fluorescence reading on the Pandex FCA have been described elsewhere . Epifluorescence of each well was read at 485/535 nm excitation/emission wavelengths for BCECF using the automated Pandex FCA. An internal standard was also run at 590/620 nm. Specific percent cytotoxicity was calculated as follows: Spontaneous release - Experimental fluorescence x loo Spontaneous release - Maximum release RESULTS
NK cell activity in infected and non-infected mice A uniform pattern of NK cell activity was seen for all 3 E/T ratios, therefore data for the 50: 1 ratio are presented. In the saline-injected, non-infected control mice, the NK cell activity did not significantly vary over time, the mean activity being 14.6 ? 1.4% cytotoxicity over the 16-h study. The retrovirally infected control mice showed an increased NK cell activity (23.9 + 2.1%) compared to the non-infected control mice, which also did not vary over time (Fig. 1). NK cell activity in the cocaine-treated animals increased dramatically over controls within 20 min of cocaine injection. In the non-infected mice, the NK cell activity was
Treatment &c+.ps Retrovirally
Fig. 1. Percent NK cell activity in female C57BLl6 mice infected with LP-BM5 retrovirus and injected with 20 m&g cocaineh~dr~h~~~de (o>~ or saline (m), and in non-infected mice injected with saline (i), or injected with 20 mgikg cocaine hydrochloride (~1.
40.4rt3.9%at20min post-injection, reaching a maximum value of 41.7 f 2.7% at 40 min post-injection. The retrovirally infected mice showed much higher levels of NK cell activity. Peak levels of activity were also reached faster: after 20 min the activity was 53.3 + 3.3% and after 40 min the values dropped slightly to 47.4 + 3.5%. Interestingly, after 1 h the NK cell activities for the retrovirally infected mice were closely comparable to the activity found for the non-infected mice after 20 min (Fig_ 1). The NK cell activity for both the infected and non-infected mice decreased back to control levels over the remainder of the 16-h time course. DISCUSSION
This study reveals interesting associations between cocaine, retroviral infection and NK cell activity. Increased NK cell activity was seen in the retrovirally infected control mice over the non-infected control mice. Other studies have reported similar findings in which NK cell activity peaks during the initial stages of viral infection and then begins to be suppressed as disease becomes established [13-20]. This has also been shown to be the case in HIV infection of humans f20]. NK cells are involved in multiple effector, regulatory and developmental activities of the immune system f5]. NK cells play an important role in the killing of virusinfected cells. However, increases in NK cell activity and numbers can also lead to decreased antibody formation and decreased B-cell activation . Compromised or absent NK cell activity has been linked to development and progression of cancer as well as chronic and acute viral infections including AIDS [8,20,22,23]. Both infected and non-infected mice showed an immediate elevation in NK cell activity after cocaine administration. Associations between cocaine and increased NK cell activity have been reported before. Van Dyke et al.  a~~n~stered cocaine
to normal subjects and observed an immediate increase in NK cell activity. We also observed a similar cocaine-associated increase in NK cell activity in polydrug users specifically screened for cocaine [Pillai et al., data submitted for publication]. The higher NK cell activity may be due to increased serum y-interferon caused by the LP-BM5 retrovirus infection . Tumor necrosis factor was also higher in the retrovirally infected mice, with further stimulation by cocaine injection . Thus, stimulation of cytokines which regulate NK and other cellular functions may help explain increased NK cell activities. The cocaine-modulated increase in NK cell function is suggested to be due to a neuroendocrine-system-mediated process [ 1,241. Cocaine administration results in multiple neurochemical actions including altered re-uptake of neurotransmitters such as serotonin, dopamine and norepinephrine [1,26,27]. Cocaine also stimulates the release of /I-endorphin [3,27,28]. /I-Endorphin and norepinephrine are potent stimulators of NK cell function [29,30]. The retrovirally infected mice also show an increase in serum cocaine concentrations, particularily at the intermediate time points [Poet et al., data submitted for publication]. While the reasons for this increase in serum cocaine levels are unknown, the higher cocaine concentrations may correlate with the increased NK cell activity. The suggestion that drugs of abuse such as cocaine may serve as co-factors in the development of AIDS makes the elucidation of the immunotoxicology of cocaine of vital importance . Abnormal stimulation of NK cell activity may have a role in the immunological abnormalities associated with HIV infection and AIDS in users of cocaine. Studies into the various aspects of such stimulation of NK cells, including changes in levels of /%endorphin, target-binding cells and recycling capacity of NK cells may provide more answers to this question. ACKNOWLEDGEMENT
Supported in part by grants AA08037 and DA04827. REFERENCES 1 Watzl, B. and Watson, R.R. (1990) Immunomodulation by cocaine: a neuroendocrine mediated approach. Life Sci. 46, 1319-1329. 2 Chaisson, R.E., Bacchetti, P., Osmond, D., Brodie, B., Sande, M.A. and Moss, A.R. (1989) Cocaine use and HIV infection in intravenous drug users in San Francisco. J. Am. Med. Assoc. 261, 561-565. 3 Watson, R.R. (1989) Cofactors in HIV 1 Infection and AIDS. CRC Press, Boca Raton, FL, pp. l-246. 4 Pillai, R. and Watson, R.R. (1990) In vitro immunopharmacology and immunotoxicology: studies on drugs of abuse. Toxicol. Lett. 53, 269-283. 5 Peterson, P.K., Gekker, G., Chao, C.C., Schut, R., Molitor, T.W. and Balfour, H.H. (1991) Cocaine potentiates HIV 1 replication in human peripheral blood mononuclear cell cdcultures. J. Immunol. 146, 81-84. 6 Herbermann, R.B. (1981) Immunoregulation by natural killer cells. Mol. Immunol. 19, 1313-1317. 7 Abruzzon, L.V. and Rowley, D.A. (1983) Homeostasis of the antibody response: immunoregulation by NK cells. Science 222, 581-582.
8 Young, H.A. and Ortaldo, J.R. (1987) One-signal requirement for interferon production by human large granular lymphocytes. J. Immunol. 139,724727. 9 Trinchieri, G. (1989) Biology of natural killer cells. Adv. Immunol. 47, 187-376. 10 Trinchieri, G., Granato, D. and Perussia, B. (1981) Interferon induced resistance of fibroblasts to cytolysis by NK cells: specificity and mechanism. J. Immunol. 126, 335-339. 11 Good, R.A., Ogasawara, W.T., Lorenz, E. and Day, N.K. (1990) Immunosuppressive actions of retroviruses. Lymphology 23, 5659. 12 Letvin, N.L. (1990) Animal models for AIDS. Immunol. Today 11, 322-326. 13 Watson, R.R. (1989) Murine models for acquired immune deficiency syndrome. Life Sci. 44, iii-xv. 14 Mosier, D.E., Yetter, R.A. and Morse III, H.C. (1985) Retroviral induction of acute lymphoproliferative disease and profound immunosuppression in adult C57BL/5 mice. J. Exp. Med. 161, 766. 15 Mufti, S.I. and Watson, R.R. (1990) Effect of chronic ethanol consumption and retroviral infection on carcinogenesis and its possible inhibition by an immunostimulant, canthaxanthin: definition of a murine model. In: D. Seminara, R.R. Watson and A. Pawlowski (Eds.), Alcohol Immunomodulation and AIDS, Alan R. Liss, Inc., New York, pp. 283-304. 16 Pillai, R., Balaram, P., Abraham, T., Padmanabhan, T.K. and Nair, M.K. (1988) Natural cytotoxicity and serum blocking in malignant cervical neoplasia. Am. J. Reprod. Immunol. Microbial. 16, 159-162. 17 Wierda, W.G., Mehr, D.S. and Kim, Y.B. (1989) Comparison of fluorochrome labelled and 51Cr labelled targets for natural cytoxicity assay. J. Immunol. Methods 122, 15-24. 18 Biron, C.A., Turgis, L.R. and Welsh, R.M. (1983) Increase in NK cell number and turn over during acute viral infection. J. Immunol. 131, 1539-l 541. 19 Stitz, L., Althage, A., Hengartner, H. and Zinkemagel, R. (1985) Natural killer cells vs cytotoxic T cells in the peripheral blood of virus infected mice. J. Immunol. 131, 1539-l 542. 20 Sirianni, M.C., Tagliaferri, F. and Aiuti, F. (1990) Pathogenesis of the natural killer cell deficiency in AIDS. Immunol. Today 11,81-82. 21 Coomes, T., Clonfet, G., Jourdan, M., Bataille, R. and Klein, B. (1990) Human natural killer cells suppress the proliferation of B cells. Immunol. Lett. 24, 57-61. 22 Robertson, M.J. and Ritz, J. (1990) Biology and clinical relevance of human natural killer cells. Blood 12, 2421-2438. 23 Aoki, T., Usuda, Y., Miyakoshi, H., Tamura, K. and Herbermann, R.B. (1987) Low natural killer syndrome: clinical and immunologic features. Nat. Immun. Cell Growth Regul. 6, 116128. 24 Van Dyke, C., Stesin, A., Jones, R., Chunthpri, A. and Seamen, W. (1986) Cocaine increases natural killer cell activity. J. Clin. Invest. 77, 1387-1490. 25 Chen, G.J. and Watson, R.R. (1991) Modulation of tumor necrosis factor and gamma interferon production by cocaine and morphine in aging mice infected with LP-BM5, a murine leukemia virus. J. Leukocyte Biol. 50, 349-355. 26 Mendelson, J.H., Mello, N.K., Teoh, S.K., Ellingboe, J. and Cochin, J.A. (1989) Cocaine affects pulstaile secretion of anterior pituitary, gonadal and adrenal hormones. J. Endocrinol. Methods 69, 1256-1260. 27 Moldow, R.L. and Fischmann, A.J. (1987) Cocaine induced secretion of ACTH, beta endorphin and corticosterone. Peptides 8, 819-823. 28 Levy, S.M., Femstrom, J., Herbermann, R.B., Whiteside, T., Lee, J., Ward, M. and Massoudi, M. (1991) Persistently low natural killer cell activity and circulating levels of plasma beta endorphin: risk factors for infectious disease. Life Sci. 48, 107-l 12. 29 Mathews, P.M., Froleich, C.J., Sibbit, W.L. and Bankhurst, A.D. (1983) Enhancement of natural cytotoxicity by beta endorphin. J. Immunol. 130, 1658-1662. 30 Kraus, L., Locke, S., Kutz, I., Edbril, S., Philips, K. and Benson, H. (1983) Altered natural killer cell activity during norepinephrine infusion in humans. Paper presented at: Annual Meeting of American Psychomatic Society, New York, 12.6.91. 31 Pillai, R. and Watson, R. (1991) AIDS: disease progression and immunomodulation by drugs of abuse and alcohol. AIDS Med. Rep. 4, 25-36.