Int. J. lmmunopharmac., Vol. 14, No. 7, pp. 1153-1163 , 1992. Printed in Great Britain.

0 1 9 2 - 0 5 6 1 / 9 2 $5.00 + .00 Pergamon Press Ltd,

",c) 1992 International Society for lmmunopharmacology.

MODIFICATION OF SPLEEN CELL SUBSETS BY CHRONIC COCAINE ADMINISTRATION AND MURINE RETROVIRUS INFECTION IN

NORMAL AND PROTEIN-MALNOURISHED MICE

MARIA C. LOPEZ,* GUAN-JIE CHEN, t DENNIS S. HUANG, YUEJIAN WANG and RONALD R. WATSON*

Department of Family and Community Medicine, Arizona Health Sciences Center, The University of Arizona, Tucson, AZ 85724, U.S.A.

(Received 23 January 1992 and in f inal form 11 May 1992)

Abstract - - We have developed an experimental mouse model to study the effect of daily cocaine administration on the immune system during an acquired immune deficiency syndrome (AIDS). Mice were infected with LP-BM5 murine leukemia virus, a retrovirus which causes immunosuppression with the development of functional murine AIDS. Increasing doses of cocaine given by daily intraperitoneal injection for 11 weeks reduced body weight. A daily cocaine injection in some mice as well as a saline injection in others showed a decrease in the percentage of Thy 1.2 +, CD4 + and CD8 ÷ cells, while both treatments increased the percentage and absolute numbers of B-cells per spleen. Saline and cocaine treatment induced an increase in y-IFN and TNF-a production by splenocytes. Cocaine treatment favored a decrease in slL-2R secretion. Saline and cocaine treatment had slightly different effects on the splenocytes of protein- malnourished mice. Cocaine treatment induced an increase in the percentage of CD8 ~ cells. Saline and cocaine treatments decreased the number of Mac 1 + cells in the spleen. Moreover, saline- and cocaine-treated protein-malnourished mice splenocytes did not present the increase in y-IFN production as well-nourished mice splenocytes showed. Retrovirus-infected mice showed a decrease in the percentage of Thy 1.2 ~ and CD8 ÷ cells and an increase in the percentage and absolute numbers of CD4 + , IL-2R ÷, Mac 1 - and B-cells. Cocaine partially prevented the enlargement of lymphoid organs due to lymphoid cell proliferation induced by murine retrovirus infection, but had little effect on the elevated percentage of CD4 + cells or B-cells or the depressed numbers of CD8 ÷ cells associated with virus infection. However, cocaine did reduce the number of activated IL-2R + cells and macrophages (Mac 1 +) in addition to reducing the total number of cells per spleen in all subsets in retrovirus-infected mice, but not in uninfected controls. Cocaine treatment and retrovirus infection alone or in combination suppressed the release of slL-2R into supernatant fluid during in vitro culture of splenocytes. These data illustrate that cocaine treatment modulates cell proliferation in retrovirus- infected mice and thus modifies the absolute number of cells in those subsets already altered by retrovirus infection. Retrovirus-infected and retrovirus-infected cocaine-treated protein-malnourished mice showed similar results.

Cocaine is an alkaloid extracted f rom the leaves of E r y t h r o x y l u m coca. Daily cocaine injection for short periods of time, less than 14 days, has been shown to cause reduced body, spleen, and thymus weight in experimental animals (Watzl & Watson, 1990; Holsapple & Munson , 1985), while short- term cocaine exposure increases the responsiveness of lymphocytes to phytohemagglut inin (PHA) and concanavalin A (Con A) and suppresses the

B-lymphocyte response to l ipopolysaccharide (LPS) (Watzl & Watson, 1990; Holsapple & Munson, 1985). It also affects resistance to infection and tumor cell growth in experimental animals (LPS) (Watzl & Watson, 1990). On the other hand, short- term exposure to high doses of cocaine with its short half-life in vivo (less than 1 h) does not adequately mimic the effects o f chronic cocaine exposure in humans .

*Fellow from CONICET (Consejo Nacional de Investigaciones Cientificas y Tecnicas) Argentina. tVisiting Scholar from Railway Medical College, Shanghai, People's Republic of China. *Author to whom correspondence should be addressed at: Department of Family and Community Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724, U.S.A.

1153

1154

Although, the proportion of cocaine users (occasional and frequent) in the United States has decreased significantly over the past few years to about 2.4 million, the number of frequent users has increased (National Institute of Drug Abuse, 1988). The increasing number of people interested in consuming drugs favor increases in prices making it difficult for the drug abuser to get enough money to pay for the drug and food. This makes difficult the fulfilment of the nutritional requirements; therefore undernutrition is a common feature among drug abusers. Moreover, cocaine itself was shown to regulate appetite in a fashion that leads to malnutrition (Watson & Mohs, 1989). Intravenous cocaine users are prone to infection with human immunodeficiency virus (HIV) which causes an acquired immune deficiency syndrome (AIDS). Little is known about the combined effects on immunomodulation of cocaine injection and retro- virus infection (Watzl & Watson, 1990; Watson, 1989), and of retrovirus infection and cocaine injection when malnutrition is another cofactor. Recently, it was demonstrated that cocaine applied in vi tro to infected T-cells increased HIV replication in v i tro via a TGF-/~-mediated mechanism (Peterson, Gekker, Chao, Schut, Molitor & Balfour, 1991). Murine leukemia virus (MuLV) infection in mice partially models retrovirus-induced immunodeficien- cy after HIV infection in humans (Mosier, Yetter & Morse, 1985; Watson, 1989). This is an experimental model that helps us to understand the effects of retrovirus infection on the immune system and the ongoing development of AIDS in humans (Watson, 1989). Although CD4 ~ cells are required for the development of routine AIDS (Yetter et al. , 1988; Chattopadhyay, Makino, Hartley & Morse, 1989), they are not destroyed by murine retrovirus infection. Mice chronically depleted of CD4 ~ cells by treatment with a monoclonal antibody against CD4 antigen did not develop the syndrome characterized by lymphadenopathy, splenomegaly, hypergamma- globulinemia, and in vi tro deficient B-cell responses to T-independent antigens (Yetter et al. , 1988; Chattopadhyay et al. , 1989). Thus, the presence of functional T-cells is required for MuLV-infected mice to develop lymphoproliferation and B-cell abnormalities (Mosier, Yetter & Morse, 1987). Mature B-cells are also required for the development of the syndrome, since mice treated from birth with anti-mouse IgM antibodies could not develop MAIDS after MuLV infection (Cerny et al. , 1990).

This murine AIDS model (Mosier et al. , 1985, 1987; Yetter et al. , 1988; Watson, 1989; Cerny et al. , 1990) was used in association with a drug abuse

M. C. LOPEZ et al.

model that resembles human drug abuse with increasing doses of cocaine given over a long period of time. The combined effects of cocaine injection and retrovirus infection on the percentage and total number of splenic lymphoid cells of normal and protein-malnourished female C57BL/6 mice are reported. Moreover, we evaluated the production of two key cytokines (y-IFN and TNF-a) and the release of soluble interleukin 2 receptor (sIL-2R) after in vi tro culture of splenocytes.

EXPERIMENTAL PROCEDURES

A n i m a l s a n d d ie ts

Female C57BL/6 mice, 4 weeks old, were obtained from the National Cancer Institute (Frederick, MD). They were fed the AIN-76A synthetic diet (Petro & Watson, 1982) with standard protein content (20% casein) (Dyets, Bethlehem, PA), or a moderately low protein diet (4°70 casein) adjusted with starch to be isocaloric with a normal protein diet (20% casein). This feeding procedure began 3 weeks before, and continued during the 11 weeks of cocaine injection. Animals were cared for as required by the University of Arizona College of Medicine Committee on Animal Research, which adheres to the Guide for the Care and Use of Laboratory Animals.

C oca ine t r e a t m e n t

Cocaine hydrochloride in a 0.9% saline solution was administered by intraperitoneal (i.p.) injection. Mice designated absolute controls did not receive i.p. treatment, while the saline controls received vehicle only (saline). Cocaine-injected mice were treated with increasing doses: week 1, 5 mg/kg body weight/day; week 2, 20 mg/kg/day; week 3, 35 mg/kg/day; and weeks 4 - 11, 40 mg/kg/day.

L P - B M 5 M u L V in f ec t ion

After 3 weeks of diet consumption, one-half of the mice were injected once intraperitoneally with 0.1 ml of a LP-BM5 MuLV inoculum which had an ecotropic titer (XC) of 4.5 log,~ PFU/ml which induced the disease with a time course comparable with that described previously (Hartley, Fredrickson, Yetter, Makino & Morse, 1989). Infection of C57BL/6 mice with LP-BM5 MuLV leads to the rapid induction of clinical symptomatology of murine AIDS with virtually no latent phase (Mosier, 1986; Mosier et al. , 1987; Yetter et al. , 1988;

Cocaine, Murine AIDS and Immunomodulation

Chattopadhyay et al., 1989; Cerny et al., 1990). LP-BM5 MuLV-infected C57BL/6 mice live 5 - 6 months before succumbing to constriction of airways by expanding lymphoid tissues (Watson, 1989). The virus inoculum was a kind gift of Dr R . A . Yetter, Veterans Administration Medical Center, Baltimore, MD.

Lymphocyte and macrophage subpopulations measurement

Spleens were individually collected after sacrifice under ether anesthesia. Mononuclear cells were obtained by gently teasing the spleens in RPMI-1640 medium plus 10% fetal bovine serum with tweezers. Spleen red blood cells were lysed by ammonium chloride solution (Sigma Chemical Co., St. Louis, MO). The remaining cell suspensions were washed twice with cold media, the number of viable cells determined by trypan blue exclusion and cells adjusted to give the desired viable cell concentrations ( 1 - 2 × 106/0.1 ml/tube) for lymphocyte and macrophage surface marker determinations.

T-cell subpopulations were quantified by indirect immunofluorescence staining using monoclonal antibodies (Becton Dickinson Company, San Jose, CA) specific for total T-cells (rat IgG2b anti-mouse Thy 1.2 antigen), T-helper cells (L3T4; rat IgG2b anti-mouse CD4 antigen, GK1.5 clone) or T-suppressor/cytotoxic cells (Lyt-2; rat IgG2a anti- mouse CD8 antigen). The total number of macrophages was characterized with rat anti-Mac l antigen monoclonal antibody (clone M 1/70) from Boehringer (Indianapolis, IN). The numbers of cells expressing IL-2R were determined by using a rat IgG2a monoclonal antibody, clone AMT-13, (Boehringer, Indianapolis, IN). Cells expressing Ia were characterized using IgG2a rat monoclonal antibody anti-mouse H - A I - A subregion (clone YE 2/36 HLK) from Sera-Lab (Accurate Chemicals, Westbury, NY). Fluorescein isothiocyanate-labeled goat anti-rat (heavy and light chain specific) immunoglobulin (Southern Biotechnology Asso- ciates, Birmingham, AL) was used as a second antibody. B-cells that express surface IgG or IgM were characterized using an indirect assay, affinity- purified rabbit anti-mouse IgG + IgM (heavy and light chain specific) as the first antibody and a fluorescein-conjugated affinity-purified goat anti- rabbit IgG, Fc fragment specific as the second antibody (Accurate Chemicals, Westbury, NY).

The staining was performed as follows, 1 × l0 s spleen cells in 100/al of media were incubated in the presence of the primary monoclonal antibodies in the

1155

appropriate dilutions for 30 min at 4°C in an ice bath, final volume 140/al. Each tube was washed with 1.5 ml cold PBS, centrifuged 400 g for 10 min. Supernatants were carefully discarded, and then the secondary antibodies were added to each sample. Samples were incubated for 30 min at 4°C. After the incubation period and PBS washing, samples were spun down, fixed with 1.0 ml of 2°70 paraformal- dehyde (pH 7.4). Samples were stored at 4 - 8 ° C until they were analysed by an Epics C or a FacStar flow cytometer.

The percentage of positive cells was obtained using the Immunofluorescent Analysis Program included in the Easy 2 software version 1.3 provided by the Epics Division of the Coulter Company (Hialeah, FL) when the Epics C was used and using the Consort 40 software when the FacStar (Becton Dickinson, Mountain View, CA) was used. Ten thousand cells were counted. The absolute number of cells labeled with the specific monoclonal antibody was calculated by multiplying the total number of cells by the percentage of positive cells.

Splenocyte cultures

Splenocytes (1 × 106 cells/well) were cultured in the presence of 2/ag/ml of Con A for 72 h at 37°C for y-IFN detection, in the presence of 5/ag/ml of PHA for slL-R determination or in the presence of 10/ag/ml of LPS during 72 h for TNF-a evaluation.

ELISA assay for gamma interferon (y-IFN). y-IFN was measured as described previously (Chen & Watson, 1991). Briefly, 50 ~1 of rat anti-mouse y-IFN monoclonal antibody (Lee Molecular Research Lab, San Diego, CA) diluted 1 : 100 in carbonate buffer, pH 9.6, were added to each well of an ELISA plate (Dynatech Immulon 2) (Dynatech Laboratories, Inc., Chantilly, VA), plates were incubated at 4°C overnight. The plates were washed with P B S - T w e e n (0.01 M PBS, 0.05070 Tween 20) and dried, 50 tal of the standards (recombinant mouse y-IFN, AMGEN, Thousand Oaks, CA) dissolved in RPMI-1640 medium + 10% FBS, or 50 ~1 of cell culture supernatant were added to each well; plates were incubated for 1 h at 37°C. Plates were washed and 50/al of rabbit anti-mouse y-IFN polyclonal antibody (a gift from Dr Philip Scuderi, Arizona Cancer Center) diluted 1 : 100 in P B S - T w e e n were added, and plates incubated at 37°C for 1 h. Plates were washed and goat anti- rabbit IgG-HRP (American Qualex, La Mirada, CA, diluted 1 : 5000 in PBS-Tween) was added to each well (50/A/well). After incubation at 37°C for 1 h, plates were washed three times with P B S - T w e e n

1156 M. C. LOPEZ et al.

and once with PBS. Finally 100 ~d of substrate solution (ABTS) (0.548 g of 2,2-Azino-bis(3-ethyl- benzthiazoline), 6-sulfonic acid and 21.0 g of citric acid monohydrate in 1 liter water, pH 4.2, plus 0.03% H2Oz) were added to each well. After 30 min the optical density was measured on a Titertek Multiskan (Flow Labs, McLean, VA) at 405 nm (standard range from 0.39 to 50 ng/ml, detection limit: 20 pg/well).

ELISA assay f o r tumor necrosis factor alpha (TNF-a). TNF-~ was measured as described previously (Chen & Watson, 1991). Briefly, 50/A of hamster anti-mouse TNF-a monoclonal antibody (Genzyme, Cambridge, MA), diluted 1 : 5 0 0 in carbonate buffer pH 9.6, were placed into each well of an ELISA plate (Dynatech Immulon 2), and incubated at 4°C overnight. Plates were washed as in the IFN assay. One hundred microliters of 2% BSA (bovine serum albumin, in 0.01 M PBS) were added to each well and incubated at 37°C for 1 h, plates were washed and dried. Fifty microliters of the standards (recombinant mouse TNF-a, Genzyme) prepared in RPMI-1640 with 10% FBS, or of cell culture supernatants, were added to each well and incubated at 37°C for 1 h. Plates were washed, and 50/A rabbit anti-mouse TNF-a polyclonal antibody (a gift from Dr Philip Scuderi, Arizona Cancer Center) diluted 1 : 167 in PBS-Tween were added to each well. Plates were incubated at 37°C for 1 h, washed and 50 tal of HRP-conjugated goat anti- rabbit IgG (American Qualex, La Mirada, CA) diluted 1 : 500 were added to each well. From here onward the assay followed the same procedure as for the y-IFN assay (standard range from 0.156 to 10 ng/ml, detection limit: 10 pg/well).

ELISA fo r slL-2R

sIL-2R was determined according to a procedure described previously (Lopez, Colombo, Huang, Wang and Watson (1992), Clin. Immun. Immuno- path., in press). Briefly, 96-well plates were coated with 50/A/well of rat anti-mouse IL-2R monoclonal antibody diluted 1 : 2 5 in carbonate buffer. The antibody (IgM) was produced by clone 7D4 obtained from the American Type Culture Collection (ATCC, Rockville, MD). Plates were washed three times with PBS-Tween and 50/al of each sample, or the standards (two-fold serial dilutions of cell-free splenocyte supernatants) diluted in media were added to the wells. The plates were incubated for 1 h at 37°C and rinsed twice with PBS-Tween . Fifty microliters of rat-anti-mouse IL-2R diluted 1 : 20 in PBS (rat IgG2a clone AMT-13, Boehringer,

Indianapolis, IN) were then added to each well and plates incubated for 1 h at 37°C and rinsed twice with P B S - Tween. After plates were washed, 50/al of rabbit-anti-rat IgG Fc horseradish peroxidase conjugated (Jackson Immunoresearch Lab. Inc., Westgrove, PA) diluted 1 :5000 in PBS-Tween were added to each well. Plates were incubated for 1 h at 37°C, after which they were washed three times with PBS - Tween and once with PBS. Finally, 100 tal of substrate solution were added to each well and the optical density was measured as was described above. The absorbance values of the samples were converted into arbitrary slL-2R units by comparing the absorbance of the samples to the absorbance of the internal laboratory standard consisting of cell-free supernatant fluid of 1 x 106 spleen cells/well from normal C57BL/6 mice, with an arbitrary assigned value of 1000 units of sIL-2R/ml.

Statistical analysis

Results are presented as mean _+ standard deviation. Data were analysed using the one-way analysis of variance, and the multiple range test. Data analysis was performed by the Biostatistical Service of the Arizona Health Sciences Center.

RESULTS

Effects o f retroviral infection and cocaine injection on body and spleen weight

Daily cocaine injection for 11 weeks produced a decrease in body weight when compared with the untreated, or saline-treated mice (Table 1). There was an increase in spleen weight and cell number in saline- and cocaine-injected mice which was not statistically significant. Body weight, spleen weight and cell number increased significantly (P< 0.05) in mice infected with the retrovirus for 11 weeks. Cocaine injection did not further modify the body weight of retrovirus-infected mice. While retrovirus infection provoked an enlargement of lymphoid organs (especially spleen and peripheral lymph nodes), cocaine decreased the weight of the thymus (data not shown) and spleen (Table 1). Spleen weight increased about 10 times in the MuLV-infected group, but only increased 5 times in those retrovirus- infected mice that also received cocaine (Table 1). The total number of cells per spleen was also found to increase in the retrovirus-infected group, but cocaine treatment largely prevented this increase (Table 1).

Cocaine, Murine AIDS and Immunomodulation

Table 1. Effects of cocaine injection and retrovirus infection on body and spleen weight in mice

1157

Treatment

Body weight* Spleen

Initial Final Weight Cell number (g) (g) (mg) ( × 10 7)

None 18.8 _+ 0.9 22.2 _+ 1.2 75.4 + 9.2 5.2 + 1.5 Saline 17.8 _+ 1.4 22.7 _+ 1.7 156.6 + 38.6 t 7.1 _+ 2.4 Cocaine 18.6 +_ 1.1 21.0 _+ 1.0 t* 135.5 + 22.7 6.0 _+ 1.1 Retrovirus 19.5 + 0.8 26.2 _+ 3.1 t* 1050.1 _+ 961.7 t* 10.4 +__ 7.7 t Cocaine and retrovirus 19.4 _+ 0.9 24.1 _+ 1.5 499.7 _+ 229.5 ~ 6.9 _+ 3.1

*Values are mean _+ S.D. for 10 mice per group. Initial weight = body weight at beginning of cocaine and/or retrovirus treatment. *Significantly different from untreated controls (P<0.05). *Significantly different from saline-injected controls (P<0.05).

Table 2. Effects of cocaine injection and retrovirus infection on body and spleen weight in mice fed low protein diet

Body weight Spleen

Initial* Final Weight Cell number Treatment (g) (g) (mg) (X 10 7)

None 17.2 __. 1.1 19.6 _+ 1.7 77 _+ 21 4.4 _+ 1.4 Saline 17.6 +_ 0.8 19.3 ___ 1.7 86 _+ 18 2.9 _+ 1.0 Cocaine 16.8 _+ 0.8 14.5 _+ 1.9 t* 49 _+ 28 2.3 _+ 1.8 Retrovirus 16.6 _+ 1.1 22.3 -+ 1.9 408 _+ 94** 9.3 _+ 2.3 t* Cocaine and retrovirus 17.6 _+ 0.6 18.1 _+ 1.2 213 _+ 61 t* 7.6 _+ 3.4 t*

*Time at which LP-BM5 infection and/or cocaine treatment began. *Significantly different from untreated controls (P<0.05) *Significantly different from saline-injected controls (P<0.05). Values are mean _+ S.D. for 10 mice per group with a mean weight of 14.9 g prior to initiation of protein-deficient diets administration.

Effects of retroviral infection and cocaine injection on body and spleen weight in mice fed the low protein diet

Mice tha t consumed the 4°7o casein diet for 3 weeks had lower body weights t han those fed the diet with a no rma l pro te in con ten t (20o70 casein diet) (P<0.01) , even t h o u g h they con t inued to grow and gain weight. Mice tha t consumed the 4°70 prote in diet dur ing the 11 weeks of cocaine in jec t ion showed a d ramat i c decrease in body weight (Table 2). The body weight increased in mice infected with re t rovirus for 11 weeks wi th a d ramat i c en la rgement in lymphoid organs, especially the spleen (Table 2). The increase in body weight of MuLV-infec ted mice was prevented in the p ro te in -malnour i shed , cocaine- t rea ted mice.

Cocaine , as well as saline t r ea tmen t , p rovoked a non-s igni f icant decrease in the n u m b e r of splenocytes (Table 2) in non- infec ted mice. Dur ing re t rovi rus infect ion, spleen weight increased five-

fold in virus-infected mice and 2.5 t imes in the virus- infected group tha t was also injected with cocaine (Table 2). Cocaine t r ea tmen t o f the retrovirus- infected mice yielded a s ignif icant (P<0.05) decrease in spleen weight compared with re t rovirus- infected mice as well as a tendency towards fewer spleen cell numbers .

Percentage and numbers of spleen lymphoid cells as modified by cocaine treatment

Saline- and cocaine- t reated mice showed a decrease in the percentage of to ta l T-cells, when compared with the cont ro l g roup tha t did no t receive any daily in jec t ion (Table 3). Moreover , a s ignif icant decrease in the percentage of CD4 + and CD8 ÷ cells was also observed (P<0.05) (Table 3). Daily saline and cocaine inject ions produced a s ignif icant increase in the percentage and absolute number s of B-cells per spleen (Tables 3 and 4).

1158 M . C . LOPEZ et al.

Table 3. Effects of cocaine injection on the percentage of cells expressing surface antigenic markers in the spleen

Treatment Thy 1.2 + CD4 ÷ CD8 ÷ IL-2R + Ia ÷ Mac 1 + B +

None 59.5 _ 7.0 25.1 _+ 2.0 12.9 __. 4.0 0.79 _+ 0.40 9.0 __. 7.4 4.9 _+ 1.0 16.2 _+ 7.7 Saline 39.0 + 12.6" 19.7 ___ 2.7* 8.4 _+ 3.3* 0.76 __. 0.43 13.2 _+ 9.9 3.4 _+ 0.7 34.3 _+ 7.0* Cocaine 37.9 _+ 9.8* 17.7 ___ 3.5* 6.1 _+ 1.6" 0.89 __. 0.29 12.1 _+ 7.1 3.7 ___ 1.2 41.5 _+ 9.9* Retrovirus 22.1 ___9.0 *t 38.7_+9.4 *t 5.3___4.1 *+ 4 . 4 + 6 . 1 *t 11.3_+6.9 10.9_+ 7.4 *t 52 .3_ 7.5 *t Cocaine and retrovirus 26.3 + 6.9 *t 37.2 ___ 7.8 *t 4.7 _+ 2.8 *t 0.93 + 0.46 I0.1 __. 5.9 7.7 + 3.1 49.7 +_ 13.0 *t

*Significantly different from untreated controls (P<0.05). tSignificantly different from saline-injected controls (P<0.05). Values are mean _+ S.D. for 10 mice per group. B + = slgG * + slgM +.

Table 4. Number of cells expressing surface antigenic markers per spleen of cocaine injected and retrovirus-infected mice

Treatment Thy 1.2 + CD4 + CD8 + IL-2R ÷ Ia + Mac 1 ~ B +

None 33.9 _+ 11.6 12.8 + 3.8 7.2 + 3.0 0.38 __. 0.19 2.6 ___ 3.4 2.7 + 1.0 9.0 __. 6.6 Saline 26.0 +__ 9.1 13.4 + 4.1 6.0 ___ 2.4 0.55 + 0.44 7.3 _ 5.7 4.7 _+ 5.9 25.9 _+ 12.4" Cocaine 22.6 + 6.3 10.6 ___ 2.8 3.7 _+ 1.2 t 0.55 __. 0.26 7.0 _+ 3.8 2.2 _+ 0.9 25.2 + 8.7* Retrovirus 25.6 __. 32.9 44.6 _+ 44.7 ** 6.3 __. 8.1 6.4 _+ 9.2 *t 15.0 +_ 21.0 14.8 _+ 20.0 *t 52.3 _+ 36.8 *t Cocaine and retrovirus 17.4 _+ 7.6* 25.6 __. 17.1 t 3.2 _+ 2.5 t 1.3 _ 2.0 6.5 __. 4.7 5.1 + 3.0 32.4 ___ 11.6"

*Significantly different from untreated controls (1°<0.05). tSignificantly different from saline-injected controls (P<0.05). Values are mean x 10 6 _ S.D. for 10 mice per group. B + = slgG ~ + slgM +.

Af te r 11 weeks o f M u L V infec t ion there was a

s ignif icant (P<0.05) decrease in the percen tage o f

T h y 1.2 + and CD8 + cells c o m p a r e d wi th un infec ted

con t ro l s (Table 3). C o n c o m i t a n t l y , increases in the

percen tage o f CD4 +, Mac 1 + and IL-2R ÷ cells, and

B-cells ( s IgM ÷ + s I g G ÷) were also detected due to

in fec t ion (Table 3). Coca ine a d m i n i s t r a t i o n

a b r o g a t e d the increase in IL-2R ~ cells that was

obse rved in re t rov i rus - in fec ted mice. In add i t ion , the

increase in the percen tage o f Mac 1 + cells (main ly

m a c r o p h a g e s ) due to re t rov i rus infec t ion was

p reven ted by cocaine exposure . H o w e v e r , there was

still a s ignif icant d i f fe rence (P<0.05) be tween saline-

injected con t ro l s and cocaine- injected, r e t rov i rus -

infected mice. Final ly, an increase in the abso lu t e

n u m b e r o f CD4 ÷, IL-2R +, s IgG + s lgM + and

Mac 1' cells was obse rved in the re t rov i rus - in fec ted

mice. Coca ine in ject ion was associa ted wi th a

decrease in the abso lu te n u m b e r o f T h y i .2 ÷ , CD4 + ,

CD8 +, IL-2R +, Ia +, Mac 1 ~ and B-cells per spleen

c o m p a r e d wi th re t rov i rus - in fec ted mice (P<0.05)

(Table 4).

Percentage and absolute number o f ceils expressing surface antigenic markers in the spleens o f retro- virus-infected mice

The analysis o f the expe r imen t s presented in

Tables 3 and 4 sugges ted that cocaine by itself did

no t m o d i f y the pheno typ i c express ion o f splenic

l ymphocy te s fu r the r t h a n the in jec t ion- induced stress

did. The re fo re , we decided to dissect the d i f ferent

effects o f saline and cocaine in jec t ions on re t rovi rus-

infected mice (Table 5). We obse rved tha t saline

in jec t ion decreased the percen tage and abso lu te

n u m b e r o f CD8 ÷ and the percen tage o f Ia + cells in

the spleen w h e n c o m p a r e d wi th re t rovi rus- infec ted

non - t r ea t ed mice. This decrease was no t observed in

re t rov i rus - infec ted coca ine- t rea ted mice. The

percen tage o f B-cells was increased in re t rovi rus-

infected cocaine- t rea ted mice w h e n c o m p a r e d with

the sal ine- t reated mice. The abso lu te n u m b e r o f

IL-2R + cells was decreased in re t rov i rus - infec ted

coca ine- t rea ted mice w h e n c o m p a r e d wi th re t rovi rus-

infected sal ine- t reated mice.

Changes in the percentage and number o f splenic lymphoid cells by protein undernutrition and cocaine injection

Daily saline inject ions for 11 weeks caused no

changes in the percen tage or n u m b e r o f T- and

B-cells, bu t s ignif icant ly (P<0.05) decreased the

n u m b e r o f Mac 1 + cells (Table 7). A s ignif icant

increase (P<0.05) in the percen tage o f CD8 + cells

wi th a s i m u l t a n e o u s decrease in the n u m b e r o f

Mac 1 ÷ cells (Tables 6 and 7) was observed in

p r o t e i n - u n d e r n o u r i s h e d mice injected wi th cocaine

fo r 11 weeks. H o w e v e r , w h e n c o m p a r e d with saline-

Coca ine , Mur ine A I D S and I m m u n o m o d u l a t i o n 1159

Tab le 5. Pe rcen tage and abso lu te n u m b e r of cells express ing surface an t igen ic ma rke r s in the spleens of re t rovi rus-

infec ted mice

T r e a t m e n t Thy 1.2 + CD4 ÷ CD8 ÷ IL-2R + Ia ÷ Mac 1 ÷ B +

Re t rov i rus 29.3 _+ 10.0 27.2 _+ 11.7 9.1 _+ 2.5 6.9 _+ 4.3 19.7 _+ 8.3 16.2 _+ 5.7 53.9 _+ 3.7

Re t rov i rus + sal ine 24.4 _+ 9.3 27.5 _+ 10.8 4.6 +__ 1.4" 5.3 _+ 1.6 12.2 _+ 7.2* 12.2 _+ 6.6 53.5 _+ 5.7

Re t rov i rus + cocaine 28.5 +__ 13.2 30.7 +_ 8.6 8.5 _+ 2.9 t 3.5 _+ 1.5 19.4 _+ 6.5 t 12.3 _+ 5.0 62.0 +_ 3.4 f

Abso lu t e n u m b e r of cells ( × 10 -6)

Ret rov i rus 11.9 +_. 5.1 11.3 _+ 5.8 3.4 +__ 1.4 2.8 ___ 2.0 7.9 + 4.4 6.6 + 3.1 20.4 _+ 5.7

Re t rov i rus + sal ine 10.3 _+ 4.9 11.7 + 5.8 1.7 _+ 0.6* 2.2 ___ 1.0 4.8 _+ 2.7 5.2 + 3.1 22.6 _+ 7.1

Re t rov i rus + cocaine 10.8 _+ 7.3 11.0 _+ 5.9 2.6 _+ 1.7 1.2 _+ 1.1 + 5.7 _+ 3.2 3.8 _+ 2.7 21.7 _+ 11.1

*Signi f icant ly d i f fe ren t f rom re t rov i rus 1°<0.05. tS ign i f i can t ly d i f fe ren t f rom re t rov i rus + sal ine P<0 .05 .

Tab le 6. Effec ts of coca ine in jec t ion and M u L V infec t ion on the pe rcen tage of splenic cells express ing specif ic an t igenic

ma rke r s in mice fed low pro te in diet

T r e a t m e n t Thy 1.2 + CD4 + CD8 ÷ IL-2R + la ÷ Mac 1 ÷ B +

None 49.6 _+_ 9.7 25.5 _+ 6.6 13.7 _+ 4.5 0.74 _+ 0.73 11.0 _+ 6.0 6.3 + 6.9 27.6 _+ 12.1 Sal ine 39.8 _ 7.2 22.6 _ 1.8 11.8 _ 2.6 1.0 + 1.2 15.7 _+ 8.7 4.9 _+ 3.3 39.1 _+ 9.3

Coca ine 51.4 _+ 12.6 24.4 _+ 5.4 18.4 _+ 8.5 + 1.3 + 1.2 18.9 _+ 6.4 5.0 _+ 2.4 39.0 + 15.8 Re t rov i rus 27.2 _+ 9.6 *+ 45.7 _+ 5.1 *t 4.1 + 1.2 *t 0.82 _+ 0.16 11.1 _+ 2.7 9.7 _+ 4.3 52.3 _+ 13.9 *t

Coca ine and re t rov i rus 38.5 + 11.0 49.9 _+ 9.5 *+ 4.2 _+ 2.7 *t 0.55 _+ 0.26 11.0 _+_ 5.3 6.9 +__ 3.7 56.6 _+ 7.56 *t

*Signi f icant ly d i f fe ren t f rom un t rea t ed cont ro ls (P<0.05) .

+Signif icant ly d i f fe ren t f rom sa l ine- in jec ted cont ro ls (P<0.05) . Values are m e a n _+ S.D. for 10 mice per g roup .

Tab le 7. A b s o l u t e n u m b e r of splenic cells express ing specif ic an t igenic marke r s as modi f i ed by cocaine and re t rov i rus

infec t ion in p ro t e in -unde rnou r i shed mice

T r e a t m e n t Thy 1.2 ~ CD4 ÷ CD8 ÷ IL-2R + l a ÷ Mac 1 + B +

N o n e 21.3 + 5.6 11.4 _+ 5.4 5.7 __. 1.3 0.32 _+ 0.27 5.1 _+ 6.0 6.5 _+ 6.9 11.9 _+ 5.2

Sal ine 27.2 _+ 29.0 7.5 _+ 3.8 4.0 _+ 2.4 0.36 + 0.42 5.7 + 4.6 1.6 _+ 1.2" 11.6 _+ 5.8 Coca ine 27.8 _+ 18.4 5.0 + 3.7 3.1 _+ 2.0 0.33 _+ 0.42 5.7 _+ 3.9 1.4 _+ 1.3" 10.9 _+ 10.3 Re t rov i rus 24.4 _+ 7.4 42.0 +_ 8 .3 '* 3.8 _+ 1.4 0.75 +__ 0.22 10.4 _+_ 2.5 9.8 _+ 4.3 t 48.9 + 19.4 *t

Coca ine and re t rov i rus 28.6 +__ 14.4 37.7 ___ 18.2 *t 2.9 + 1.0 0.39 _+ 0.15 8.7 + 5.9 5.2 + 3.4 42.5 _+ 18.5 *t

*Signi f icant ly d i f fe ren t f rom un t rea ted cont ro ls (P<0.05) .

eSigni f icant ly d i f fe ren t f rom sa l ine- in jec ted cont ro ls (P<0.05) . Values are m e a n × 106 _+ S.D. for 10 mice per g roup .

t r e a t e d c o n t r o l s , t h e r e w a s n o c h a n g e in t h e

p e r c e n t a g e o f M a c 1 + ce l l s , b u t t h e r e w a s a n i n c r e a s e

in t h e p e r c e n t a g e o f C D 8 + ce l l s .

Modulatory effects of cocaine in protein- undernourished mice infected with MuL V

A f t e r 11 w e e k s o f M u L V i n f e c t i o n a s i g n i f i c a n t

( P < 0 . 0 5 ) d e c r e a s e i n t h e p e r c e n t a g e o f t o t a l T - c e l l s

( T h y 1.2 +) a n d C D 8 + ce l l s w a s o b s e r v e d ( T a b l e 6).

C o n c o m i t a n t l y , a n i n c r e a s e i n t h e p e r c e n t a g e o f

C D 4 + a n d B-ce l l s ( s I g M + + s I g G ÷) w a s d e t e c t e d

( T a b l e 6). T h e t o t a l n u m b e r o f C D 4 ÷ ce l l s a n d

B - c e l l s p e r s p l e e n c h a n g e d s i m i l a r l y i n t h e r e t r o v i r u s -

i n f e c t e d a n i m a l s ( T a b l e 7). T h e s e m i c e a l s o s h o w e d

a n i n c r e a s e i n t h e t o t a l n u m b e r o f ce l l s p e r s p l e e n

e x p r e s s i n g t h e M a c 1 + a n t i g e n ( T a b l e 7). S i m i l a r

1160 M. C. LoPEZ et al.

Table 8. Gamma-interferon and alpha-tumor necrosis factor production and slL-2R release by splenocytes after in vitro culture

y-IFN TNF-a slL-2R Treatment Casein (ng/ml) (ng/ml) (units/ml)

None 2.35 _+ 1.29 0.46 _ 0.15 82.95 _+ 40.27 Saline 20o70 19.75 + 8.19" 0.81 +__ 0.12' 111.45 _+ 32.80 Cocaine 17.34 _+ 5.21" 0.91 _+ 0.10" 46.10 _+ 26.91" Retrovirus 1.05 ___ 0.62 *+ 0.73 + 0.42 26.15 _+ 23.00 *+ Cocaine and retrovirus 1.55 +_+_ 1.69 + 1.17 _+ 0.19 *+ 14.4 _+ 16.08 *+

None 9.00 +_ 6.60 0.35 +_ 0.29 105.33 _+ 32.86 Saline 4o70 5.82 _+ 4.50 0.96 _+ 0.03* 61.83 _+ 18.25" Cocaine 5.79 _+ 5.11 1.09 _+ 0.21" 64.0 + 18.08" Retrovirus 0.22 _+ 0.08* 1.6 _+ 0.10" 11.6 _+ 16.13" Cocaine and retrovirus 1.54 _+ 1.07 *+ 1.34 _+ 0.40* 0.0 _+ 0.0 *+

Ten mice per group were studied. *P<0.05 significantly different from non-treated mice. +P<0.05 significantly different from saline-treated mice.

results were observed in the animals infected with re t rovirus and also injected with cocaine (Table 6). The increase in the percentage of Mac 1 + and the decrease in the percentage of Thy 1.2 + cells were lost dur ing cocaine t rea tment . W h e n the retrovirus- infected mice were t rea ted with cocaine, there was no increase in the n u m b e r of IL-2R+ cells (Tables 6 and 7).

y-IFN, TNF-a production and sIL-2R release after in vitro culture o f normal splenocytes

Saline and cocaine t r ea tmen t signif icantly increased the p roduc t ion of y-IFN by spleen cells. Saline t r ea tment induced a non-s igni f icant increase in sIL-2R release (Table 8) while cocaine t r ea tmen t significantly decreased the levels of sIL-2R release. Splenocytes f rom MuLV-infec ted mice showed a d iminished p roduc t ion of y-IFN and sIL-2R when compared with the non- t rea ted controls . Spleen cells f rom retrovirus- infected, cocaine- injected mice showed a lower p roduc t ion of y-IFN and sIL-2R, compared with splenocytes f rom sal ine-treated controls .

Spleen cells f rom saline- and cocaine- t reated mice as well as those f rom retrovirus- infected, cocaine- t rea ted mice showed an increase in T N F - a p roduc t ion af ter culture.

y-IFN and TNF-a production and sIL-2R release after in vi t ro culture o f splenocytes f rom protein- undernourished mice. Non- t rea ted , prote in-mal- nour i shed mice p roduced significantly h igher amoun t s of y-IFN than the unt rea ted , wel l -nourished

mice (9.00 _+ 6.60 vs 2.35 + 1.29, P<0.01) . Secretion of y-IFN by splenocytes f rom saline- and cocaine- t reated mice did not differ f rom unt rea ted controls (Table 8). However , splenocytes f rom retrovirus- infected and re t rovirus- infected cocaine-injected animals secreted significantly (P<0.05) lower levels of y-IFN than splenocytes f rom non- t rea ted and sal ine-treated mice.

Splenocytes f rom saline- and cocaine-injected mice, as well as re t rovirus- infected and virus-infected cocaine- t reated mice all p roduced more T N F - a than cells f rom non- t rea ted mice (Table 8). Saline and cocaine t r ea tmen t decreased the secretion of sIL-2R by spleen cells dur ing in vitro incubat ion . Retrovirus infect ion, and re t rovirus infect ion plus cocaine- t rea tment , suppressed the secretion of sIL-2R by splenocytes f rom pro te in -malnour i shed mice.

DISCUSSION

The exper imental an imal models designed previously to s tudy the effect of cocaine on the immune system generally evaluated shor t periods of t r ea tmen t (Watzl & Watson , 1990). We developed a mur ine model s imulat ing the effects of long-term, chronic cocaine use a lone or combined with mur ine AIDS. Both cocaine use and re t rovirus infect ion are c o m m o n in h u m a n in t ravenous drug users. A n exper imenta l mur ine system model ing h u m a n chronic cocaine addic t ion was developed to s tudy the effects of cocaine inject ion on T- and B-cell subsets

Cocaine, Murine AIDS

in the spleens of well-nourished and protein- malnourished mice. Protein malnutrition was also evaluated because general malnutrition is common among drug abusers and because cocaine indirectly favors undernutrition by regulating the apetite (Watson & Mohs, 1989). Cocaine administration for 11 weeks reduced the body weight in both groups in agreement with previous studies of short-term cocaine administration (Watzl & Watson, 1990; Holsapple & Munson, 1985) that also showed changes in lymphoid cell functions.

Long-term (11 weeks) cocaine daily administration provoked a significant decrease in the percentage of Thy 1.2 ÷ , CD4 + and CD8 + cells in the spleen of well- nourished mice when compared with untreated mice (Table 3). Concomitantly, there was an increase in the percentage and total number of B-cells per spleen of these mice and a decrease in the absolute number of CD8 + cells in cocaine-treated mice (Table 4). These results are in agreement with those obtained in rats given low doses of cocaine for short periods of time where a decrease in the percentage of T-cells and an increase in the percentage of B-cells was observed (Bagasra & Forman, 1989). Saline-injected mice showed similar results. Therefore, changes in the percentage of T-cells may be the consequence of stress provoked by daily intraperitoneal injection and handling (Moynihan, Brenner, Koota, Breneman, Cohen & Ader, 1990). Such stress may be partially responsible for the changes observed on T- and B-cell markers in the spleen after cocaine administration. Repeated handling reduced IgG antibody production and T-cell mitogenesis (Moynihan et al., 1990). Nevertheless, the authors could not demonstrate that handling produced any change in lymphocyte subsets in the spleen (Moynihan et al., 1990). Thus, while handling may not be the cause of the changes we observed in spleen cell subsets, the effect of the stress of injection plus handling for a long period of time could induce some of the changes we observed. Nevertheless, the main difference between saline and cocaine treatment was a decrease in the absolute number of CD8 + cells in the spleens of cocaine-treated mice. Protein-mal- nourished mice did not show changes in the percen- tage or in the absolute number of Thy 1.2 +, CD4 + or B-cells per spleen after saline or cocaine treatment (Tables 6 and 7). Nevertheless, there was an increase in the percentage of CD8 ÷ cells in the spleens of cocaine-treated, protein-malnourished mice.

Cocaine and saline injections also had similar effects on the production of y-IFN and TNF-a by murine splenocytes, increasing the production of these cytokines similarly. These findings suggest that

and Immunomodulation 1161

T-cells and macrophages were able to respond to mitogens after saline or cocaine exposure. T-cells synthesize y-IFN. TNF-a is synthesized by T-cells, but more particularly by macrophages (Carding, Hayday & Bottomly, 1991). In experimental animals, the increase of serum slL-2R after IL-2 therapy was associated with an increase in spleen weight. T-cells are the source of serum slL-2R, as nude mice showed the increase in spleen weight but not the increase in slL-2R levels (Wagner, Wong, Gately & Nelson, 1990). The release of slL-2R after in vitro culture was suppressed in cocaine-treated mice but was not altered in saline-treated ones that received a normal diet. This suggests that certain early events in T-cell activation are altered by chronic cocaine treatment, although IL-2R expression was not modified. Among mature T-cells, IL-2R expression is always linked to an activation process dependent on antigens or mitogen binding (Hemler, 1984; Cantrell & Smith, 1983).

The effects of cocaine on the immune system could be mediated by actions on the neuroendocrine system with hormones directly affecting lymphoid cells. In vivo studies would favor the latter assumption since increased plasma levels of ACTH, beta-endorphin and corticosterone were observed after cocaine injection (Forman & Estilow, 1988). Here we observed changes in cytokine production by murine lymphocytes isolated after chronic exposure to cocaine in vivo that may have significant regulatory actions on T-cell development in vivo. Therefore, spleen lymphoid cells are more likely an indirect target of cocaine administration, due to cocaine effects on the neuroendocrine system.

MuLV infection induced several changes in the numbers and functions of splenic T-celts, B-cells, and macrophages that resemble HIV infection in humans (Hartley et al., 1989; Morse et al., 1989). Recently AZT (azidothymidine), a major drug for treating human retrovirus disease, was shown to significantly reduce MuLV infection progression, strengthening it as a model of human AIDS (Morse et al., 1989; Basham, Rios, Holdener & Merigan, 1990). Retrovirus infection provoked dramatic changes in T- and B-cell subsets (Morse et al., 1989). Those changes not only include functional impairment of the immune system (Morse et al., 1989), but also surface phenotype changes (Morse et al., 1989; Holmes, Morse, Makino, Hardy & Hayakawa, 1990). Our results indicate that there is a decrease in the percentage of Thy 1.2 + and CD8 + cells as well as an increase in the percentage of CD4 ÷ cells, independent of the diet used. In uninfected mice the combined percentage of CD4 ÷ cells and

1162 M. C. LOPEZ et al.

CD8 ÷ cells is equivalent to the percentage of Thy 1.2 + cells ( total T-cells). However , in retrovirus- infected mice the percentage of Thy 1.2+ is less t han the combined percentage of CD4 ÷ and CD8 ÷ cells. Our results suggest the presence of a Thy 1- CD4+ cell popu la t ion in the re t rovirus- infected mice because the percentage of CD4 ÷ cells is even higher t han tha t of Thy 1.2 + cells. This f inding is in agreement with recent da ta demons t r a t ing the expans ion of a Thy 1- CD4 ÷ cell subset in M u L V infected mice (Holmes et al. , 1990). Pe rhaps the more i m p o r t a n t difference between AIDS and M A I D S is the decrease in the n u m b e r of CD4 ÷ cells in AIDS, tha t does not occur in M A I D S , where the cells persist a l though they are no t funct ional .

The percentage and the absolute n u m b e r of cells per spleen tha t express IL-2R (CD25) was increased in MuLV-infec ted mice. These cells were not able to release the receptor af ter in vitro i ncuba t ion in cont ras t to cells f rom uninfec ted mice. This suggests the presence of immatu re act ivated cells in the per iphery tha t canno t complete their d i f fe ren t ia t ion af ter re t rovirus infect ion. These results dif fer part ial ly f rom the ones ob ta ined in h u m a n ret rovirus infect ion since HIV- infec ted pat ients present an increase in serum s lL-2R levels (Kloster et al. , 1987). This f inding was correla ted with a decrease in the n u m b e r of CD4 lymphocytes (Sprickett & Dalgleish, 1988). However , HIV- infec ted cells did not produce elevated levels of T A C (IL-2R) in culture, suggesting tha t soluble CD25 found in vivo resulted f rom act ivat ion of T-cells t h r ough oppor tun i s t i c infect ion ( H o n d a et al., 1989). Moreover , pat ients with H I V

infect ion showed a lower percentage of CD25+ cells when compared with no rma l subjects (Zola, Koh, Martz ior is & Rhodes , 1991). These results also suggest some difference between HIV infect ion in h u m a n s and re t rovirus infect ion in mice.

W h e n the relative p ropor t ion of cells was analysed, cocaine t r ea tment of re t rovirus- infected mice did not modify changes in lymphoid cell subsets induced by M u L V infection. W h e n the absolute n u m b e r of cells per spleen tha t bear specific antigenic markers was analysed, the results suggested that cocaine was able to modula te prol i fera t ion by decreasing the absolute n u m b e r of Thy 1.2+, CD4+ and CD8 ÷ cells in re t rovirus- infected mice (Table 4). However , this effect was not observed in protein- ma lnour i shed mice. Moreover , cocaine (but not saline) normal ized the n u m b e r of IL-2R + and Mac 1 + cells, which were changed by retrovirus infect ion a lone (Tables 4 and 5). Nonetheless , cocaine t r ea tment could not normal ize the secretion of s lL-2R by splenocytes in culture, perhaps due to the fact tha t coca ine- t rea tment to normal mice also in terfered with this process. We do not know if these phenotypic changes induced by cocaine in mur ine AIDS-mice were able to improve or impai r fur ther the course of the disease.

Acknowledgements - - The support of ADAMHA grants DA 04827 and AA08037 is recognized. The authors greatly appreciate the technical assistance of Pamela Schubart, Dave Pena, Amy Horn, Thuy Nguyen, Dan Nelson and Jim Logan with the Epics C flow cytometer, under the direction of Dr M. J. Hicks; and Barbara Carolus with the FacStar.

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