Modification of thymic cell subsets induced by long-term cocaine administration during a murine retroviral infection producing AIDS

Modification of thymic cell subsets induced by long-term cocaine administration during a murine retroviral infection producing AIDS

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Vol. 65, No. 1, October, pp. 45-52, 1992 Modification of Thymic Cell Subsets Induced by Long-Term Cocain...

940KB Sizes 2 Downloads 962 Views





Vol. 65, No. 1, October, pp. 45-52,


Modification of Thymic Cell Subsets Induced by Long-Term Cocaine Administration during a Murine Retroviral Infection Producing AIDS MARIA Department


of Family









LP-BM5 murine leukemia virus (MuLV) infection and cocaine administration are known to impair the murine immune system. We have developed a murine model to study the effect of daily cocaine administration and retrovirus infection on the lymphoid cell populations of the thymus. C5’7BLE female mice were studied following chronic cocaine administration for 11 weeks with simultaneous LPBM5 MuLV infection. Cocaine administration reduced body and thymus weight, significantly reduced the number of CDS+ cells in the thymus, and partially prevented thymus enlargement due to lymphoid cell proliferation induced by LP-BM5 MuLV infection. Retrovirus infection was associated with a decrease in the percentage and absolute number of Thy 1.2+, CD4+, and CD8’ cells in the thymus, an effect potentiated by cocaine administration. Therefore cocaine impairs thymic function by altering the number of cells expressing T cell differentiation markers in MAIDS. o 1992 Academic Press, Inc.


Prolonged cocaine use is associated with an increased rate of infections including HIV (human immunodeficiency virus) leading to AIDS (acquired immune deficiency syndrome) (1). This suggests that cocaine administration could modulate disease resistance, therefore facilitating infection by opportunistic pathogens (2). Animal studies are needed to model complex immunomodulatory events resulting from the interaction of cocaine use and retrovirus infection. Cocaine has been shown to suppress the responses of mouse and human cells to mitogens in vitro (3). Animals that received a daily cocaine injection for 14 days had reduced thymus weight (4). A single cocaine injection of 5 mg/kg affected the phagocytosis of sheep red blood cells by peritoneal macrophages 24 hr later (51, suggesting that cocaine was able to induce changes in immune cell functions. Cocaine injected to rats for 10 days reduced the number of T cells, increased B cells, and had no effect on macrophages (6). Obviously, shortterm exposure at high doses of cocaine may only partially model long-term human use, increasing the in-



WANG, University

AND RONALD of Arizona,

R. WATSON Tucson,

Arizona 85724

terval of cocaine exposure may alter the effect observed. We used an experimental design by which mice received the drug daily in increasing doses for 11 weeks in an attempt to resemble human chronic drug addiction. The onset of immunodeficiency postinfection with LP-BM5 murine leukemia virus (MuLV) has been intensively studied in susceptible strains of mice (7). LPBM5 MuLV infection induces changes in the phenotype and in vitro response to alloantigens and selfantigens of T and B cells and macrophages in the spleen and peripheral lymph nodes (8-12). The presence of functional T cells is necessary for the induction of lymphoproliferation and appearance of B cell abnormalities (9). Mice chronically depleted of CD4’ T cells did not develop lymphadenopathy, splenomegaly, hypergammaglobulinemia, in vitro deficient B cell responses to T-independent antigens, or depressed allogeneic cytotoxic T-lymphocyte responses indicating the requirement of CD4+ T cells for induction of the immunodeficiency syndrome (101. The presence of mature B cells is also a requirement for the development of the disease since mice treated from birth with anti-mouse IgM antibodies did not develop murine AIDS (11). Thus LP-BM5 MuLV infection in mice could be an useful model to study the consequence of HIV infection in humans (7-12). The alteration in T cell subsets observed after retrovirus infection or cocaine treatment in human peripheral blood cells or mouse spleen cells could be the consequence of changes at the thymus level. Pro-thymocytes traffic from the bone marrow to the thymus where they find the appropriate environment to complete their differentiation and become functional T cells (13). Mature T-lymphocytes express CD4 or CD8 antigens and originate from CD4- CD8- bone marrow-derived cells. They appear in the thymus after several differentiation steps which yield CD4+ CD8+ cells (14). These immature thymocytes after extensive positive (15,161 and negative (17, 18) selection become single positive, mature T-lymphocytes (19). It was shown that 50% of Lyt2-/L3T4cells in the adult thy-

45 0090-1229/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.



mus express interleukin-2 receptors (IL-2R) (20). This expression marks a step in thymocyte differentiation previous to CD3 and TCR expression (21). Among mature T cells, IL-2R expression is always linked to an activation process dependent on antigens or mitogen binding (22, 23). Sera soluble interleukin-2 receptor (sIL-2R) levels were increased in cancer patients after therapy with recombinant IL-2 (24). The same finding was obtained in experimental animals where this increase was associated with an increase in spleen weight; it was also shown that T cells were the source of sIL-2R in sera because nude mice showed the increase in spleen weight but not the increase in sIL-2R levels in sera cm.

The percentages of Thy 1.2+ and CD8+ cells were decreased in the spleens of cocaine-treated mice especially in those infected with LP-BM5 MuLV (43). The alterations in T cell subsets that we observed in the spleen may be the consequence of an impairment of thymic differentiation. Chronic cocaine administration could also modify the onset or time course of murine AIDS. In this paper we evaluated the combined effects of cocaine treatment and retrovirus infection on the thymus cell subpopulations by studying the expression of differentiation markers. Moreover, we studied the production of interferon-y (IFN-y) and the release of sIL-2R after in vitro culture. MATERIALS


Animals and diets. Female C57BW6 mice, 4 weeks old, were obtained from National Cancer Institute (Frederick, MD). They were fed the AIN-76A synthetic diet (Dyets, Bethlehem, PA) (26) containing 20% casein. The diet was commenced 3 weeks prior to onset of cocaine treatment. Animals were cared for as required by the University of Arizona College of Medicine Committee on Animal Research. Cocaine treatment. Cocaine was administered as cocaine hydrochloride in 0.9% saline solution by intraperitoneal (ip) injection. Mice received 0.2 ml of saline or cocaine solution. Control mice were divided into two groups, one that received no treatment and saline controls that received vehicle solution (saline) by ip injection. Cocaine was dissolved in saline 15 min before ip administration. Cocaine-injected mice were treated as follows: Week 1 5 mg/kg body wtlday Week 2 20 mg/kg body wtlday Week 3 35 mglkg body wtlday Weeks 4-11 40 mg/kg body wt/day LP-BMS murine leukemia virus infection. Animals were assigned to one of two groups. The first group of mice was injected once intraperitoneally with 0.1 ml of a LP-BM5 MuLV inoculum which had an ecotropic ti-

ter (xc) (this test gives us an idea of the numbers of infective virus that we have in the preparation) of 4.5 log,, PFU/ml which induced the disease with a time course comparable to that previously published (27). Infection of adult C57BL/6 mice with LP-BM5 MuLV leads to the rapid induction of clinical symptomatology of AIDS with virtually no latent phase (7-12, 27). LPBM5 MuLV-infected C57BW6 mice live 5 to 6 months before succumbing to constriction of airways by expanding lymphoid tissues (8). Virus inoculum was a kind gift of Dr. R. Yetter, Veterans Administration Medical Center (Baltimore, MD). Lymphocyte subpopulations measurement. Thymuses were individually collected after sacrifice under ether anesthesia and mononuclear cells obtained by gently teasing with tweezers in RPMI-1640 medium + 10% fetal calf serum (FCS). The cell suspensions were washed twice with cold medium. Viability was always greater than 95% as determined by trypan blue exclusion. Cell suspensions were adjusted to give the desired viable cell concentrations (l-2 x 106/0.1 ml/tube) for lymphocyte surface marker determinations. T cell subpopulations were determined by indirect immunofluorescence staining using monoclonal antibodies (Becton-Dickinson Company, San Jose, CA) specific for total T cells (rat IgG2b antimouse Thyl.2 antigen), T helper cells (L3T4; rat IgG2b antimouse CD4 antigen, GK1.5 clone), or T suppressor/cytotoxic cells (Lyt-2; rat IgG2a antimouse CD8 antigen). The number of cells expressing IL-2R was determined using rat IgG2a monoclonal antibodies (clone AMT-13) (Boehringer, Indianapolis, IN). Cells expressing Ia were enumerated with IgG2a rat monoclonal antibody anti-mouse H-A I-A subregion (clone YE 2/36 HLK) (Sera-Lab, Accurate Chemicals, Westbury, NY). Fluorescein isothiocyanate-labeled goat anti-rat immunoglobulin (heavy and light chain specific) (Southern Biotechnology Associates, Birmingham, AL) was used as a second antibody. Briefly, 1 x lo6 thymocytes in 100 pl of RPM1 were incubated in the presence of the primary monoclonal antibodies in the appropriate dilutions for 30 min at 4°C in an ice bath, final volume 140 ~1. Each tube was washed with 1.5 ml cold PBS and centrifuged (9OOg, 10 min). Supernates were carefully discarded, cell pellets were resuspended in media, and secondary antibodies were added to each sample (30 min, 4°C). After incubation each cell suspension was washed once with PBS, centrifuged, and finally fixed with 1.0 ml of 2% paraformaldehyde (pH 7.4). Samples were stored at AC for up to 1 week until they were analyzed by an EPICS C flow cytometer. Our preliminary study showed that cells could be kept in these conditions for 1 month without observing changes in the percentages obtained. The percentage of positive cells was obtained by using the Immunofluorescent Analysis Program included in the Easy 2 software version 1.3 provided by Epics




Division of Coulter Co. (Hialeah, FL). Ten thousand cells were counted: The absolute number of cells per thymus labeled with the specific monoclonal antibody was calculated by multiplying the total number of cells per thymus by the percentage of positive cells. For the double-staining studies directly conjugated antibodies were used: FITC-labeled anti-mouse Thy 1.2, FITC-labeled anti-mouse L3T4 (CD4), PE-labeled anti-mouse L3T4 (CD4), and PE-labeled anti-mouse Ly-2 (CD8a) from PharMingen (San Diego, CA). Fixed samples were analyzed by a FacStar (BectonDickinson, Mountain View, CA) that includes a Consort 40 program. Graphs presented in this paper were obtained with the Reproman analysis software (True Facts Software, Inc., Seattle, WA). Thymocytes (1 x lo6 cell/well) Thymocyte cultures. were cultured in the presence of 2 bg/ml of concanavalin A for 72 hr at 37°C for IFN-?I detection or sIL-2R determination. ELBA for interferon-y. The assay was performed as described previously (28). Briefly, 50 ~1 of rat antimouse IFN-7 monoclonal antibody (Lee Molecular Research Lab, San Diego, CA) diluted 1:lOO in carbonate buffer, pH 9.6, was added to each well of an ELISA plate (Dynatech Immulon 2, Dynatech Laboratories, Inc., Chantilly, VA) and plates were incubated overnight at 4°C. The plates were washed with PBS-Tween (0.01 M PBS, 0.05% Tween 20) and dried, 50 $well of standards (recombinant mouse IFN-7, Amgen, Thousand Oaks, CA) in RPMI-1640 medium (+ 10% FCS), or 50 ~1 of cell culture supernatant was added to each well; plates were incubated for 2 hr at 37°C. Plates were washed and dried. Then, 50 Fl/well of rabbit antimouse IFN-y polyclonal antibody (a gift from Dr. Philip Scuderi, Arizona Cancer Center) diluted 1:lOO in PBS-Tween was added and plates were incubated at 37°C for 2 hr. Plates were washed, and goat anti-rabbit IgG-HRP conjugate (American Qualex, La Mirada, CA), diluted 1:5000 in PBS-Tween, was added to each well (50 pi/well). After incubation at 37°C for 1 hr, plates were washed three times with PBS-Tween and once with PBS. Finally, 100 ~1 of substrate, ABTS (0.55 g of 2,2-azino-bis(3-ethylbenzthiazoline, 6-sulfonit acid and 21.0 g of citric acid monohydrate in 1 liter water, pH 4.2, before use adding 10 ~1 of H,O, in 10 ml of ABTS) was 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 rig/ml; detection limit, 20 pglwell). ELISA for soluble interleukin-2 receptor. The 96well plates were coated with 50 ~1 of rat anti-mouse sIl-2R monoclonal antibody (IgM) diluted 1:25 in carbonate buffer. The antibody was produced by clone 7D4 obtained from the American Type Culture collection (Rockville, MD). Plates were washed twice with PBS-Tween and 50



~1 of sample, or the standards (twofold serial dilutions of cell-free spleen-cell supernates) diluted in media were added to the wells. The plates were incubated for 1 hr at 37°C and rinsed twice with PBS-Tween. Then, 50 l.~lof rat anti-mouse IL-2R diluted 1:20 in PBS (rat IgG2a clone AMT-13, Boehringer) was added to each well. The plates were incubated for 1 hr at 37°C and rinsed twice with PBS-Tween. Then, 50 t.~l of rabbit anti-rat IgG Fc peroxidase-conjugated horseradish (Jackson Immunoresearch Lab, Inc., Westgrove, PA) diluted l/5000 in PBS-Tween was added to each well. Plates were incubated for 1 hr at 37°C. After the incubation period, the plates were washed three times with PBS-Tween and once with PBS. Finally, 100 ~1 of the substrate ABTS was added to each well and optical density measured as described above. The absorbance values of the samples were corrected into arbitrary sIl2R units by comparing the absorbance of the samples to the absorbance of the internal laboratory standard: cell-free supernates of 1 X lo6 cells/well of a pool of normal C57BL16 mice which was arbitrarily assigned as 1000 units of sIL-2Rlml. Statistical analysis. Results are presented as means + standard deviation. Data were analyzed using the one-way analysis of variance and the multiple range test. Data analysis was performed by the Biostatistical Service at the Arizona Health Sciences Center. RESULTS

Modification of thymus weight and cell numbers by retrovirus infection and cocaine treatment. C57BL/6 female mice treated daily for 11 weeks with increasing doses of cocaine showed a decreases in body weight (Table 1). A nonsignificant decrease in thymus weight and cell number was also observed. LP-BM5 MuLVinfected mice showed a significant (P < 0.05) increase in body and thymus weight and surprisingly, a 50% decrease in thymus cell number. When retrovirusinfected mice also received cocaine treatment the retrovirus-induced body weight increase was less marked. Moreover, cocaine treatment potentiated the retroviTABLE 1 Modification of Body and Thymus Weight by Cocaine Administration and Retroviral Infection Treatment None Saline Cocaine Virus Cocaine

and virus

Body weight” w

Thyrnus weight (mg)

22.5 22.8 20.9 26.2 24.1

54.8 56.0 43.2 104.4 16.1

2 r + + 2

a Values are means + SD for b Significantly different from ’ Significantly different from d Significantly different from

1.5 1.7,C 3.1*,’ 1.5

2 ‘f + t

8.2 8.6 9.0b,’ 28.16~’ 22.3b.c,d

Thymus cell number ( x 10”) 15.3 15.5 12.9 8.2 5.1

10 mice per group. untreated controls fP < 0.05). saline-injected controls (P < 0.05). virus group (P < 0.05).

k + 2 ‘f

3.7 3.1 3.8 4.5’~’



i-us-induced decrease in thymus cell number (Table 1). Cocaine treatment slightly reduced the thymus/body weight ratio while retrovirus infection tended to increase it (Table 1). Effects of cocaine and LP-BM5 MuLV infection on the percentage and absolute number of lymphocytes in the thymus. The stress produced by daily saline injection did not modify the number of thymus cells (Table 1). Cocaine treatment for 11 weeks did not modify either the percentage or the absolute numbers of Thy 1.2 + and CD4+ cells compared to saline-injected controls (Tables 2 and 3). Nonetheless, a significant decrease in the absolute number of cells bearing the CD8 antigen was observed (Table 3). LP-BM5 MuLV infection was associated with a statistically significant decrease (P < 0.05) in the percentage and number of CD4+ and CD8+ cells. The alteration in the CD8+ subset was more marked. The number of CD8+ cells in the retrovirus-infected mice decreased to one-fourth that of the controls (Table 3). In addition, a decrease in the absolute number of Thy 1.2+ cells was observed. By comparing the number of cells bearing Thy 1.2 and CD4 antigens in the control groups (nontreated and saline-treated mice), we observed that they had twice as many cells per thymus as the infected mice. Mice that were retrovirus infected and that also received cocaine treatment showed an even more marked decreased in the absolute number of Thy 1.2 +, CD4+, and CD8+ cells. No statistically significant changes were observed in the percentage and absolute number of ILB-R+ and Ia+ cells in any of the studied groups. Virus-infected mice showed a nonsignificant increase in the absolute numbers of Ia+ cells. When both virusinfected groups were compared, we found that cocaine injection to retrovirus-infected mice decreased the absolute number of Thy 1.2+, CD4+, IL-2R+, and Iaf cells. Double-labeling studies in control and virus-infected mice thymocytes. In Fig. 1 we showed the doublestaining studies performed on normal and MAIDS-

infected mice thymocytes. The value for CD4+ CD8+ cells in normal mice represented 88.6% while in virusinfected mice, this proportion dropped to 16.6%. Double-stained Thy 1 CD8 cells comprised 87% of normal thymocyte while in MAIDS-mice this percentage dropped to 27.7%. In a similar fashion Thy l+ CD4+ cells that represented a 76.0% of the total thymocytes in normal mice dropped to 56.3% in MAIDS-infected mice. Production ofIFN-y and release of sIL-2R by thymocytes. Thymocytes from nontreated, saline-, and cocaine-treated mice produced very small amounts of IFN-y. On the contrary, thymocytes from virusinfected and virus-infected cocaine-treated mice produced significantly higher amounts of IFNq (Table 4). Cocaine treatment tended to induce lower levels for sIL-2R and retrovirus infection tended to increase these levels. DISCUSSION

Cocaine can potentiate HIV-l replication in human peripheral blood mononuclear cell cultures (29) and may modulate lymphoid cell development (2), particularly in retrovirus-infected people. Drugs of abuse have been suggested as cofactors accelerating development of AIDS after HIV infection by their immunosuppression (30-33). Therefore we evaluated in an experimental model the effects of cocaine on in vivo immunomodulation during retrovirus infection. Chronic cocaine administration for 11 weeks reduced body and thymus weight. These results are in agreement with previous studies of short-term cocaine administration that showed a reduction in both parameters (4, 5). Previous animal models were designed to study the effect of short-term, not chronic, heavy cocaine use on the immune system (2) for 4 (5) to 14 days (3). In our experimental model, cocaine administration for 11 weeks produced a nonsignificant decrease in the percentage and in the absolute numbers of cells bearing specific T cell markers (Thy 1.2, CD4, and CD81 in mice spleens (34). Moreover, a decrease in the absolute num-


Effect of Cocaine Administration

and Retrovirus Infection on the Percentage of Thymic Cells Bearing Specific Differentiation

Treatment None” Saline Cocaine Virus Cocaine and virus a Values are means 2 b Significantly different c Significantly different d Significantly different

Thy 1.2+ 82.1 84.6 81.1 73.2 67.1

f f + f +

18.7 12.7 15.2 11.8 14.7**’

Antigens CD8+

CD4+ 71.5 84.8 78.8 70.1 57.9

k f ” + +

20.0 8.6 12.5 4.5’ 9.6b*c,d

SD for 10 mice per group. from untreated controls (P < 0.05). from saline-injected controls (P < 0.05). from virus group (P < 0.05).

74.9 75.6 70.4 31.1 21.5

+ f + + 2

5.5 9.3 11.6 17.6’,’ 15.6’~”

IL-2R + 0.79 0.68 0.56 1.1 0.55

” + + ” +

0.88 0.66 0.32 0.8 0.38

Ia+ 1.5 0.3 0.9 7.8 5.0

+ ” + + f

2.2 0.2 0.9 7.5’ 3.0’


THYMOCYTES, COCAINE ADMINISTRATION, AND MAIDS Effect of Cocaine Administration Treatment

None” Saline Cocaine Virus Cocaine and virus

TABLE 3 and Retrovirus Infection on the Absolute Number of Thymic Cells Bearing Specific Differentiation Antigens

Thy 1.2+ 123.7 132.3 104.2 58.8 34.4

k * * * k

36.9 36.7 37.9 30.1’,’ 17.3b,‘,d

CD4’ 117.4 133.2 103.1 58.0 29.7

* 32.5 -+ 35.5 ? 39.5 f 34.36,’ * 15.0b*‘,d

CD8+ 114.7 120.9 91.3 27.2 12.0

f 5 4 * ?

28.3 27.7 33.6’ 28.4b~c 10.6b,c

IL-2R+ 1.00 1.20 0.72 0.63 0.25

k f ” 5 2

0.92 1.0 0.46 0.26 0.17’rd

Ia+ 2.5 0.39 1.0 4.3 2.3

f 3.7 t 0.31 * 0.84 ” 2.5” +- 1.5d

a Values are 2 x lo6 2 SD for 10 mice per group. b Significantly different from untreated controls (P < 0.05). c Significantly different from saline-injected controls (P < 0.05). d Significantly different from virus group (P < 0.05).

bers of cells bearing these markers in the spleens of the malnourished mice was observed: as well, an increase in the percentage and absolute numbers of B cells in these spleens was detected (43). These results were in agreement with those obtained in rats with different doses of cocaine where a decrease in the percentage of T cells and an increase in the percentage of B cells in the spleen were observed (6). As changes in T cell phenotype distribution observed in the spleen may be the consequence of alterations in T cell differentiation, several T cell subsets were analyzed in the thymus of cocaine-treated mice. Chronic, long-term cocaine injection for 11 weeks did not cause a significant reduction of numbers of Thy 1.2 + and CD4 + cells in the thymus, yet long-term cocaine administration induced a statistically significant reduction in the numbers of CD8+ cells in the thymus. This finding suggests that after long periods of exposure to cocaine an alteration in thymocyte differentiation of CD8+ cells can be induced. This would be expected to produce an impairment in cytotoxic T cell function and host resistance (30-33). LP-BM5 MuLV infection in mice can be used to evaluate potentially useful anti-retrovirus drugs that would be further tested for human use in HIV infections. It has been shown that several drugs such as zidovudine (AZT) can alter the time course of murine AIDS (35). Moreover, this model can be used to evaluate, in uiuo, how drugs of abuse such as cocaine could potentiate the impairment of the immune system induced by the retrovirus. The data on the changes in thymic subsets in the thymus induced by LP-BM5 expand the understanding of this model. Although thymus weight increases in virus-infected animals, the number of cells recovered from those thymuses is lower. Our preliminary hystological studies showed the disappearance of defined areas (like the cortex and medulla) in these thymuses. Moreover, there are empty spaces between groups of cells that do not correspond to collagen fibers. We are performing other studies to further classify the hystological alterations in the thymus of retrovirus-infected mice. We

observed a significant decrease in the percentages of CD4’ and CD8+ cells which were not equal for both populations in the thymus. In other words, murine retrovirus infection which has progressed to murine AIDS alters T cell differentiation by changing the normal overlapping of CD4+ CD8’ cells. These doublepositive cells represent 70-90% of the cells in the thymus of normal uninfected mice. However, this population dropped to only 30% and another population, CD4 + CD8-, whose function is unknown, appears and represents 40% of thymocytes in LP-BM5 MuLVinfected mice. These results suggests that LP-BM5 MuLV infection alters T cell differentiation. Moreover, when double-labeling studies were performed, we confirmed the appearance of higher percentages of CD4+ CD8- and CD4- CDS+ cells as well as Thy 1.2+ CD8and Thy 1.2+ CD4- cells. These data suggest a breakdown in the early steps of T cell differentiation that is currently under investigation in our lab. When virus-infected mice also received cocaine the percentages of CD8+ and CD4+ cells were decreased, indicating that cocaine could potentiate the effect of LP-BM5 MuLV on the immune system. Moreover, cocaine treatment further reduced the numbers of CD4+ and CD8+ in the thymus of retrovirus-infected mice. Interestingly, this has not resulted in a comparative decrease of CD4+ and CD8+ cells in the spleens of similarly treated mice (M. C. Lopez, G.-J. Chen, D. S. Huang, and R. R. Watson, unpublished data). This may be due to a peripheral expansion of these cells after leaving the thymus. We can conclude that LPBM5 retrovirus infection’s immunomodulation is exacerbated by chronic, long-term cocaine treatment. It is probable that in an attempt to fight retrovirus infection, thymocytes increased the production of IFN-y (Table 4) after in vitro culture. IFN-y is mainly synthesized by T cells (Tnl cells) but can also be produced by NK cells. Increased resistance to parasitic infection appears to be associated with a higher production of IFN-7 (36). Treatment with anti-IFN-y monoclonal antibody greatly enhanced oocyst shedding during Cryptosporidium infection in an adult mouse





















40 FL1











FL1 (CD4) n19lnou2sl74




1 20







m192natd1112 60-




--VT c.4






THY 1.2 FIG. 1. Double labeling of normal and MAIDS-infected thymocytes. A, C, and E show results obtained in normal mice, while B, D, and F are the virus-infected counterparts. (A) CD4+ CDS+, 88.65%; CD8+, 1.14%; CD4+, 7.67%. (B) CD4+ CDS+, 16.6%; CDB+, 15.5%; CD4+, 33.98%. (C) Thy 1.2+ CD8+, 87.57%; Thy 1.2+ 12.11%; CD8+, 0.03%. (D) Thy 1.2+ CD8+, 27.68%; Thy 1.2+, 44.68%; CD8+, 2.51%. (E) CD4+ Thy 1.2+, 76.03%; Thy 1.2+, 5.33%; CD4+, 1.28%. (F) Thy 1.2+ CD4+, 56.31%; Thy 1.2+, 40.10%; CD4+, 0.80%.

model (37). Nevertheless, the increased production of IFN-r after in vitro culture may not represent higher levels of IFN-r in ho. We have demonstrated that it is possible to infect LP-BM5 MuLV-infected mice with Cryptosporidium parvum (38).

HIV-infected patients present an increase in sera sIL-2R levels (39). This finding was correlated with a decreased in the number of peripheral CD4 lymphocytes (40); however, HIV-infected cells did not produce elevated levels of TAC (CD25 = IL-2R) in culture sug-




TABLE4 Interferon-y

Production after

and Soluble IL-2 in Vitro Culture


Release 9.

Treatment None Saline Cocaine Virus Cocaine and virus a Significantly b Significantly

IFN--r (rig/ml) 0.019 0.011 0.028 1.640 1.701

* 0.025 2 0.003 ” 0.034 ? 1.810” If: 1.970”,b

sIL-2R (units/ml) 22.6 23.0 12.6 70.3 31.8

k 23.0 -+ 19.6 k 6.4 t 92.1 k 28.8

different from untreated controls (p < 0.05). different from saline-treated controls (p < 0.05).

gesting that soluble CD25 found in uiuo resulted from activation of T cells through opportunistic infection

(41). Moreover, patients with HIV infection showed a lower percentage of CD25 + cells when compared with normal subjects (42). We observed (unpublished results) that there were no changes in the levels of sera sIL-2R in MAIDS mice. Our results did not show changes in the expression of IL-2R on virus-infected mice thymocytes, or in the production of sIL-2R by thymocyte cultures, suggesting a difference between human and mouse retrovirus infection.



12. 13.



ACKNOWLEDGMENTS 16. 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 Epics C flow cytometer under the direction of Dr. M. J. Hicks, and Barbara Carolus with the FacStar. M.C.L. is a Fellow from CONICET (Consejo National de Investigaciones Cientificas y Tecnicas) Argentina.

17. 18. 19. 20.

REFERENCES 1. Chaisson, R. E., Bacchetti, P., Osmond, D., Brodie, B., Sande, M. A., and Moss, A. R., Cocaine use and HIV infection in intravenous drug users in San Francisco. JAMA 261,561-565,1989. 2. Watzl, B., and Watson, R. R., Immunomodulation by cocaine-a neuroendocrine mediated response. Life Sci. 46, 1319-1329, 1990. 3. Klein, T. W., Newton, C. A., and Friedman, H., Suppression of human and mouse lymphocyte proliferation by cocaine. Adu. Biochem. Psychoparmacol. 44, 139-143, 1988. 4. Holsapple, M., and Munson, A., Immunotoxicology of abused drugs. In “Immunotoxicology and Immunopharmacology.” (J. Dean, Ed.), p. 381, Raven Press, New York, 1985. 5. Ou, D. W., Shen, M-L., and Luo, Y-D., Effects of cocaine on the immune system of Balblc mice. Clin. Zmmunol. Zmmunopathol. 52, 305-312, 1989. 6. Bagasra, O., and Forman, L., Functional analysis of lymphocytes subpopulations in experimental cocaine abuse. I. Dosedependent activation of lymphocytes subsets. Clin. Exp. Zmmunol. 77, 289-293, 1989. 7. Mosier, D. E., Yetter, R. A., and Morse, H. C., III, Retroviral induction of acute lymphoproliferative disease and profound immunosuppression in adult C57BL/6 mice. J. Erp. Med. 161,766784, 1985. 8. Chattopadhyay, S. K., Makino, M., Hartley, J. W., and Morse, H. C., III, Pathogenesis of MAIDS, a retrovirus induced immu-



23. 24.






nodeficiency disease in mice. In “Immunodeficient Animals in Experimental Medicine.” (B-q. Wu and J. Zheng, Eds.1, pp. 1218, Karger, Basel, 1989. Mosier, D. E., Yetter, R. A., and Morse, H. C., III, Functional T lymphocytes are required for a murine retrovirus-induced immunodeficiency disease (MAIDS). J. Exp. Med. 165, 1737-1742, 1987. Yetter, R. A., Buller, R. M. L., Lee, J. S., Elkins, K. L., Mosier, D. E., Fredrickson, T. N., and Morse, H. C., III, CD4’ T cells are required for development of a murine retrovirus-induced immunodeficiency syndrome, MAIDS. J. Exp. Med. 168, 623-635, 1988. Cerny, A., Hugin, A. W., Hardy, R. R., Hayakawa, K., Zinkernagel, R. M., Makino, M., and Morse, H. C., III, B-cells are required for induction of T cell abnormalities in a murine retrovir-us-induced immunodeficiency syndrome. J. Exp. Med. 171,315320,1990. Watson, R. R., Murine models for acquired immune deficiency syndrome. Lifi Sci. 44, iii-xv, 1989. Scollay, R., Wilson, A., D’Amico, A., Kelly, K., Egerton, M., Pearse, M., Wu, L., and Shortman, K., Developmental status and reconstitution potential of subpopulations of murine thymocytes. Zmmunol. Rev. 104, 81-120, 1988. Lesley, J., Trotter, J., Schulte, and Hyman, R., Phenotypic analysis of the early events during repopulation of the thymus by bone marrow prothymocyte. Cell. Zmmunol. 128, 63-78, 1990. Teh, H. S., Kisielow, P., Scott, B., Kishi, H., Uematsu, Y., Bluthmann, H., and von Boehmer, H., Thymic major histocompatibility complex antigens and the T-cell receptor determine the CD4/ CD8 phenotype of T cells. Nature 335, 229-233, 1988. Benoist, C., and Mathis, D., Positive selection of the T cell repertoire: Where and when does it occur? Cell 58,1027-1033,1989. Kappler, J. W., Roehm, N., and Marrack, P., T cell tolerance by clonal elimination in the thymus. Cell 49, 273-280, 1987. Schwartz, R. H., Acquisition of immunologic self-tolerance. Cell 57, 1073-1081, 1990. Smith, L., CD4+ murine T-cells develop from CD8+ precursors in vivo. Nature 326, 798-800, 1987. Ceredig, R., Lowenthal, J. W., Nabholz, M., and MacDonald, H. R., Expression of interleukin-2 receptors as a differentiation marker on intrathymic stem cells. Nature 314, 98-100, 1985. Pearse, M., Wu, L., Egerton, M., Wilson, A., Shortman, K., and Scollary, R., A murine early thymocyte development sequence is marked by transient expression of the interleukin-2 receptor. Proc. Natl. Acad. Sci. USA 86, 1614-1618, 1989. Hemler, M. E., Antigenic stimulation regulates the level of expression of interleukin 2 receptor on human T cells. Proc. Natl. Acad. Sci. USA 81, 2172-2175, 1984. Cantrell, D., and Smith, K. A., Transient expression of interleukin-2 receptors. Consequences for T cell growth. J. Exp. Med. 158, 1895-1911, 1983. Lotze, M. ., Custer, M. C., Sharrow, S. O., Rubin, L. A., Nelson, D. L., and Rosenberg, S. A., In vivo administration of purified human interleukin-2 to patients with cancer: Development of interleukin-2 receptor positive cells and circulating soluble interleukin-2 receptors following interleukin-2 administration. Cancer Res. 47, 2188-2195, 1987. Wagner, D. K., Wong, H. L., Gately, M. K., and Nelson, D. L., Cellular source of soluble interleukin-2 receptors in serum of mice after recombinant interleukin-2 administration. Cytokine 2, 337343, 1990. Petro, T. M., and Watson, R. R., Resistance to L1210 mouse leukemia cells in moderately protein-malnourished BALD/c mice treated in vivo with thymosin fraction V. Cancer Res. 42, 21392145,1982. Hartley, J. W., Frederickson, T. N., Yetter, R. A., Makino, M.,






32. 33.



LOPEZ ET AL. and Morse, H. C., III, Retrovirus-induced murine acquired immunodeficiency syndrome: Natural history of infection and different susceptibility of inbred mouse strains. J. Viral. 63, 12231231,1989. Chen, G. J., and Watson, R. R., Modulation of tumor necrosis factor and gamma interferon production by cocaine and morphine in aging mice infected with LP-BMB, a murine retrovirus. J. Leuk. Biol. 50, 349-355, 1991. Peterson, P. K., Gekker, G., Chao, C. C., Schut, R., Molitor, T. W., and Balfour, H. H., Jr., Cocaine potentiates HIV-l replication in human peripheral blood mononuclear cell cocultures. J. Immunol. 146, 81-84, 1991. Watson, R. R., and Wallace, C. L., Drugs of abuse as cofactors in the progression of HIV infection to AIDS. In “Cofactors in HIV-l Infection and AIDS,” pp. 97-102, CRC Press, Boca Raton, FL, 1989. Pillai, R. M., and Watson, R. R., In vitro immunotoxicology and immunopharmacology: Studies on drugs of abuse. Toxicol. Lett. 53, 269-283, 1990. Pillai, R. M., and Watson, R. R., AIDS: Disease progression and immunomodulation by drugs of abuse and alcohol. AZDS Med. Report 4, 25-36, 1991. Pillai, R. K., Somasekharan, B., and Watson, R. R., AIDS, drugs of abuse, and the immune system: A complex immunotoxicolog ical network. Archiu. Toxicol. 65, 609-617, 1991. Lopez, M. C., Huang, D. S., Watzl, B., Chen, G. J., and Watson, R. R., Splenocyte subsets in normal and protein malnourished mice after long-term exposure to cocaine or morphine. Life Sci. 49, 1253-1262,1991. Basham, T., Rios, C. D., Holdener, T., and Merigan, T. C., Zidovudine (AZT) reduces virus titer, retards immune dysfunc-

Received December 12, 1991; accepted with revision June 13, 1992





40. 41.



tion, and prolongs survival in the LP-BM5 murine induced immunodeficiency model. J. Infect. Dis. 161, 1006-1009, 1990. Scott, P., and Kaufmann, S. H. E., The role of T-cell subsets and cytokines in the regulation of infection. Immunol. Too!ay 12, 346-348,1991. Ungar, B. L. P., Kao, T.-C., Burris, J. A., and Finkelman, F. D., Cryptosporidium infection in an adult mouse model. Independent roles for IFN- and CD4+ T lymphocytes in protective immunity. J. Zmmunol. 147, 1014-1022, 1991. Darban, M., Enriquez, J., Sterling, C. R., Lopez, M. C., Chen, G. J., Abbaszadegan, M., and Watson, R. R., Cryptosporidiosis facilitated by murine retroviral infection with LP-BMB. J. Znfeet. Dis. 164, 741-745, 1991. Kloster, B. E., John, P. A., Miller, L. E., Rubin, L. A., Nelson, D. L., Blar, D. C., and Tomar, R. M., Soluble interleukin-2 receptors are elevated in patients with AIDS or at risk of developing AIDS. Clin. Immunol. Zmmunopathol. 45, 440-446, 1987. Sprickett, G. P., and Dalgleish, A. G., Cellular immunology of HIV infection. Clin. Exp. Immunol. 71, l-7, 1988. Honda, M., Kitamura, K., Matsuda, K., Yokota, Y., Yamamoto, N., Mitsuyasu, R., Chermann, J. C., and Tokunga, T., Soluble IL-2 receptor in AIDS. Correlation of its serum level with the classification of HIV-induced diseases and its characterization. J. Immunol. 142, 4246-4255, 1989. Zola, H., Koh, L. Y., Mantzioris, B. X., and Rhodes, D., Patients with HIV infection have a reduced proportion of lymphocytes expressing the IL-2 receptor ~55 chain (TAC, CD25). Clin. Zmmunol. Immunopathol. 59, 16-25, 1991. Lopez, M. C., Chen, G.-J., Huang, D. S., Wang, Y., and Watson, R. R. Znt. J. Immunopharmucol. 1992, in press.