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Originally published online as doi:10.1189/jlb.0405219 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1198-1203.)
© 2005 by Society for Leukocyte Biology

Cocaine and {sigma}-1 receptors modulate HIV infection, chemokine receptors, and the HPA axis in the huPBL-SCID model

Michael D. Roth*, Katherine M. Whittaker{dagger}, Ruth Choi{dagger}, Donald P. Tashkin* and Gayle Cocita Baldwin{dagger},1

* Divisions of Pulmonary and Critical Care Medicine and
{dagger} Hematology-Oncology, Department of Medicine, David Geffen School of Medicine at University of California Los Angeles

1 Correspondence: Division of Hematology-Oncology, 11-934 Factor, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1678. E-mail: gbaldwin{at}mednet.ucla.edu

ABSTRACT

Cocaine is associated with an increased risk for, and progression of, clinical disease associated with human immunodeficiency virus (HIV) infection. A human xenograft model, in which human peripheral blood mononuclear cells were implanted into severe combined immunodeficiency mice (huPBL-SCID) and infected with a HIV reporter virus, was used to investigate the biological interactions between cocaine and HIV infection. Systemic administration of cocaine (5 mg/kg/d) significantly increased the percentage of HIV-infected PBL (two- to threefold) and viral load (100- to 300-fold) in huPBL-SCID mice. Despite the capacity for cocaine to increase corticosterone and adrenocorticotropic hormone levels in control mice, the hypothalamic-pituitary-adrenal axis was suppressed in HIV-infected animals, and corticosterone levels were further decreased when animals were exposed to HIV and cocaine. Activating huPBL in vitro in the presence of 10–8 M cocaine increased expression of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) coreceptors. Expression of CCR5 was also increased at early time-points in the huPBL-SCID model following systemic exposure to cocaine (54.1±9.4% increase over control, P<0.01). This effect preceded the boost in viral infection and waned as HIV infection progressed. Cocaine has been shown to mediate immunosuppressive effects by activating {sigma}-1 receptors in immune cells in vitro and in vivo. Consistent with these reports, a selective {sigma}-1 antagonist, BD1047, blocked the effects of cocaine on HIV replication in the huPBL-SCID mouse. Our results suggest that systemic exposure to cocaine can enhance HIV infection in vivo by activating {sigma}-1 receptors and by modulating the expression of HIV coreceptors.

Key Words: CCR5 • CXCR4 • corticosterone • AIDS • xenograft

INTRODUCTION

Human immunodeficiency virus (HIV) infection and the clinical progression to AIDS are complex biological processes, which can be affected by several variables including viral tropism, the presence and function of coreceptors, the integrity of host immunity, and a variety of other factors [1 , 2 ]. The role that drugs of abuse play in this process is controversial and may be multifactorial. Cocaine users are at increased risk for viral transmission through needle-sharing and high-risk sexual behavior [3 ]. Crack cocaine has also been described as an independent risk factor for the development and progression of HIV [4 , 5 ]. This may be related to its capacity to activate the hypothalamic-pituitary-adrenal (HPA) axis, resulting in steroid-associated immune suppression [6 , 7 ]. Cocaine has also been shown to impair the function of antigen-presenting cells and T cells, modulate cytokine production, and disrupt host defenses [8 9 10 11 ]. Finally, in vitro exposure of peripheral blood leukocytes (PBL) to cocaine has been shown to accelerate HIV replication directly [12 , 13 ].

To investigate the in vivo interaction between cocaine and HIV in greater detail, we adapted a hybrid human-mouse model in which human PBL (huPBL) are implanted into the peritoneal cavity of mice with severe combined immunodeficiency (SCID) mice [14 , 15 ]. As these mice lack mature B and T cells, they accept human cell xenografts, and various strains of HIV can be subsequently introduced via intraperitoneal (i.p.) injection. This produces an environment in which huPBL are susceptible to HIV in vivo, recapitulating many aspects of early HIV infection [16 , 17 ]. Mosier and colleagues [18 ] initially developed this xenotransplant model, referred to as the huPBL-SCID mouse, as a relevant small animal model for in vivo assessment of HIV pathogenesis. Using this approach, we previously reported that concurrent administration of cocaine and HIV resulted in a significantly higher percentage of huPBL becoming infected with HIV in vivo (38.8% vs. 18.5%). The number of CD4+ cells recovered following peritoneal lavage from HIV-infected/cocaine-treated animals was significantly lower than from mice infected with HIV alone (6.5x104 vs. 19x104) and was associated with a lower CD4:CD8 ratio (2.7 vs. 6.9). We also reported that exposure to cocaine alone did not affect the implantation of PBL nor the number or viability of human CD4+ or CD8+ cells recovered from the peritoneum, suggesting a specific interaction between cocaine and HIV infection [14 ]. The goal of the current study was to investigate potential pathways and mechanisms responsible for the enhanced HIV infection occurring in cocaine-treated huPBL-SCID mice.

MATERIALS AND METHODS

Preparation of huPBL
PBL were purified from normal donor leukopacks by density gradient centrifugation and resuspended at 1–2 x 108 cells/ml in 0.9% saline. A single donor was used for each set of experiments, and no donors were used twice.

Monoclonal antibodies (mAb) and flow cytometry
Fluorochrome-conjugated mAb, specific for human CD4, CD8, CD45, CCR5, CXCR4, and the murine heat stable antigen (HSA; mCD24) were obtained from BD Biosciences (San Jose, CA). Conjugated isotype control mAb (BD Biosciences) were used to control for nonspecific binding. For each analysis, 3–5 x 105 human cells, recovered following peritoneal lavage of huPBL-SCID mice or from in vitro experiments, were labeled with mAb, fixed with 2% paraformaldehyde, and analyzed by multiparameter flow cytometry using a FACStarplus flow cytometer and CELLQuest software (BD Biosciences). Forward- versus side-scatter analysis of sham-infected implants was used to gate on the live lymphocytes, and gating on human anti-CD45-stained cells was used to exclude contaminating murine cells.

HIV viral stocks
The construction of a replication-competent CXC chemokine receptor 4 (CXCR4)-tropic HIV reporter construct for use in the huPBL-SCID model was described previously [14 , 15 , 19 ]. In brief, the CXCR4-tropic reporter virus, NL-r-HSAS, was constructed by cloning the full-length gene encoding murine HSA (mCD24) into the partially deleted vpr gene region of the CXCR4-tropic strain, HIV-1NL4–3 [15 ]. The resulting construct was further digested with PflMI and EcoRI to liberate the new vpr/HSA region. This 588-base pair fragment was then ligated into HIV-1NFN-SX to generate the CC chemokine receptor 5 (CCR5)-tropic reporter virus. NL-r-HSAS (R4-tropic) and NFN-SX-HSAS (R5-tropic) are cytopathic replication-competent reporter viruses, which express murine HSA on the surface of infected cells. The vpr gene was selected as the site for insertion of the reporter construct, as unlike nef deletion mutants of NL4-3, vpr deletion mutants replicate within and are cytotoxic for CD4-bearing cells with kinetics similar to that of wild-type virus [15 ]. Virus stocks were prepared by electroporation of mycoplasma-free HeLa-tat cells. Tris buffer was used as a negative control, and supernatants were collected 2–3 days post-electroporation. Virus production was quantitated by p24 Gag enzyme-linked immunosorbent assay (ELISA; Coulter, San Francisco, CA), and expression of mCD24 on the surface of infected cells was determined by flow cytometry. Infectious units (IU) were determined by limiting dilution on phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) or approximated on the basis of p24 antigen release. Viral supernatants were stored at –70°C.

Administration of cocaine and HIV in the huPBL-SCID model
CB-17 scid/scid (SCID) mice were bred and maintained under laminar flow conditions in the Mouse/Human Chimera Core Facility at the University of California Los Angeles (UCLA). No antibiotics were administered. Acidified water, autoclaved bedding, and a sterilized mouse diet were supplied ad libitum, and the institutional Animal Research Committee approved all animal protocols.

Eight- to 12-week-old SCID mice were implanted with 1–2 x 107 huPBL by a single i.p. injection. Immune reconstitution was documented by serial measurements of human serum immunoglobulin (by isotype-specific ELISA), which reached a plateau by 2 weeks after implantation and remained stable for up to 3 months as described previously [16 , 20 ]. At 12–15 days post-implantation, animals were infected with an i.p. injection containing 300–400 IU of the reporter virus diluted in 0.1 ml saline. Mock infections of huPBL-SCID mice were performed using supernatants from mock-electroporated HeLa-tat cells that were diluted in the same manner as virus stocks. Each experimental animal group consisted of five mice unless otherwise stated.

Cocaine hydrochloride (5 mg/ml in saline) was obtained from the National Institute on Drug Abuse [NIDA; National Institutes of Health (NIH), Bethesda, MD] and diluted in saline prior to use. Cocaine (5 mg/kg/d for 5–12 days) was delivered by i.p. injection beginning 2–5 days postinfection (14–17 days post-PBL implantation) or in some studies, on the day of infection, as described in each figure legend. This dose of cocaine was selected on the basis of prior dose/response experiments (0.1, 5, or 10 mg/kg), in which the 5-mg/kg dose was shown to have no effects on engraftment, yet enhanced HIV infection similar to that observed for the 10-mg/kg/d dose [14 ].

At the conclusion of individual experiments, prior to euthanasia, a puncture of the retro-orbital venous plexus was performed to collect peripheral blood for viral load analysis. Mice were then euthanized, and previously implanted PBL were recovered by peritoneal lavage. To determine if the effects of cocaine were mediated through the {sigma}-1 receptor, HIV-infected and uninfected huPBL-SCID animals were treated with the {sigma}-1 antagonist BD1047 (20 mg/kg, Tocris Cookson, Inc., Ellisville, MO) or PRE-084 (20 mg/kg, Tocris Cookson, Inc.), a {sigma}-1 receptor agonist, alone or in combination with cocaine (5 mg/kg/d) for 9–10 days postinfection.

Serum measurements of adrenocorticotropic hormone (ACTH) and corticosterone
To determine dose-response relationships, the response of serum ACTH and corticosterone to acute administration of cocaine was first modeled in 9- to 11-week-old female Balb/c mice (Jackson Labs, Bar Harbor, ME), congenic for the SCID animals. Three hours following a single-dose i.p. injection of cocaine (0, 1, 5, or 10 mg/kg), blood was recovered via puncture of the retro-orbital venous plexus, serum was recovered, and hormone levels were determined with 125iodine radioimmune assays specific for ACTH (Nichols Institute Diagnostics, San Clemente, CA) or corticosterone (MP Biomedicals, Orangeville, NY). In experiments using huPBL-SCID animals, uninfected and HIV-infected animals were treated daily for 8–9 days with cocaine (5 mg/kg) or saline. On the final day of the experiment, 3 h after drug injection, retro-orbital venous samples were collected and assayed for ACTH and corticosterone in the same manner.

In vitro exposure of huPBL to cocaine and HIV infection
Normal donor PBL were activated in vitro for 48 h with PHA (2.5 µg/ml) and interleukin (IL)-2 (5 U/ml, Roche Diagnostics, Indianapolis, IN) in the presence or absence of 10–8 M cocaine and then screened for HIV coreceptor expression via flow cytometry as described. For in vitro infections, 4 x 106-activated PBL were infected with NL-r-HSAS at 18 ng/106 cells in a 1-ml volume of RPMI-1640 medium (Life Technologies, Rockville, MD), supplemented with 20% fetal bovine serum (Gemini Bioproducts, Woodland, CA) and PHA/IL-2. Viral absorption was allowed to proceed for 2 h at 37°C, followed by addition of fresh media (final cell concentration 1x106/ml). Cells were then cultured with cocaine (10–6–10–12 M) or saline for 5 days, and virus production was quantitated by p24 Gag ELISA (Coulter). The frequency and phenotype of HIV-infected cells (expressing mCD24) were also determined by flow cytometry.

Quantitative viral polymerase chain reaction (PCR)
The Roche Amplicor HIV-1 monitor PCR assay (Roche Diagnostics) was used to measure viral copy number in the plasma obtained from animals at the time of sacrifice, according to the manufacturer’s instructions.

Data analysis
Comparisons between treatment groups (in vivo studies) and culture conditions (in vitro studies) were performed using an unpaired Student’s t-test. A P value ≤0.05 was considered significant for all comparisons.

RESULTS

In vivo administration of cocaine significantly enhances HIV replication
The huPBL-SCID model was adapted for these studies as a mechanism for evaluating the interaction between cocaine and HIV in vivo. Immune-reconstituted huPBL-SCID animals were infected with the NFN-SX-HSAS (R5-tropic) or NL-r-HSAS (R4-tropic) reporter virus or supernatants from mock-electroporated HeLa-tat cells (control; Fig. 1A ). Two to 5 days postinfection, animals were treated daily with 5 mg/kg cocaine. Although cocaine had no independent effect on PBL implantation, cell viability, or the number of human CD4+ or CD8+ cells (data not shown), the percentage of peritoneal cells expressing mCD24 (HIV-positive) increased significantly (average 2.9-fold over control) following co-administration of cocaine and HIV. Systemic viral load also increased by ~150-fold (Fig. 1B) , consistent with our prior experience with this model [14 ].



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  		Figure 1. (A) The huPBL-SCID model. Eight- to 12-week-old CB-17 scid/scid (SCID) mice were implanted with 1–2 x 107 huPBL by i.p. injection. After allowing 2 weeks for immune reconstitution, huPBL-SCID animals were infected i.p. with 300–400 IU of a reporter virus, NFN-SX-HSAS (R5-tropic) or NL-r-HSAS (R4-tropic), or supernatants from mock-electroporated HeLa-tat cells (control). Two to 5 days later, daily i.p. injections of cocaine hydrochloride (5 mg/kg) or saline alone (control) were started and continued until the experiment’s end (5–10 days). Outcome measures included quantitative PCR to measure systemic viral load in peripheral blood, serum concentrations of ACTH, corticosterone by 125iodine-radioimmune assays, and flow cytometry to determine the impact of treatment on cell subsets and HIV infection in cells recovered by peritoneal lavage. (B) Coadministration of cocaine and HIV enhances infection and increases viral load in huPBL-SCID animals. At 10–12 days postinfection, cocaine-treated animals (5 mg/kg/d) exhibited a significant increase in the percentage of human CD45+ peritoneal cells, which expressed the HIV reporter construct (mCD24+) compared with control animals (mCD24/CD45+, left panel), as well as a significant increase in systemic viral load (right panel). Results represent mean values ± 1 SE for five (right panel) or seven (left panel) experiments, each with three to four animals/group. *, P < 0.01, compared with control.

 
In vivo effects of cocaine on the HPA axis
In preliminary studies, a single injection of cocaine to normal Balb/c mice produced a dose-dependent increase in ACTH and corticosterone (Fig. 2A ). Administration of cocaine by itself to huPBL-SCID animals (5 mg/kg/d for 10 days) had similar effects on these neuroimmune modulators, significantly increasing corticosterone production by 1.5- to twofold (Fig. 2B) . However, the administration of cocaine to animals that were infected with HIV had the opposite effect. HIV infection alone, in the absence of cocaine, suppressed corticosterone, and levels decreased even further in animals concurrently exposed to cocaine and HIV. A similar decrease in ACTH levels was also found (not shown), making it unlikely that these immunosuppressive agents act as a mechanism by which cocaine enhances HIV replication in the huPBL-SCID model.



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  		Figure 2. (A) Cocaine mediates a dose-dependent increase in serum ACTH and corticosterone in Balb/c mice. Animals were injected with a single dose of cocaine (1–10 mg/kg) and evaluated 3 h later for serum ACTH and corticosterone by radioimmune assay. Results represent mean values ± 1 SE for three separate experiments, each with six mice per group. (B) Cocaine suppresses serum corticosterone levels in huPBL-SCID animals. Mice were infected with the HIV reporter construct, NFN-SX-HSAS, and then treated daily for 7–9 days with cocaine (5 mg/kg) or saline. On day 10, 3 h after drug injection, serum was collected and assayed for corticosterone. Results represent mean values ± 1 SE for two experiments, each with three animals/group. *, P < 0.01, compared with control; {dagger}, P < 0.01, compared with cocaine treatment; {ddagger}, P < 0.01, compared with control or cocaine treatment.

 
Cocaine increases expression of HIV coreceptors in vitro and in vivo in huPBL-SCID mice
Cocaine has been reported to up-regulate CCR5 expression on microglial cells [21 ], and we hypothesized that similar effects on PBL might contribute to its effects on viral replication. Consistent with this hypothesis, exposing activated PBL to 10–8 M cocaine in vitro significantly increased expression of CCR5 on CD13+ monoyctes and CXCR4 on CD3+ lymphocytes (Fig. 3A ). Exposing activated PBL to cocaine in vitro also enhanced HIV replication with a dose-dependent (10–6–10–12 M) increase in HIV replication, with exposure to 10–8 M cocaine producing a 3.5-fold increase in HIV-p24 (5.2 ng p24/ml vs. 1.5 ng p24/ml in controls).



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  		Figure 3. (A) Cocaine enhances chemokine coreceptor expression in vitro. Human PBL were purified from buffy coats and cultured in the presence or absence of 10–8 M cocaine for 48 h, and expression of the HIV coreceptors, CCR5 and CXCR4, was determined in different cell populations by flow cytometry. Results are the mean ± 1 SE for three experiments. (B) Up-regulation of the HIV coreceptor CCR5 precedes the increase in HIV infection in huPBL-SCID mice. Peritoneal cells were harvested from HIV (NFN-SX-HSAS)-infected huPBL-SCID animals at 5, 7, or 14 days postinfection. Animals were treated with cocaine (5 mg/kg/d) or saline (control) starting on the day of HIV infection (cells harvested at Day 5) or 2 days after infection (7-day harvest) or 4 days after infection (14-day harvest). Cells were examined for the presence of CD45 to identify the human cells and then gated on this population to determine the percentage of CD45+ cells, which expressed CCR5 or mCD24 (the HIV reporter construct). Results are expressed as the percent increase in cocaine-treated animals as compared with saline controls. Results represent the mean values ± 1 SE for two to three experiments per time-point, five to six animals/group. *, P < 0.01, compared with control.

 
To relate these observations to HIV infection in vivo, huPBL-SCID mice were infected with a CCR5 (R5)-tropic or CXCR4 (R4)-tropic HIV reporter virus and then treated daily with cocaine (5 mg/kg/day) or saline. Peritoneal cells were harvested at various times following cocaine exposure and examined for expression of CCR5, CXCR4, and mCD24 (HSA), the HIV reporter gene (Fig. 3B) . At 5 days postinfection, exposure to cocaine had enhanced the number of CCR5+ cells significantly (29.1±8.1% CCR5+ cells) compared with saline-treated controls (13.3±2.8% CCR5+ cells). The mean fluorescence intensity for CCR5, a measure of receptor density, was also enhanced 69.9 ± 8.4% over control levels (not shown). At this early time-point, however, there was no detectable effect of cocaine on the percentage of HIV-infected cells. When the same parameters were evaluated at Day 7, CCR5 expression was still increased in cells recovered from cocaine-treated, HIV-infected animals (20.6±5.8% vs. 11.1±2.3 CCR5+ for control animals), but the percentage of HIV-infected cells was increased 28.6 ± 7.7% over control. Finally, at Day 14 (10 days postinfection), in vivo exposure to cocaine significantly increased the percentage of cells infected by HIV (35.3±9.8% vs. 17.1±3.6 for control animals), but up-regulation of the CCR5 coreceptor was no longer apparent (Fig. 3B) . Therefore, similar to our in vitro findings, systemic administration of cocaine to huPBL-SCID animals in the absence of HIV infection also enhanced CCR5 expression (2.7- to 3.1-fold; not shown). Taken together, these results suggest that up-regulation of CCR5 precedes the increase in viral infection, providing a potential mechanism by which cocaine enhances HIV infectivity.

In contrast to enhanced expression of CCR5, in vivo administration of cocaine to huPBL-SCID mice resulted in more modest effects on CXCR4 expression (8–10% increase over control levels), regardless of whether the animals were also HIV-infected. Although these effects did not reach statistical significance, it is possible that cocaine-mediated changes in CXCR4 were masked by cell loss as a result of HIV infection and/or effects of HIV infection on the continued expression of coreceptors. Notably, the R4 (NL-r-HSAS)- and R5 (NFN-SX-HSAS)-tropic HIV reporter constructs resulted in similar increases in HIV infection in cocaine-treated animals. As such, it is likely that other mechanisms, in addition to coreceptor modulation, contribute to the impact of cocaine on HIV replication.

The effects of cocaine on HIV replication are mediated through the {sigma}-1 receptor
The capacity for cocaine to interact with {sigma}-1 receptors has been shown to modulate immune function in vivo and in vitro [22 , 23 ], promoting a T helper cell type 2 immune response associated with increased production of IL-10 and transforming growth factor-ß (TGF-ß) [11 , 24 ]. As modulation of IL-10 and TGF-ß are important events in the pathogenesis of HIV [9 , 25 ], we hypothesized that an interaction of cocaine with {sigma}-1 receptors might also contribute to its effects in the huPBL-SCID model. To test this hypothesis, huPBL-SCID mice were infected with HIV and then treated daily for 10 days with saline, cocaine alone, BD1047 as a selective {sigma}-1 antagonist, or the combination of BD1047 with cocaine. Administration of BD1047 alone had no positive or negative significant effect on HIV replication. However, co-administration of BD1047 with cocaine almost completely blocked the impact of cocaine on HIV replication (Fig. 4 ). Consistent with this finding, administration of PRE-084, a selective {sigma}-1 agonist, also increased HIV infection in the huPBL-SCID model, when compared to control animals (35.5±8.7% infected cells vs. 24.9±9.25%, P<0.05).



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  		Figure 4. A {sigma}-1 receptor antagonist blocks the effect of cocaine in HIV-infected huPBL-SCID mice. Animals were infected with the HIV reporter construct NFN-SX-HSAS and treated daily for 10 days with saline, cocaine alone (5 mg/kg), a selective {sigma}-1 antagonist (BD1047, 20 mg/kg), or the combination of BD1047 and cocaine. Peritoneal cells were harvested, and flow cytometry was used to assess the presence of HIV-infected human PBMC (percent of CD45+ cells expressing mCD24). Results represent mean values ± 1 SE for three experiments, each with four to five animals/group. *, P < 0.01, compared with control; {dagger}, P < 0.01, compared with cocaine treatment.

 
DISCUSSION

The impact of cocaine on the development and progression of HIV infection is difficult to assess by examining isolated cells in culture [1 , 2 , 12 , 13 ]. Furthermore, clinical correlations between cocaine abuse and HIV infection have been problematic as a result of concurrent behavioral issues, intermittent patterns of drug use, and polypharmacy [3 ]. To overcome some of these obstacles, we adapted the huPBL-SCID model so that animals could be exposed to cocaine under controlled conditions and the effects on viral pathogenesis examined in vivo. As reported previously [14 ], administration of cocaine alone had no significant impact on the viability or number of engrafted huPBL. However, HIV infectivity, viral replication, and the loss of CD4 cells were increased markedly when cocaine was administered in the presence of HIV, suggesting a dynamic interaction between drug exposure and viral infection.

There are several mechanisms by which cocaine might mediate these outcomes, including changes in host immunity, viral susceptibility, and the capacity to control viral replication [9 ]. HIV infection and cocaine have been reported to disrupt the HPA axis, leading to secondary effects on host immunity [6 , 7 , 26 ]. Consistent with other reports, we found that cocaine increased the production of ACTH and corticosterone in normal mice and in huPBL-SCID animals, which were not HIV-infected. However, the opposite effects were observed when cocaine was administered in combination with HIV, leading to a marked fall in ACTH and corticosterone. This paradoxical interaction between cocaine and HIV warrants further investigation but suggests that immunosuppressive levels of adrenal hormones do not play a major pathogenic role in the HIV-infected huPBL-SCID model.

The coordinated expression of CD4 in conjunction with specific chemokine coreceptors plays an essential role in the capacity for HIV to enter target cells [1 , 2 , 27 ]. Nair et al [28 ] found that expression for CCR5 mRNA was increased several fold when huPBL were activated in vitro in the presence of cocaine. Similar effects were recently observed when microglial cells were cultured with cocaine in vitro [21 ], encouraging us to examine coreceptor expression in our model. Exposure to cocaine significantly increased expression of CCR5 on normal PBL in vitro, in PBL recovered from control huPBL-SCID mice, and when cocaine and HIV were administered together in this same model. Furthermore, there was a temporal relationship between changes in CCR5 expression, which occurred early, and the stimulatory effects of cocaine on viral infection, which were evident later. However, not all of our findings are consistent with a causative relationship between these events. Although the impact of cocaine on CXCR4 expression was relatively modest, infection by an R4-tropic virus was still enhanced dramatically following cocaine administration. As it is clear that this magnitude of change in coreceptor expression cannot completely account for the 150-fold increase in systemic viral load, it would appear that several mechanisms are likely to be operative in this complex in vivo model.

Consistent with this theory, cocaine has also been shown to modulate the production of inflammatory and immunosuppressive cytokines [8 9 10 11 12 , 24 , 25 ]. Cocaine acts as a {sigma}-1 receptor ligand and, in this respect, can simultaneously suppress the production of immunoprotective cytokines such as interferon-{gamma} and promote the release of immunoregulatory factors including IL-10 and TGF-ß [11 , 24 ]. A similar pattern of cytokine changes occurs in response to HIV infection and contributes to its immunopathogenesis [25 ]. Although we have not yet confirmed the role of cocaine on cytokine production, it is clear from the {sigma}-1 ligand studies that this receptor pathway contributes to the effects observed in the huPBL-SCID model.

Overall, our findings suggest that cocaine can act as a cofactor and promote HIV infection in vivo. Mechanistically, the biological consequences of cocaine and HIV appear to interact at several levels with effects on host immunity, cell susceptibility, and {sigma}-1 receptors contributing to the observed changes in viral proliferation. However, the huPBL-SCID model has limitations that need to be considered. Implanted PBL react to the host environment and are chronically activated as a result of the graft-versus-host response [17 , 29 ]. Furthermore, the magnitude and extent of human engraftment are limited, with no substantial repopulation of lymphoid organs. As a result, our findings are limited to cells recovered from the peritoneal cavity. Other xenotransplant models in which the human immune system is more extensively reconstituted and integrated with that of the mouse need to be considered to address these issues [17 , 29 ]. The timing and sequence of events may also be important. In the present study, animals were first infected with HIV and then exposed to cocaine, or exposed to both simultaneously. However, alternative patterns of drug administration or differences in the sequence of HIV infection and cocaine warrant further investigation, as they could produce different results. Furthermore, whether the same interactions occur between cocaine and HIV in seropostive individuals and whether these changes impact on the course and pathogenesis of human HIV infection remain to be determined.

ACKNOWLEDGEMENTS

Funding was provided by NIH/NIDA Grant R01 DA08254. The Jonsson Comprehensive Cancer Center/UCLA and the UCLA AIDS Institute Center for AIDS Research (CFAR; NIH A128697) provided core facilities for flow cytometry (Flow Cytometric Core Laboratory), virological assessments (Virology Core Laboratory), and huPBL-SCID experimentation (Human/Mouse Chimera Core Laboratory).

Received April 24, 2005; revised September 1, 2005; accepted September 12, 2005.

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