Morphine inhibits CD8?T cell-mediated, noncytolytic, anti-HIV
activity in latently infected immune cells
Xu Wang,* Ning Tan,†Steven D. Douglas,* Ting Zhang,* Yan-Jian Wang,* and Wen-Zhe Ho*,1
*Division of Allergy and Immunology, Joseph Stokes, Jr., Research Institute at The Children’s Hospital of
Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine; and†Department of
Medicine, The People’s Hospital of Guangdong Province, Guangzhou, People’s Republic of China
function of human immune cells and are a possible
cofactor in the immunopathogenesis of human im-
munodeficiency virus (HIV) disease. We investi-
gated the impact of morphine on CD8?T cell-
mediated, noncytotoxic, anti-HIV activity in la-
tently infected human immune cells. Morphine
inhibited the noncytotoxic, anti-HIV activity of
CD8?T cells in HIV latently infected cells (U1 and
J1.1). Naltrexone abrogated the morphine-medi-
ated, inhibitory effect on the noncytotoxic, anti-
HIV activity of CD8?T cells. Interferon-? (IFN-?),
a potent antiviral cytokine produced by CD8?T
cells, was partially responsible for CD8?T cell-
mediated, noncytotoxic, anti-HIV activity. The
anti-HIV activity of IFN-? was also compromised
by morphine treatment. Further, morphine atten-
uated CD8?T cell-mediated suppression of the
HIV long-terminal repeat promoter activation.
Morphine also inhibited CD8?T cell-induced ex-
pression of the signal transducer and activator of
transcription-1, an important transcriptional fac-
tor in the IFN signaling pathway. These data pro-
vide additional evidence to support the notion that
opioids play a role in impairing the anti-HIV func-
tion of the immune system. J. Leukoc. Biol. 78:
Opiates have profound effects on the
Key Words: IFN-? ? PBMC ? U1 cells ? J1.1 cells
Injection drug use (IDU) is a significant risk for acquiring
human immunodeficiency virus (HIV) infection  and con-
tributes to the spread of the virus . Approximately 30% of
patients with HIV infection and AIDS have a history of IDU,
which frequently involves opiate abuse. Opioids have profound
effects on the functions of human immune cells [3–5] and are
implicated as a cofactor in the immunopathogenesis of HIV
disease [1, 2, 5, 6]. In the present study, we examined the
impact of opioids on the anti-HIV function of CD8?T cells,
which have important direct and indirect antiviral effects on
the control of HIV replication. CD8?T cells suppress HIV
replication in latently infected, immune cells . Although the
direct killing of virus-infected cells by antigen-specific cyto-
toxic T lymphocytes is the dominant mechanism of virus sup-
pression, a noncytotoxic, soluble factor(s) released by the
CD8?T cell also plays a critical role in inhibiting HIV
replication [8–11]. As opioid abuse has the ability to impair
the host defense mechanisms against HIV infection, we spec-
ulated that morphine, the active metabolite of heroin, compro-
mises CD8?T cell-mediated suppression of HIV replication in
latently infected human immune cells.
MATERIALS AND METHODS
The promonocytic (U1) and T (J1.1 and 1G5) cell lines were obtained through
the AIDS Research and Reference Reagent Program, Division of AIDS, Na-
tional Institutes of Health (NIH; Bethesda, MD). U1 is a cloned cell line
derived from U937 cells surviving acute HIV infection . J1.1 is a subclone
of the Jurkat E6.1 (J/W) T cell line, which is latently infected with HIV .
1G5 is a Juckat T cell line that harbors two copies of a stable transfected
plasmid, containing the luciferase reporter gene downstream of the HIV
long-terminal repeat (LTR) . J1.1, U1, and 1G5 cells were cultured as
Morphine sulfate was obtained from Elkins-Sinn, Inc. (Cherry Hill, NJ). Tumor
necrosis factor-? (TNF-?), mouse monoclonal antibody against CD3 receptor,
mouse monoclonal anti-human interferon-? (IFN-?) antibody, and recombinant
IFN-? were purchased from R&D Systems (Minneapolis, MN). Naltrexone and
rabbit polyclonal antibody against actin were obtained from Sigma Chemical
Co. (St. Louis, MO). Rabbit polyclonal antibody against signal transducer and
activator of transcription-1 (STAT-1) was obtained from Santa Cruz Biotech-
nology (CA). Horseradish peroxidase-conjugated goat anti-rabbit immunoglob-
ulin G was purchased from Jackson ImmunoResearch Labs (West Grove, PA).
Preparation of CD8?T cell culture
Peripheral blood samples were obtained from five asymptomatic, HIV-infected,
adult subjects with CD4?T cell counts ranging from 400 to 1200/mm3. The
Institutional Review Board of the Children's Hospital of Philadelphia (PA)
approved this investigation. Informed consent was obtained from the subjects.
Peripheral blood mononuclear cells (PBMC) were processed as described
previously  using lymphocyte separation medium (Amersham Pharmacia
Biotech, Uppsala, Sweden). PBMC were subjected to CD8?T lymphocyte
1Correspondence: Division of Allergy and Immunology, The Children’s
Hospital of Philadelphia, 34th Street & Civic Center Boulevard, Philadelphia,
PA 19104. E-mail: firstname.lastname@example.org
Received March 28, 2005; revised May 26, 2005; May 27, 2005; doi:
0741-5400/05/0078-0001 © Society for Leukocyte Biology
Journal of Leukocyte Biology
Volume 78, September 2005
Uncorrected Version. Published on July 6, 2005 as DOI:10.1189/jlb.0305167
Copyright 2005 by The Society for Leukocyte Biology.
purification using magnetic cell sorter CD8 microbeads, according to the
manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). CD8?T cell cul-
ture SN were prepared as follows: Purified CD8?T lymphocytes were culti-
vated in a 48-well plate precoated with the anti-CD3 antibody (3 ?g/ml) in
RPMI-1640 media containing interleukin-2 (50 U/ml) for 72 h. SN were
collected from the cell cultures and stored at –70°C.
Treatment with morphine, naltrexone, and/or
U1 and J1.1 cells plated in triplicate (2?104cells/well) in a 96-well culture
plate were incubated with or without morphine (10?10–10?6M) for 2 h before
the addition of CD8?SN (25%, v/v) for 2 h. For the experiments using
naltrexone, the cells were treated with naltrexone (10?8M), first for 30 min
prior to the addition of morphine and/or CD8?SN as stated above. Naltrexone
was not washed out before the addition of morphine. The latent HIV in these
cells was activated by treatment with TNF-? (2 ng/ml). HIV reverse transcrip-
tase (RT) activity was measured in culture SN collected 48 h post-TNF-?
treatment. 1G5 cells in 48-well culture plates (2?105cells/well) were treated
for 2 h with or without morphine (10?8M) before the addition of CD8?SN
(25% v/v) for 16 h. The lysed cells were subjected to luciferase assay 8 h after
TNF-? (10 ng/ml) stimulation. CD8?T cell-depleted PBMC were stimulated
with phytohemagglutinin (PHA) for 72 h and then seeded into 48-well plates
(106cells/well), precoated anti-CD3 antibody (1 ?g/ml). The cells were incu-
bated at 37°C with or without morphine for 2 h before the addition of CD8?SN
(25%, v/v). HIV RT activity was measured in culture SN collected at Day 9
HIV RT assay and luciferase assay
HIV RT activity was determined based on the technique of Willey et al. 
with modification . The luciferase activity was determined using a lucif-
erase assay kit (Promega Biotec, Madsion, WI), according to the manufactur-
Western blot analysis
U1 and J1.1 cells were washed with phosphate-buffered saline and lysed by
lysis buffer (Promega Biotec). The cell lysates were collected, and protein
concentration was determined using the Bio-Rad dendritic cell protein assay
kit (Bio-Rad Laboratories, Hercules, CA). Approximately, 10 ug protein was
resuspended in NuPAGE lithium dodecyl sulfate sample buffer (Novex, San
Diego, CA) and heated for 3 min at 100°C, and the equal amounts of protein
for each sample were analyzed by Western blot as described previously .
All variables were tested in triplicate, and all experiments were repeated at
least three times. Triplicate wells had variability of less than 15%. One-way
ANOVA was used to test for difference in means, and the post-hoc t-test was
used for comparisons. Differences were considered significant if P ? 0.05.
To determine whether morphine has the ability to inhibit the
anti-HIV activity of CD8?SN, PBMC from HIV-infected sub-
jects, U1 and J1.1 cells, were treated with CD8?SN in the
presence or absence of morphine. The anti-HIV activity of
and PBMC (Fig. 1). As U1 and J1.1 cells express ?-opioid
receptors (data not shown), we speculated that the morphine
action on the anti-HIV activity of CD8?SN is mediated
through the ?-opioid receptor. Thus, we determined whether
naltrexone, a pan-opioid receptor antagonist, has the ability to
block the morphine action. Naltrexone abrogated the inhibitory
effect of morphine on the anti-HIV activity of CD8?SN in U1,
J1.1, and PBMC (Fig. 1, B and C), and the addition of naltrex-
?SN was impaired in morphine-treated U1 and J1.1 cells
one alone to the cell cultures had no impact on HIV replication
(Fig. 1, B and C).
As CD8?T cells are the primary source of IFN-?, a potent,
antiviral cytokine , we determined whether IFN-? contrib-
utes, at least in part, to the anti-HIV activity of CD8?SN.
Antibody to IFN-? partially reversed the anti-HIV activity of
CD8?SN (Fig. 2A). To further confirm that IFN-? plays a role
in CD8?SN-mediated inhibition of HIV replication, we used
exogenous IFN-? to treat U1 and J1.1 cells. IFN-? significantly
inhibited (up to 80%) TNF-?-induced HIV replication (Fig.
2A). The activation of HIV promoter LTR is responsible for the
virus replication in the latently infected cells. We speculated
that morphine-mediated inhibition of the anti-HIV activity of
CD8?T cells is associated with its ability to activate HIV
promoter LTR. We used 1G5 cells , which contain a stably
integrated HIV LTR linked to a luciferase gene, to examine
this speculation. Although morphine had no direct effect on
TNF-?-induced HIV LTR activation (Fig. 2B), morphine treat-
ment inhibited CD8?SN-mediated suppression of HIV LTR
activation (Fig. 2B). Morphine also reduced exogenous IFN-?-
mediated suppression of HIV LTR activation (Fig. 2B). The
antibody to IFN-? partially neutralized the inhibitory effect of
CD8?SN-mediated suppression of HIV LTR activation (Fig.
As soluble factor(s) secreted from CD8?T lymphocytes
inhibit HIV replication through activation of STAT-1, a critical
transcriptional factor in IFN signaling pathways , we in-
vestigated whether morphine has the ability to reduced CD8?
SN-mediated activation of STAT-1. CD8?SN activated
STAT-1 expression in U1 and J1.1 cells (Fig. 3). This CD8?
SN-mediated activation of STAT-1, however, was inhibited by
morphine (Fig. 3).
Opioids have a cofactor role in the immunopathogenesis of HIV
disease, as demonstrated by in vitro [15, 21–23] and in vivo [2,
24, 25] studies. In the present study, we focused on the impact
of morphine on the noncytotoxic, anti-HIV function of CD8?T
cells, as CD8?T cells have a critical role in suppressing HIV
replication in latently infected, immune cells . The noncy-
totoxic, anti-HIV activity is demonstrated using activated
CD8?T cell culture SN . We demonstrated that morphine
reduced the anti-HIV activity of CD8?SN in the latently
infected immune cells. This action of morphine is specific, as
naltrexone abrogated the morphine-mediated, inhibitory effect
on the noncytotoxic, anti-HIV activity of CD8?SN.
IFN-? has pleiotropic effects on HIV replication in macro-
phage and T cell lines [27–29]. The loss of CD4?T cells is
associated with a deficiency of IFN-? production by CD8?T
cells in HIV-infected subjects with advanced disease .
These findings prompted us to investigate whether morphine
has the ability to suppress the anti-HIV activity of CD8 T
cell-produced IFN-? in latently infected immune cells. We
showed that the antibody to IFN-? partially neutralized CD8?
SN anti-HIV ability (Fig. 2). We also observed that the anti-
body to IFN-? partially reversed the inhibitory effect of CD8?
SN on HIV LTR activation (Fig. 2). This finding is in agree-
2Journal of Leukocyte Biology
Volume 78, September 2005
ment with the study by Chang et al.  showing that the
antibodies to IFN-? and IFN-?, but not the antibody to IFN-?,
modestly reversed the CD8 T cell-mediated inhibition of HIV
LTR activation in Hela cells. The role of CD8?T cell-released
IFN-? in suppressing HIV and its promoter activation was
further supported by our data, showing that exogenous IFN-?
inhibited TNF-mediated HIV and HIV LTR activation in U1,
J1.1, and 1G5 cells. Collectively, these observations support
the studies showing that CD8?T cell-released, soluble factors
inhibit HIV RNA transcription, particularly at the step of HIV
LTR-driven gene expression [31–33]. The antagonizing effect
of morphine on CD8?SN-mediated inhibition of HIV and LTR
is not a result of its direct effect on the activation of the HIV
LTR promoter, as morphine alone had little impact on the
promoter (Fig. 2). The lack of a direct effect of morphine on
HIV and its promoter LTR in our cell system provided us with
the opportunity to examine whether morphine has a negative
impact on CD8?T cell-mediated, noncytotoxic, anti-HIV ac-
tivity on HIV replication. Our findings, however, are contra-
dictory to the report showing that morphine directly transacti-
vated the HIV LTR promoter in human neuroblastoma cells
. The discrepancies between the observations of Squinto et
al  and our study may be attributable to different target
cells used in the studies. Morphine also inhibits CD8?T
cell-induced expression of the STAT-1, which constitutes a
mechanism responsible for morphine’s inhibitory action on the
noncytotoxic, anti-HIV activity of CD8?T cells (Fig. 3).
Taken together, our study provides compelling evidence that
morphine has the ability to impair the noncytotoxic, anti-HIV
activity of CD8?T cells. These findings, in conjunction with our
earlier reports [15, 23] showing that morphine enhances HIV
infection of human immune cells, support the notion that opiates
indeed have a cofactor role in promoting HIV disease. It is most
important that understanding the impact of opiates on the host
immune system against HIV infection is of great interest for
developing future strategies of controlling the virus and reconsti-
tuting the immune system in HIV-infected opiate abusers.
This work was supported by NIH Grants DA 12815, DA16022
(to W-Z. H.), and MH 49981, AA13547 (to S. D. D). X. W. and
N. T. contributed equally to this study.
Fig. 1. Effect of morphine on the anti-HIV activity of CD8?SN in U1 and
J1.1 cells and latently infected PBMC. (A) A dose-dependent effect of mor-
phine on CD8?SN-mediated, anti-HIV activity. U1 and J1 cells were incu-
bated with or without morphine at indicated concentrations for 2 h before the
treatment with CD8?SN (25%, v/v) for an additional 2 h. TNF-? (2 ng/ml) was
then added to the cultures to induce HIV replication. SN were collected for
measurement of RT activity 48 h post-TNF-? induction. The data are ex-
pressed as HIV RT activity relative (percent) to untreated control, which is
defined as 100%. (B) Effect of morphine and/or naltrexone on CD8?SN-
mediated, anti-HIV activity in U1 and J1.1 cells, which were incubated with
or without naltrexone (10?8M) for 30 min and then morphine (10?8M) for 2 h
before the addition of CD8?SN (25%, v/v) to the cultures. TNF-? (2 ng/ml)
was added to the cultures to induce HIV replication 2 h after treatment with
CD8?SN, and SN were collected for measurement of HIV RT activity 48 h
after TNF-? stimulation. The data are expressed as HIV RT activity relative
(percent) to untreated control, which is defined as 100%. (C) Effect of mor-
phine and/or naltrexone on CD8?SN-mediated anti-HIV activity in PBMC
from HIV-infected patients. CD8?T cell-depleted PBMC were stimulated with
1% PHA (v/v) for 72 h. Cells then were incubated with or without CD8?SN,
morphine (10?8M), and/or naltrexone (10?8M) as indicated. Cultures were
refed with fresh medium containing the indicated reagent every 3 days. Day 9
culture SN were collected for measurement of RT activity. The data are
expressed as HIV RT activity relative (percent) to untreated control, which is
defined as 100%. The results (A and B) shown are the mean ? SD of HIV RT
activity in triplicate cultures, representative of five independent experiments.
The result (C) shown is the mean ? SD of HIV RT activity in triplicate cultures,
representative of three independent experiments using PBMC from three
different HIV-infected subjects. ?, In the presence; –, in the absence.
Wang et al.
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Fig. 2. Effect of morphine on CD8?SN- or IFN-?-mediated inhibition of
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TNF-? (10 ng/ml) 16 h after treatment with CD8?SN or IFN-?. Cell lysates
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additional 2 h. TNF-? (2 ng/ml) was then added to the cell cultures. Cellular
proteins were extracted from the cells 48 h post-TNF-? treatment and then
subjected to immunobloting assay using the antibodies to STAT-1 and actin.
The arrows indicate the position of STAT-1 (91 kD) or actin (42 kD). The insets
below the panels show the signal intensities [density scan unit (DSU)] of
protein bands of the representative blot, expressed as densitometry scanning
units. The results shown are representative of three independent experiments.
CD8 SN, CD8?SN.
4 Journal of Leukocyte Biology
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Morphine inhibits CD8?T cell5