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From the
Received for publication, February 14, 2000, and in revised form, July 6, 2000
All HIV-1 strains studied to date use CCR5,
CXCR4, or both receptors to enter cells. Simian immunodeficiency virus
(SIV) infection of non-human primates has served as a useful model for
understanding AIDS pathogenesis in humans. Research on several
genetically divergent SIV isolates has revealed that SIV uses CCR5, and
not CXCR4, for entry. CEM x174, a human lymphoid cell line, has been
routinely used to cultivate and maintain various SIV strains. However,
questions have arisen about how CEM x174, which reportedly was unable
to express detectable amounts of CCR5 transcripts, efficiently
supports the growth of SIV. In searching for an answer, we
resorted to a sensitive competitive reverse transcriptase-polymerase
chain reaction procedure in an attempt to detect as well as quantify the amount of CCR5 expression. Here we present our findings, which indicate that CEM x174 indeed expresses CCR5 and that the amount of
CCR5 is increased in cells pretreated with morphine. These results
correlate well with our previous observations that morphine treatment
causes CEM x174 cells to be more susceptible to SIV infection. Similar
morphine effect was not observed on CEM x174 cells infected with simian
retroviruses, which do not depend on CCR5 for entry. These findings
suggest a plausible mechanism whereby opiate drug users render
themselves more susceptible to HIV infection, thereby explaining the
vast prevalence of HIV infection among endemic drug use populations.
Opiate users constitute a large portion of the patient population
contracting AIDS. The feasibility and success of human studies have
always been hampered by the complexity of an individual's history of
intravenous drug use. Thus, rhesus monkey treated with opioids and
infected with simian immunodeficiency virus
(SIV)1 provides an excellent
animal model for studying drug abuse and AIDS under a controlled manner
(1). Using this animal system we have previously found that monkeys
dependent upon opioid administration and subsequently infected with SIV
will have a faster rate of viral replication in comparison with
drug-naive, virus-infected monkeys (1). CEM x174 is a human lymphoid
cell line (2) commonly used to co-cultivate SIV isolated from infected
monkeys. The syncytium formation of SIV-infected CEM x174 cells was
found to be significantly enhanced in the presence of morphine sulfate, with a concomitant increase in the activity of cellular reverse transcriptase and the expression of SIV p27 core antigen (3). Chemokines are small 6-10-kDa polypeptides that are synthesized in
response to infection and that function mainly as chemoattractants for
phagocytic cells, recruiting monocytes and neutrophils from the
vascular system to sites of infection (4). Further studies on the
effects of opioids on immune cells revealed that the addition of
opioids to the chemokines interleukin-8, RANTES, or
MIP-1 Chemokines act on receptors that belong to the G protein-coupled
receptor family whose members contain seven transmembrane domains (4).
Activation, desensitization, and resensitization of receptor proteins
are thought to involve the activity of receptor-specific G
protein-coupled receptor kinases and arrestins (8). Chemokines such as
RANTES, MIP-1 SIVmac239 replicates most efficiently in the human transformed lymphoid
cell line CEM x174 (15). In fact, this cell line is routinely used for
preparing high titered stocks of this virus (11). It has been reported
that SIVmac239 may use CCR5, BOB (GPR15), or BONZO (STRL33) as a
coreceptor for entry into various cell types, including 293T.CD4 (16),
3T3.CD4 (16), and Cf2Th.CD4 (17). In all of these studies, CCR5
was a preferred coreceptor over BOB or BONZO; cells with CCR5 produced
a greater amount of virus than cells with BOB or BONZO. However, CCR5
has not been identified in CEM x174 (11-13). In the course of a study
to quantify virus production in CEM x174 cells, we serendipitously
found that addition of morphine sulfate to CEM x174 cell cultures
significantly increases the replication of SIVmac239 (3). The present
study determines which coreceptor is responsible for the observed
morphine effect on CEM x174 cells.
Cell Line--
The CEM x174 cell line, a hybrid of the human B
cell line 721.174 and human T cell line CEM (2), was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated bovine calf serum, 2 mM L-glutamine, 25 mM
HEPES, and penicillin and streptomycin. GHOST Parental Cell Line and
GHOST Hi-5 (a GHOST cell transfectant with high CCR5 expression) were
maintained in Dulbecco's modified Eagle's medium supplemented
with 10% heat-inactivated fetal bovine serum, 500 µg/ml G418, 100 µg/ml hygromycin, and penicillin and streptomycin. For GHOST Hi-5,
the medium also contained 1 µg/ml puromycin. All cells were grown at
37 °C in a CO2 incubator.
Morphine or Naloxone Treatment--
CEM x174 cells in culture
were diluted 1:3 with fresh medium every 3-4 days. At the time of
dilution, morphine sulfate, naloxone HCl, or H2O (as
controls) was added, and incubation was continued for the indicated
time. When naloxone was used together with morphine in an experiment,
cells were first treated with naloxone HCl for 30 min at 37 °C
followed by morphine treatment.
Construction of Competitor Molecules--
A PCR fragment of CCR5
(1114 bp), BONZO (797 bp), or BOB (563 bp) was cloned into pCRII
(Invitrogen), yielding plasmid CCR5/pCRII, BOB/pCRII, or BONZO/pCRII,
which was then sequenced to prove identity (Fig.
1a). To construct competitor
molecules, small internal deletions were introduced into the inserts.
CCR5/pCRII was deleted 96 bp from bases 298 to 393 (inclusive), and
BONZO/pCRII was deleted 125 bp from bases 145 to 269 (inclusive) using
the Exo mung bean deletion kit (STRATAGENE). BOB/pCRII was deleted 63 bp from bases 404 to 466 (inclusive) after double digestion with
HincII and NdeI (Fig. 1a).
Competitive RT-PCR--
Total RNA was isolated from
opioid-treated or control CEM x174 cells using TRIZOL Reagent (Life
Technologies, Inc.). In both the reverse transcription and the PCR
steps, all the reaction reagents were prepared as master mixes and then
aliquoted to each tube to provide uniform reaction conditions and
minimize inter-tube variations. To confirm the detection range of
competitive RT-PCR, the relationship between the amount of cDNA
generated and the initial concentrations of total RNA used were
determined: reverse transcription was performed on 0.5, 1, and 2 µg
of total RNA, and the reaction was for 30 min at 42 °C using 200 units of Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Inc.). The reaction mixture also contained 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, 1 mM dithiothreitol, 10 units of RNase inhibitor (Promega), 1 mM of each dNTP,
and 7.5 nM random hexamers in a final volume of 20 µl.
For PCR amplication, 20 µl of PCR Master Mix containing 0.5 µM of each primer, 0.5 unit of Taq polymerase
(Promega), 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, and 1.5 mM MgCl2 were
dispensed into tubes. To the PCR Master Mix 2 µl of the reverse
transcription reaction product and various concentrations of competitor
molecules were added. The PCR was performed in a M. J. Research
Thermocycler PTC-200, using the following conditions: after an initial
3-min incubation at 95 °C, PCR amplification was carried out for 20 cycles (BOB), 27 cycles (BONZO), or 31 cycles (CCR5) at 95 °C for
40 s, 60 °C for 40 s, and 72 °C for 1 min. The last
primer extension step was conducted for 10 min. The primers for BOB
were 5'-CATCTGCTCTTTGGTGATG-3' and 5'-GTATGGCTTATCATCAATCAGC-3',
corresponding to bases 66-85 and 607-628 of the published BOB
sequence (16). The primers for BONZO were 5'-CAGGCATCCATGAATGGGTGT-3'
and 5'-CAAGGCCTATAACTGGAACATGCTG-3', corresponding to bases 269-289
and 1041-1065 of the published STRL33 (or BONZO) sequence (16). The
primers for CCR5 were 5'-GGTGGAACAAGATGGATTAT-3' and
5'-ATGTGCACAACTCTGACTG-3', corresponding to bases 44-63 and 1139-1157
of the published CCR5 sequence (13). PCR products separated on 2%
agarose gel were visualized by ethidium bromide staining and
photographed under UV illumination (Fig. 1b). The intensity
of the bands on the image from Eagle Eye II Still Video System
(STRATAGENE) was measured with the public domain NIH Image program
(developed at the U.S. National Institutes of Health). For
analyzing the results, the log of the ratio of amplified target to
competitor products was graphed as a function of the known amount of
competitor added to the PCR reaction (Fig. 1c). CA-Cricket Graph III (Computer Associates) was used for the regression analysis and calculation of x intercepts. When the log ratio equals
zero, the concentrations of the target (originally from the RT
reaction) and the competitor are equal (Fig. 1c). The amount
of cDNA synthesized from 1 µg of the initial RNA sample was
calculated from the graph (Fig. 1c).
Flow Cytometry and Western Blot Analysis--
The expression of
CCR5 on CEM x174 cells treated with or without morphine was evaluated
by FACS. Fluorescein-conjugated mouse monoclonal anti-human CCR5 (clone
45549.111) and fluorescein-labeled mouse IgG2B antibody (clone
20116.11, as a negative control) were obtained from R & D Systems.
Procedures for sample staining followed the manufacturer's
specifications. After the final wash, cells were fixed in 1%
paraformaldehyde before FACS analysis using FACScan (Becton Dickinson,
San Jose, CA). The method of Western blotting has been described
elsewhere (18). Rabbit anti-CCR5-CT for the Western analysis was
obtained from ANASPEC Incorp. (San Jose, CA).
Infection of CEM x174 cells with SIVmac239 or SRV--
Methods
for the experimental infection of CEM x174 cells with SIVmac239 or SRV
and the subsequent assay of the reverse transcriptase activity of the
infected cultures were detailed elsewhere (3, 19). SIVmac239 was
propagated and titered in CEM x174 cells (3), and titers for SRV
serotypes 1-3 were determined by the Raji cell infectivity assay as
described previously (19). The Raji cell line was obtained from
American Type Culture Collection.
Fig. 2a is a
representative figure of the results from an RT-PCR analysis of CEM
x174 CCR5. Using primer sequences corresponding to bases 44-63 and
1139-1157 of the published human CCR5 sequence (13), CEM x174 cells
synthesized a 1114-bp segment (lane 2) that was identical in
size to the segment synthesized by GHOST Hi-5 (a GHOST cell
transfectant encoding CCR5, lane 4) and that was not found
in GHOST Parental cells (lane 6). Using primer pairs corresponding to bases 66-85 and 607-628 of the published BOB sequence (16) and bases 269-289 and 1041-1065 of the published BONZO
sequence (16), we also found that CEM x174 cells synthesized cDNA
segments of 563 and 797 bp, respectively, in length (data not shown).
The cDNA synthesized in each case was sequenced to prove identity.
It was found that CEM x174 cells express receptors with gene sequences
identical to the published human CCR5, BOB, and BONZO sequences (13,
16) (data not shown).
To further establish that the CCR5 transcripts detected in CEM x174
cells are translated into receptor proteins, we performed both flow
cytometry (Fig. 2b) and Western blot analysis (see Fig. 4b, inset) using fluorescein-conjugated mouse
monoclonal anti-human CCR5 (for flow cytometry) and rabbit polyclonal
anti-human CCR5 (for Western blot). Both procedures confirmed the
presence of CCR5 molecules on CEM x174 cells (Figs. 2b and
4b). Therefore, we demonstrated that in addition to BOB and
BONZO, CEM x174 cells indeed express significant amounts of CCR5, the
major coreceptor for SIVmac entry.
To determine coreceptor densities on CEM x174 cells, plasmids
containing segments of CCR5, BOB, and BONZO, and plasmids containing CCR5, BOB, and BONZO segments with 96-bp (CCR5), 63-bp (BOB), and
125-bp (BONZO) deletions were constructed (see "Experimental Procedures" and Fig. 1a). Plasmids with deleted segments
were used in quantitative RT-PCR as external controls for quantifying the expression of chemokine receptor genes in CEM x174. To select a
cycle number to achieve an exponential amplification phase, the
densities of amplified fragments from different cyclical amplifications were measured after gel electrophoresis and ethidium bromide staining. Cycle numbers 31, 20, and 27 were selected for experiments on CCR5,
BOB, and BONZO, respectively (data not shown). To confirm the detection
range of competitive RT-PCR, the relationship between the amount of
cDNA generated and the initial concentrations of total RNA used
were determined. For each RT reaction of 0.5, 1.0, or 2.0 µg of total
RNA, five different concentrations of competitors were used for PCR
amplification (Fig. 1b). The results showed that the amount
of cDNA generated was in proportion to the amount of initial RNA
used (Fig. 1c). To investigate the effect of morphine treatment on the gene expression of CCR5, BOB, and BONZO, the amount of
cDNA amplified by competitive RT-PCR from cells treated with
morphine sulfate was compared with that of untreated cells. Morphine
treatment, if used, was either 10 µM or 10 nM; these are physiological morphine concentrations in
morphine-dependent animals (20). Samples were taken 0, 12, 24, and 36 h post-morphine treatment for analysis. It was found that
after the dilution of the cultures at time 0 (see "Experimental
Procedures"), the cells synthesized an increasing amount of CCR5
(Fig. 3a) or BOB (Fig. 3b), with the amount reaching a plateau 12-24 h
post-dilution. However, it was also found that in comparison with the
control cells at each time point, 10 µM morphine
treatment increased CCR5 expression 207% by 12 h post-treatment,
whereas 10 nM morphine treatment induced a 240% increase
of CCR5 by 24 h post-treatment (Fig. 3a). On the
contrary, morphine treatment did not affect the expression of BOB (Fig.
3b) or BONZO (Fig. 3c) in CEM x174 cells. Further
experiments showed that the morphine-induced increase in CCR5
expression correlated with the amount of CCR5 proteins on the cell
surface, as determined by flow cytometry (Fig.
4a) as well as Western blot
analysis (Fig. 4b) and that the effect was opioid
receptor-mediated, because it could be completely abolished when
cells were pretreated with naloxone, a µ opioid receptor antagonist
(Fig. 5).
Morphine Induces Gene Expression of CCR5 in Human CEM x174
Lymphocytes*
,,
Department of Medical Pharmacology and
Toxicology, the § Section of Molecular and Cellular
Biology, and the ¶ Department of Medical Microbiology and
Immunology, University of California, Davis, California 95616
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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INTRODUCTION
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reduces the ability of monkey neutrophils and monocytes to
migrate toward these chemokines (5). The reaction occurs
instantaneously, without the inclusion of a cell opioid preincubation
step (6). This suggests that the presence of opioids during SIV/HIV
infection immediately disrupts the body's first line of defense
against harmful external pathogens by disrupting the chemotaxis ability of immune cells toward harmful pathogens. Such observations may provide
an indirect mechanism to explain why primates or humans dependent upon
intravenous drug administration have a higher probability of developing
into a full-blown disease than non-drug users when exposed to a viral
challenge (1, 7).
, and MIP-1 have been implicated in the pathogenesis of HIV disease; they may be selectively secreted from
infected individuals and induce inhibition of different strains of
HIV-1, HIV-2, and SIV (9). It was later found that chemokine receptors
(especially CCR5 and CXCR4) are coreceptors for HIV or SIV entry
(10-13). However, regions in CCR5 or CXCR4 required for ligand
(chemokine) binding and coreceptor activity are not identical and only
partially overlap (10). It was further established that in addition to
blocking viral entry through steric hindrance, cognate ligand
interaction with chemokine receptors has been shown to result in
receptor down-regulation for CCR5 and CXCR4 (14).
EXPERIMENTAL PROCEDURES
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View larger version (30K):
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Fig. 1.
Diagram illustration of competitive
RT-PCR. a, construction of competitor molecules. A
BamHI-EcoRV fragment of CCR5 (1114 bp),
EcoRI-EcoRI fragment of BONZO (797 bp), or
EcoRI-EcoRI fragment of BOB (563 bp) was first
cloned into the plasmid pCRII. Competitor molecules for RT/PCR analysis
were constructed by introducing internal base deletions (hatched
boxes) into the fragments. The numbers shown on the diagram
indicate where the bases were deleted, assuming the first base of each
fragment is number 1. b, gel analysis. PCR products from
each RT-PCR reaction containing native (originally from 2 µl of RT
reaction) and one of the five different concentrations of competitor
molecules were separated on a 2% agarose gel, visualized by ethidium
bromide staining, and photographed under UV illumination. Three
concentrations (0.5, 1, or 2 µg) of RNA were used in the initial
samples. c, calculation of cDNA synthesized. The
intensity of each band of the gel portrayed on an Eagle Eye Still video
system was measured with an NIH Image program as described under
"Experimental Procedures." The ratio of native versus
competitor of each reaction was obtained from the intensities of the
gel bands, and the logarithm of the ratio was graphed against the
logarithm of the concentration of the competitor added to the reaction.
CA-Criket Graph III was used for the regression analysis and
calculation of x intercepts at which the log ratios equaled
zero. c is a representative figure of the experiment, drawn
from data obtained from a RT-PCR analysis of BONZO cDNA synthesized
from 0.5 µg( 
), 1.0 µg (
), or 2.0 µg (
)
RNA.
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View larger version (31K):
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Fig. 2.
CCR5 on CEM x174 as analyzed by RT-PCR
(a) or flow cytometry (b).
a, CCR5 mRNA from CEM x174 (lanes 2 and
3), GHOST Hi-5 (lanes 4 and 5), or
GHOST Parental cells (lanes 6 and 7) was
determined by RT-PCR. Lanes 3, 5, and
7, RT was omitted in the reactions. Lane 1,
100-bp DNA ladder. b, cells were stained with either
fluorescein isothiocyanate-conjugated mouse anti-human CCR5
monoclonal antibody (upper panel) or
carboxyfluorescein-conjugated mouse IgG2B isotype control
(lower panel) and subjected to FACS analysis.
FL1-H, green florescence; FL2-H, red florescence
(auto florescence measured in "red" channel). The data were
reproducible in three independent experiments.
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Fig. 3.
Effect of morphine treatment on SIV
coreceptor expression. The amount of CCR5 (a), BOB
(b), or BONZO (c) expressed in CEM x174 cells was
determined by the competitive RT-PCR after treatment of the cells with
10 µM (solid column) or 10 nM
(column with wavy lines) morphine sulfate for the indicated
time. Control (open column), H2O-treated cells.
The data were reproducible in at least four independent
experiments.
View larger version (18K):
[in a new window]
Fig. 4.
Effect of morphine treatment on cell surface
CCR5. a, CCR5 expressed on cell surface of CEM x174 cells
was analyzed by FACS after treatment of the cells with 10 µM morphine sulfate (solid column) or
H2O (open column). The y axis
represents the percentage of total cells being CCR5-positive.
b, effect of morphine treatment on the expression of CCR5
protein by CEM x174 cells was analyzed by Western blotting. Protein
isolated from CEM x174 cells was separated on a 10% SDS/polyacrylamide
gel, blotted onto a nitrocellulose filter, and incubated with rabbit
anti-CCR5-CT. Morphine treatment was for 24 h. The data were
reproducible in three independent experiments.
View larger version (21K):
[in a new window]
Fig. 5.
Effect of naloxone on morphine-induced CCR5
expression. The amount of CCR5 expressed in CEM x174 cells was
determined by the competitive RT-PCR after treatment of the cells for
12 h with 10 nM morphine, 10 nM naloxone
or a combination of naloxone and morphine (10 nM each). The
data were reproducible in three independent experiments.
To correlate the observed morphine effect on CCR5 up-regulation
with viral infectivity, CEM x174 cells with or without morphine treatment were assayed for viral susceptibility after infection with
SIVmac239 or with SRV in parallel experiments. SRV infect B cells, T
cells (CD4+ and CD8+), macrophages, and epithelial cells. Like
SIVmac239, SRV cause an acquired immunodeficiency syndrome in monkeys
(19). The receptor for SRV is not CCR5 but a neutral amino acid
transporter that has been identified and cloned (21). In this study CEM
x174 cells were treated with morphine sulfate at 0, 0.4, 4, or 10 µM and infected with SIVmac239 or three serotypes of SRV
(SRV-1, SRV-2, and SRV-3). The cells were observed for syncytia
formation and assayed for viral reverse transcriptase activities. Fig.
6 shows that on day 6 post-infection, syncytia appeared in SIVmac239-infected cultures and the number of
syncytia formation increased with increasing concentrations of morphine
in the culture (Fig. 6A and Table
I). On the contrary, none of the
SRV-infected cultures showed signs of syncytia even in the presence of
morphine treatment (Fig. 6B). Results of RT assay of the
infected cultures indicated a gradual increase of the enzyme
activity with time of infection in all cultures (Table II); however, morphine significantly
enhanced RT activities of SIVmac239-infected cells but not SRV-infected
cells (Table II). For SRV-infected CEM x174 cells, we instead observed
a slight decrease in RT activities upon morphine exposure (Table II).
Further incubation of SRV-infected cells up to 12 days in the presence of morphine had little effect on, or slightly decreased, viral infectivity, as again shown by RT assays (Table
III).
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Immune cells have been shown to express brain-like opioid
receptors (22-24). Similar to chemokine receptors, opioid receptors are also G protein coupled, seven-transmembrane domain receptors (25).
Human CEM x174 lymphocytes possess all three subtypes of opioid
receptors, µ, 
, and 
(22-24). The current study shows that
activation of opioid receptors of human lymphocytes, probably of the µ subtype, by morphine up-regulates the expression of the chemokine
receptor CCR5. The downstream molecular mechanisms induced by receptor
activation through which morphine affects CCR5 expression awaits
further investigation. Morphine, which inhibits chemokine-induced chemotaxis, nevertheless does not perturb chemokine binding to CCR5
(26). It is therefore attractive to propose that morphine, by binding
to its own cell surface receptor, initiates a series of G
protein-coupled signal transduction pathways (18) that thereby
hetero-sensitize (or up-regulate) CCR5. Many facts support this
proposal. For instance, morphine has been shown to modulate the
expression of other cellular proteins that may induce CCR5 expression.
Specifically, morphine reportedly modulates the cellular activation of
NFB and TNF- in macrophages (27) and interleukin-2 in lymphocytes
(28); activation of NFB, TNF-, and interleukin-2 has been found
to up-regulate CCR5 expression (10, 29). Alternatively, morphine may
up-regulate CCR5 by inhibiting chemokine synthesis and thus chemotaxis.
Under certain conditions, opiates such as morphine as well as opioid
peptides may down-regulate cytokine synthesis and release (30, 31).
Chemokines (chemotactic cytokines) such as RANTES, MIP-1, and
MIP-1 may induce receptor internalization and decrease cell surface
expression of CCR5 (14, 32), which ultimately contributes to anti-HIV-1
activity (9, 14). Thus, one yet-to-be-proved mechanism of
morphine-mediated up-regulation of CCR5 is through the down-regulation
of chemokine synthesis.
SRV are endemic in wild macaques of India and Indonesia and in captive macaques important for medical research (33). Five distinct neutralization serotypes (SRV1-SRV5) have been described, of which three have been molecularly cloned (SRV1-SRV3). The present study shows that unlike SIVmac239 infection, morphine treatment will not increase the infectivity of SRV 1-3 (Fig. 6). Instead, morphine may induce a "protective" effect against SRV infection (Tables II and III). Similar phenomenon has been reported for murine Friend retrovirus infection (34). Like SRV, the Friend retrovirus infection model has been described as relevant to several aspects of AIDS; in particular, there are significant changes in immune function similar to those observed in HIV infection. The receptor for such virus is again not CCR5 but murine cationic amino acid transporter 1 (35), and morphine was found to attenuate the pathological manifestations of the virus in infected animals (34). Therefore, it appears that morphine-induced CCR5 in human lymphocytes facilitates only SIVmac239 infection, for which CCR5 is a co-receptor for viral entry.
This study shows for the first time that lymphocytes express CCR5
at higher levels when treated with morphine sulfate. Previous studies
that were unable to detect CCR5 in CEM x174 may have been due to the
low levels of CCR5 expression inherent in cells; this level is below
the threshold of many conventional detection methods. These low levels
of CCR5 expression are significant nevertheless, especially when these
low levels of CCR5 expression are augmented by morphine treatment. In
addition, recent studies have shown that in cells with large amounts of
CD4, a low trace of CCR5 was sufficient for susceptibility to virus
infection (36). Therefore, in cells with low levels of CCR5 expression,
morphine treatment may bring CCR5 concentrations above threshold levels
for maximal infection. Morphine does not affect the gene expression of
BOB or BONZO (Fig. 3). In this regard, the induction of the chemokine receptor CCR5 gene expression by morphine may provide a mechanism by
which morphine sulfate enhances HIV/SIV infection and hence exacerbates
the Simian AIDS or AIDS pathogenesis.
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ACKNOWLEDGEMENTS |
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The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health: GHOST Parental Cell Line and GHOST Hi-5 (with high CCR5 expression), from Dr. Vineet N. KewalRamani and Dr. Dan R. Littman. We also thank Dr. Vineet N. KewalRamani for useful advice.
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FOOTNOTES |
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* This work was supported by Grants DA 05901 and DA 10433 from the NIDA, National Institutes of Health and by American Cancer Society Grant RPG-99-338-01-CIM.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Medical
Pharmacology and Toxicology, School of Medicine, University of California, Davis, CA 95616. Tel.: 530-752-7713; Fax:
530-752-7710; E-mail: rychuang@ucdavis.edu.
Published, JBC Papers in Press, July 7, 2000, DOI 10.1074/jbc.M001269200
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ABBREVIATIONS |
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The abbreviations used are:
SIV, simian
immunodeficiency virus;
RANTES, regulated upon activation, normal T
expressed and secreted;
MIP-1, macrophage inflammatory protein 1;
HIV, human immunodeficiency virus;
PCR, polymerase chain reaction;
RT, reverse transcriptase;
bp, base pair(s);
FACS, fluorescence-activated cell sorter;
SRV, simian type D
retrovirus.
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