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INTRODUCTION |
Long term opioid usage induces immunosuppression by modulating a
spectrum of immune activities, such as inhibition of lymphocyte proliferation, decreased production of interferon
, interleukin 2, and interleukin 4 by activated lymphocytes, enhanced synthesis of
tumor necrosis factor
and interleukin 1
in activated macrophage, enhanced production of MCP-1, RANTES (regulated on activation normal
T-cell expressed and secreted), and IP-10 in peripheral blood
mononuclear cells, and reduction in antibody production (1-5). The
fact that accelerated human immunodeficiency virus pathogenesis
occurred in patients who abuse heroin is consistent with the
immunosuppressive activity of the opioids (6). Although previous
studies indicate that opioids may regulate immune responses through
their action on central nervous system or sympathetic nervous system,
the discovery of opioid receptors on peripheral blood mononuclear cells
suggested that these opioid receptors could directly modify the
response of proinflammatory receptors (2-4, 7). Data from knock-out
mice and in vitro studies suggest that such suppression is
mediated largely by µ,
, and
opioid receptors. Opioids
receptors, members of the seven-transmembrane receptor family, perform
their function by coupling to heterotrimeric Gi/o proteins.
Their activation leads to inhibition of adenylyl cyclase by G
,
inhibition of voltage-dependent calcium channels, and
activation of G protein-coupled inwardly rectifying K+
channels by G
.
In cells expressing multiple G protein-coupled receptors
(GPCRs),1 prolonged
activation of one receptor has been shown to result in the
down-regulation of other GPCR through a process called heterologous
desensitization. Accumulating data have demonstrated that heterologous
down-regulation of GPCR is mediated by protein kinase A and protein
kinase C (PKC) (8, 9, 11). Calcium flux, a potent activator of PKC, has
been considered essential for heterologous desensitization of chemokine
receptors in peripheral blood mononuclear cells (8-10). Interaction of
chemokine receptors with their ligands activates Gi
proteins, induces the production of DAG, and releases Ca2+,
followed by activation and translocation of PKC to the plasma membrane.
This process is associated with desensitization of other GPCRs in the
same cell by phosphorylation of their consensus sites (10, 13).
The family of PKC consists of more than 12 isozymes, and each of them
exhibits a unique pattern of tissue distribution, subcellular translocation, and function (14). For instance, PKC
is essential in
mediating neutrophil chemotaxis and in regulating the polarity of
astrocytes during wound healing process (15, 16). PKC
is
predominantly expressed in lymphocytes and is recruited to the membrane
during antigen presentation to T-cells (17). The 12 PKC isozymes can be
divided into three subfamilies: classical PKCs (cPKCs), such as
,
I,
II, and
, require both Ca2+ and DAG for
activation; novel PKCs (nPKCs), such as
,
,
, and
, are
DAG-dependent but Ca2+-independent; and
atypical PKCs, such as
and
, require neither Ca2+ or
DAG. Eight PKC isozymes,
,
1,
2,
,
,
, µ, and
,
have been identified in human blood monocytes (18). However, their contribution to heterologous desensitization of chemokine receptors has
not been defined.
Preincubation with µ- or
-opioid agonists has been shown to
inhibit MIP-1
-mediated chemotaxis of monocytes and neutrophils (7,
21). Such inhibition can be blocked by the nonselective opioid
antagonist naxolone or the µ- or
-selective antagonists CTOP and naltrindole (7). Preincubation with opioids has been shown to enhance phosphorylation of CXCR1, correlating with modest impairment of chemotaxis. Compared with other chemokines, opioids are
less capable of mediating heterologous desensitization. Heterologous desensitization of chemokine receptors seems to follow a hierarchy; certain receptors are more potent in desensitizing than others (19,
20). The capacity of a receptor to cross-desensitize GPCRs has been
proposed to correlate with its ability to induce a greater
phosphoinositide hydrolysis (8). We set out to investigate the
possibility that the hierarchy may be related to the PKC isotypes.
In this study, we first show that, in monocytes, opioid-mediated
heterologous desensitization inhibits not only chemokine-induced chemotaxis but also Ca2+ mobilization in a
dose-dependent manner. Such inhibitory effects result from
a decrease in chemokine receptor affinity and their coupling efficiency
to G protein. Furthermore, we find that, unlike chemokine receptors,
opioid receptors fail to elicit a calcium flux and only activate novel
and atypical PKC, resulting in modest suppression of chemokine receptor
function. Our data suggest that the relative potency of G
protein-coupled receptors in heterologous desensitization is dependent
on which subfamily of PKC is activated. A robust heterologous
desensitization requires both calcium-dependent and
-independent kinase C. Opioids are unique and only utilize a
calcium-independent PKC pathway to phosphorylate and inactivate heterologous receptors.
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MATERIALS AND METHODS |
Chemicals and Materials--
MCP-1 and MIP-1
were obtained
from Pepro Tech (Rocky Hill, NJ); I125-MIP-1
,
3H-DAMGO, and Taq polymerase were from
PerkinElmer Life Sciences; Met-enkephalin was from Peninsula
Laboratories, Inc. (San Carlos, CA); DAMGO was from Sigma; calphostin C
and Go6976 were from Alexis Biochemicals (San Diego, CA); chemotaxis
chamber and membrane were from Neuroprobe (Gaithersburg, MD);
polyclonal anti-PKC
(sc-216), anti-PKC
,
I,
II (c-20), and
anti-PKC
(c-17) antibodies were from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA); and ECL reagents were from Pierce. All of the other
reagents were reagent grade and were obtained from standard suppliers.
Cells and Cell Culture--
Human peripheral monocytes were
obtained from healthy donor blood packs and isolated from Buffy coats
(Transfusion Medicine Department, National Institutes of Health
Clinical Center, Bethesda, MD) by iso-osmotic Percoll gradient. The
monocytes were >90% pure by nonspecific esterase staining or by
morphological analysis. Freshly isolated monocytes were suspended in
ice-cold PBS and used for experiments on the same day. HEK293,
vector/HEK293, and µ-opioid receptor/HEK293 cells were grown in DMEM
(Biowhittaker, Walkersville, MD) supplemented with 10% fetal bovine
serum (Hyclone, Logan, UT), 1 mM glutamine (Invitrogen),
100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen).
CCR1/HEK293, µ-receptor/CCR1/HEK293, and vector/CCR1/HEK293 cells
were grown in the same buffer with 400 µg/ml Geneticin (Invitrogen).
Macrophages were generated in vitro by incubating freshly
isolated monocytes at 1 × 106/ml in RPMI 1640 medium
and 10% fetal bovine serum in the presence of recombinant human
macrophage colony-stimulating factor (50 ng/ml) at 37 °C in a
humidified CO2 (5%) incubator for 7 days with the addition
of fresh recombinant human macrophage colony-stimulating factor-containing medium every 2-3 days (22).
Plasmid and Stable Cell Line Construction--
PCR fragments of
full-length CCR1 were inserted into pcDNA 3.1 to make pCCR1. pCCR1
was linearized with ScaI and electroporated into HEK293
cells. After selection with 800 µg/ml Geneticin, the CCR1 level was
screened by Western blotting, and a single colony with high expression
of CCR1 was selected to generate CCR1/HEK293. Full-length µ-opiate
receptor was inserted into pLZRS-IRES-EGFP retroviral vector to
generate pµ-opioid receptor. The empty vector was used as a
control. Phoenix-Amphotrophic Retroviral Packaging cells were plated at
1.5-2 million cells/60-mm plate in DMEM with 10% fetal bovine serum,
1% penicillin-streptomycin, 1% glutamine, 18-24 h prior to
transfection (23). Approximately 2 µg of each plasmid containing the
desired inserts (pLZRS-IRES-EGFP) were prepared for transfection into
cells by using FuGENE 6 Transfection Reagent (Roche Diagnostics
Corporation, Indianapolis, IN) according to the manufacturer's
instructions (23, 24). The LZRS vector replicates episomally via use of
the EBNA-1 protein and also contains a puromycin resistance gene.
Finally, the inclusion of the IRES-EGFP expression cassette allows for
sorting of infected target cells by flow cytometry. After transfection,
the Phoenix-Ampho cells were selected with 2 µg of puromycin for 7 days, at which time the population consisted of virtually 100%
EGFP-expressing cells. Infectious virus-containing supernatants were
prepared by growing packaging cell lines infected with recombinant
retroviral vectors as described above, in T75 flasks to ~80%
confluency and then overlaying this culture with 10 ml of complete
medium to allow cells to produce virus overnight. At the same time,
target cell populations were prepared by plating cells into suitable
tissue culture vessels and overlaying the target culture with 0.22 µm of filtered, recombinant retrovirus-infected supernatants the following
morning. Infectious supernatants were supplemented with 5 µg/ml
polybrene to assist in virus attachment. 3-5 days post-infection, the
target cells were sorted by flow cytometry and analyzed as described
under "Results."
Chemotaxis--
Chemotaxis was performed as described by the
manufacturer (Neuroprobe). In brief, the monocytes were pretreated with
PBS, MIP-1
, MCP-1, Met-enkephalin, or morphine for 30 min at
37 °C. The cells were then washed twice with binding medium (RPMI
from Biowhittaker, 1% bovine serum albumin, and 20 mM
HEPES) and loaded into the upper chemotaxis chambers. Chemokines,
diluted to various concentrations were loaded into the lower chambers.
The chambers were incubated at 37 °C, 100% humidity, and 5%
CO2 for 1 h. The 5-µm filter between the chambers
was then washed, fixed, and stained. The cells that migrating through
the filter were counted by microscopy. The chemotaxis index was the
ratio of chemotactic cell numbers in a chemokine gradient
versus the cell numbers in a medium control. For HEK293
cells, the polycarbonate filter was pretreated with 50 µg/ml of
collagen in binding medium at 4 °C overnight. Chemotaxis of HEK293
cells was assayed after 5 h. The statistical analysis of
chemotactic responses was performed by PRISM3.0.
Ligand-induced Calcium Mobilization--
Calcium flux was
measured as described by Badolato et al. (25). In brief, the
cells were incubated at 107/ml for 30 min at room
temperature in DMEM containing 1 µM Fura-2. The cells
were then washed with DMEM once, washed with Hanks' balanced salt
solution twice, and diluted into 2 × 106/ml. The
cells were then loaded into a 2-ml cuvette at 37 °C, and the
relative ratio of fluorescent emission at 510 nm when excited by 340 nm
and 380 nm was recorded by a PerkinElmer Life Sciences luminescence
spectrometer. For heteorologous desensitization experiments, the cells
were first incubated with Met-enkephalin or chemokines at 37 °C for
30 min before adding Fura-2.
Ligand Binding Analysis--
The ligand binding assays were
carried out as described by Grimm et al. (7) with
modifications. The cells were preincubated with MIP-1
, MCP-1, or
Met-enkephalin for 30 min at 37 °C, washed extensively, and
resuspended in binding medium at 107/ml. The binding assays
were carried out on ice using 0.5 nM
I125MIP-1
in the presence of increasing concentration of
competing unlabelled-MIP-1
. The cells were incubated at 4 °C for
30 min, and unbound ligands were separated from cells by a 10% sucrose gradient. The level of binding was determined in a
-counter. Nonlinear regression analysis of data was performed by a PRISM3.0 program by fitting the following equation.
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(Eq. 1)
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MIP-1
-stimulated [35S]GTP
S Binding
Assay--
The assay was performed as described by Richardson et
al. (20) with minor modifications. The cells were pretreated with MIP-1a, MCP-1, Met-enkephalin, or binding medium for 45 min at 37 °C. After three washes, the cell membranes were isolated for binding assays.
PKC Translocation Assay--
Assay of PKC translocation in
monocytes were performed as described by Laudanna et al.
(15) with minor modifications. In brief, fresh monocytes were stored in
ice-cold PBS for 90 min to decrease the membrane-bound PKC before
stimulation. Cocktails of protease inhibitors were added. The cells
were then stimulated at 37 °C, stopped by ice-cold PBS, homogenized
by sonication, and underwent ultra-centrifugation at 100,000 × g for 1 h. The supernatant was kept as the cytosolic
fraction. The precipitates were sonicated in one half volume of PBS
with 1% Triton X-100 and centrifuged at 100,000 × g
again for 30 min. The solublized precipitates were kept as the membrane
fraction. The samples were then loaded on 10% SDS-PAGE, followed by
Western blotting analysis. Confocal microscopic analysis of
immunofluorescent staining of PKC isotypes was carried out as described
by Etienne-Manneville (16). The cells were pretreated with 100 ng/ml MCP-1 or 10
7 M Met-enkephalin for 10 min, then fixed, permeablized, and stained by rabbit antibody to PKC
isotypes followed by fluorescein isothiocyanate-labeled goat
anti-rabbit antibody. The cells were visualized using a Zeiss inverted
fluorescent confocal microscope.
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RESULTS |
Opioids Induced Monocyte Chemotaxis, but Not Calcium
Flux--
First, we compared the degree of Met-enkephalin-mediated
chemotaxis of freshly isolated monocytes to those induced by
conventional chemokines, MIP-1
and MCP-1. As shown in Fig.
1A, MCP-1, presumably by
activating chemokine receptor, CCR2, induced a robust chemotaxis response, which peaked at 3 × 10
10 M.
MIP-1
, the endogenous ligand for receptor CCR1 and 5, was also a
potent chemoattractant of monocytes. However, the chemotactic response
of Met-enkephalin on monocytes, although significant, was less potent,
with an index ranging from 2 to 4.5. Morphine was also a weaker
stimulus of monocyte chemotaxis (data not shown). The lower chemotactic
activity of opioids may be due to the lower receptor expression on cell
surface or perhaps due to inefficient coupling between opioid receptors
and Gi proteins. Furthermore, although MCP-1 and MIP-1
induced monocyte chemokinesis, the background level of monocyte
motility was unchanged when treated with opiates over a wide
concentration ranging from 10
11 to 10
6
M (data not shown).

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Fig. 1.
Met-enkephalin induces chemotaxis in
monocytes but not calcium flux. A, left
panel, Met-enkephalin-mediated monocyte chemotaxis, optimal at
concentration of 10 9 M. Right
panel, MCP-1 and MIP-1 induced robust monocyte chemotaxis.
B, unlike MIP-1 and MCP-1, Met-enkephalin did not
stimulate Ca2+ flux. A, in chemotaxis assays,
each data point is an average of quadruplicates (p < 0.014).
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Because opioid receptors couple to Gi proteins and induce
chemotaxis, we determined to assess their capacity to elicit a
concomitant Ca2+ response. As shown in Fig. 1B,
both MCP-1 and MIP-1
rapidly induced a potent Ca2+ flux
in monocytes, confirming that the inositol 1,4,5-trisphosphate-induced Ca2+ flux is functional (26). However, Met-enkephalin, at
concentrations from 10
10 to 10
4
M, failed to initiate a detectable Ca2+
response in monocytes (Fig. 1B and data not shown). Morphine also did not induce any measurable Ca2+ mobilization (data
not shown). The deficiency of opioid-induced Ca2+
mobilization is likely due to insufficient production of inositol 1,4,5-trisphosphate by activated Gi-coupled PLC
. These
data further suggest that the µ- and/or
-opioid receptors are less
potent in inducing chemotactic Gi signaling in monocytes
than chemokine receptors.
Pretreatment with Opioids Inhibited Chemokine-mediated Chemotaxis
and Calcium Response in Monocytes--
To evaluate the potency of
opioid-induced desensitization, the inhibitory effects of
Met-enkephalin on MIP-1
-induced chemotaxis were compared with the
desensitizing effects of MIP-1
and MCP-1 on chemokine receptors.
Freshly isolated monocytes pretreated with 100 ng/ml MIP-1
exhibited
a reduction of more than 85% of the chemotactic response to MIP-1
,
because of homologous desensitization (Fig.
2A). This homologous
desensitization was dose-dependent (Fig. 2B).
Monocytes pretreated with 100 ng/ml MCP-1 for 30 min also showed a
marked decrease in their chemotactic response to MIP-1
, because of
heterologous desensitization of chemokine receptors (Fig. 2,
A and B). Pretreatment with 10
8
M of Met-enkephalin significantly impaired
MIP-1
-mediated chemotaxis but to a lesser degree (Fig.
2A). Met-enkephalin exhibited dose-dependent heterologous desensitization effects with optimal inhibitory activity of 50% at 10
6 M (Fig. 2B).

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Fig. 2.
Comparison of heterologous desensitization of
MIP-1 by Met-enkephalin and chemokines.
A, effects of pretreatment with Met-enkephalin on
MIP-1 -mediated chemotaxis. B, dose dependence of MIP-1
receptor desensitization by MIP-1 , MCP-1, and Met-enkephalin.
C, dose dependence of MIP-1 -induced calcium flux.
D, comparison of desensitization of MIP-1 -mediated
calcium flux by MIP-1 , MCP-1, and Met-enkephalin. (In chemotaxis
assays, each data point is an average of quadruplicates;
p < 0.027. Each calcium assay has been repeated at
least three times.)
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|
To determine the effect of opioid mediated heterologous desensitization
on Ca2+ flux, we compared the dose dependence of the
MIP-1
-elicited Ca2+ response of monocytes, which were
pretreated cells with 1000 ng/ml of MIP-1
, MCP-1, or
10
7 M Met-enkephalin. MIP-1
, at 0, 5, 15, 50, 150, and 500 ng/ml, induced Ca2+ flux in monocytes in a
dose-dependent manner (Fig. 2C and data not
shown). To directly compare homologous and heterologous
desensitization, the maximal value of each Ca2+ flux was
used to plot the dose response of the stimulus (Fig. 2D).
Monocytes pretreated with 1000 ng/ml MIP-1
for 30 min followed by
three washes failed to exhibit a detectable response to 5 and 15 ng/ml
MIP-1
(Fig. 2D). Homologous desensitization also
decreased the response to MIP-1
at 150 and 500 ng/ml. MCP-1
pretreatment severely reduced the MIP-1
-induced Ca2+
response, but the inhibitory effects decreased as the concentration of
stimulus was increased and became undetectable at 150 ng/ml of MIP-1
(Fig. 2D). Pretreatment with Met-enkephalin resulted in
moderate inhibition of Ca2+ flux (Fig. 2D).
Monocytes pretreated with 10
7 M
Met-enkephalin lost 70% of the Ca2+ response to 5 ng/ml
MIP-1
. Met-enkephalin-mediated inhibitory effects were maximal when
chemokine concentration was low and decreased as the chemokine
concentration was increased (Fig. 2D). Both chemotaxis and
calcium flux data show that the heterologous desensitization of the
MIP-1
response by Met-enkephalin was significant but less potent
than either homologous or heterologous desensitization of chemokine
receptors by chemokines.
We further examined the effect on MIP-1
-mediated chemotaxis in
in vitro macrophage colony-stimulating factor-stimulated
macrophages. As shown in Fig. 3, although
opioids also did not induce calcium flux in activated macrophage, they
induced similar heterologous desensitization of chemokine receptors in
activated macrophages.

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Fig. 3.
Effects of Met-enkephalin on function of
chemokine receptors on macrophage colony-stimulating factor-stimulated
macrophages. A, effects of Met-enkephalin pretreatment
on MIP-1 -mediated chemotaxis. B, Ca2+ flux of
macrophages upon stimulation by MIP-1 or Met-enkephalin. (In
chemotaxis assays, each data point is an average of quadruplicates;
p < 0.004. Each calcium flux has been repeated at
least three times.)
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Met-enkephalin Pretreatment Reduced Binding Affinity of MIP-1
Receptors and Coupling Efficiency between MIP-1
Receptors and G
Protein--
Desensitization of seven-transmembrane receptors may
involve internalization of receptors, decrease in ligand binding
affinity, or impaired interaction with Gi proteins (11-13,
27-31). I125-MIP-1
was used to monitor the effect of
Met-enkephalin on the total numbers of MIP-1
-binding sites and
binding affinity on monocytes. Homologous competitive binding analyses
showed a 3-fold decrease in MIP-1
binding affinity after
Met-enkephalin pretreatment, whereas the total number of binding sites
was unchanged (Fig. 4, A and
B). In contrast, preincubation with MIP-1
reduced the number of surface binding sites by over 70%, presumably because of
receptor internalization during homologous desensitization (Fig.
4C). Activation of MCP-1 receptors also decreased the number of MIP-1
receptors by more than 35% (Fig. 4C). In
contrast with our results, previously reported binding analysis,
carried out at room temperature, did not reveal any detectable affinity
change (7). Our binding assays were performed at 4 °C to prevent
MIP-1
-induced homologous desensitization.

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Fig. 4.
Effects of Met-enkephalin versus
chemokine pretreatment on MIP-1 surface
binding sites, binding affinity, and receptor-G protein coupling
efficiency. A, homologous competitive binding analysis
on the binding affinity and binding sites of MIP-1 on monocytes.
B, homologous competitive binding analysis on the binding
affinity and binding sites of MIP-1 on Met-enkephalin-pretreated
monocytes. C, changes of total MIP-1 -binding sites after
pretreated with MIP-1 , MCP-1, or Met-enkephalin. D,
comparison of MIP-1 -stimulated binding of [35S]GTP S
to membrane in monocytes pretreated with MIP-1 , MCP-1, or
Met-enkephalin. (A and B, one set of
representative data from two independent experiments. In each
experiments, each data point is the average of duplicated binding
assays. C and D, Each data point is the average
of triplicate binding assays. The data were repeated twice.)
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Upon ligand binding, chemokine receptors activate downstream
heterotrimeric G proteins by enhancing the exchange of bound GDP to
GTP. A desensitized GPCR shows a decrease in its capability to induce
the binding of [35S]GTP
S to membrane G proteins.
Pretreatment with Met-enkephalin for 30 min resulted in a 34% loss of
MIP-1
-stimulated [35S]GTP
S binding on the membrane,
indicating an impairment of the coupling efficiency between chemokine
receptors and downstream G protein (Fig. 4D). In contrast,
the capability of MIP-1
receptors to enhance membrane
[35S]GTP
S binding was severely inhibited (up to
62-70%) after pretreatment with MCP-1 or MIP-1
(Fig.
4D). The decrease in both receptor affinity and coupling
efficiency to G protein may directly contribute to the desensitization
of MIP-1
-mediated chemotaxis, calcium flux, and other signals.
Overexpression of Opioid Receptors Did Not Rescue Ca2+
Flux Defects--
The expression level of opioid receptors on
monocytes and macrophages is very low relative to that of chemokine
receptors. Less that 103 binding sites were detected using
3H-Met-enkephalin (data not shown). Thus, this lack of a
robust opioid-mediated Ca2+ response or opioid
receptor-mediated inhibition of chemokine receptor function may simply
be due to the low copy numbers of opioid receptors. To circumvent this
situation, we overexpressed the µ-opioid receptors in both HEK293 and
CCR1/HEK293 cells. Radiolabeled binding analysis showed that, in both
µ-receptor/HEK293 and µ-receptor:CCR1/HEK293 cells, the number of
binding sites for 3H-Met-enkephalin was over 3.1 × 105/cell, and the CCR1/HEK293 cells expressed 2.6 × 105 MIP-1
-binding sites. There was an undetectable level
of µ-opioid receptors in vector-transformed HEK293 cells. The
functionality of overexpressed µ-receptors was analyzed by chemotaxis
assay. Met-enkephalin at 1 nM concentration elicited robust
chemotaxis in both µ-receptor/HEK293 and µ-receptor:CCR1/HEK293
cells (data not shown). Despite the high level of surface
µ-receptors, upon Met-enkephalin or DAMGO stimulation, no
Ca2+ flux was detected in either µ-receptor:CCR1/HEK293
or µ-receptor/HEK293 cells (Fig.
5A and data not shown). In
contrast, MIP-1
elicited rapid and robust Ca2+ flux
response in both CCR1/HEK293 and µ-receptor:CCR1/HEK293 cells, even
though the expression of CCR1 is comparable with that of the
µ-receptors. Thus, the deficiency in opioid receptor-mediated calcium
flux must be based on their incomplete activation of downstream signaling pathways.

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Fig. 5.
Effects of Met-enkephalin on CCR1/HEK293
cells infected to overexpress µ-opioid
receptors. A, effects of Met-enkephalin on
Ca2+ flux in µ-receptor overexpression cells.
B, effect of Met-enkephalin on CCR1 internalization in
µ-receptor overexpression cells. C, effect of
Met-enkephalin on heterologous desensitization by µ-receptor
overexpression cells. (A-C, a set of representative data
from at least two independent experiments. B, each data
point is an average of triplicate assays; p < 0.026. C, each data point is an average of quadruplicate assays;
p < 0.041.)
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We examined the capacity of µ-opioid receptors to induce heterologous
desensitization in µ-receptor:CCR1/HEK293 cells. Consistent with
previous data from monocytes, opioids suppressed MIP-1
-induced chemotaxis up to 50% and did not induce receptor internalization (Fig.
5, B and C). These results provide further
evidence that long term activation of opioid receptors leads to a
moderate level of down-regulation of chemokine receptor function
through Ca2+-independent heterologous desensitization.
Met-enkephalin-induced Chemokine Receptor Desensitization Could Be
Blocked by Calphostin C but Not Go6976--
Second
messenger-stimulated kinases, protein kinase A and PKC, are involved in
heterologous desensitization of G protein-coupled receptors via
feedback regulation (8, 9, 11). Previous studies have repeatedly shown
that, upon chemokine binding, receptors elicit a Ca2+ flux
response, which in turn activates PKC, resulting in heterologous desensitization of other types of chemokine receptors. However, because
Met-enkephalin did not induce a detectable Ca2+ response,
it therefore presumably used another mechanism. We used calphostin C,
an inhibitor of nPKCs and cPKCs, to evaluate whether PKCs play any role
in opioid receptor-mediated inhibition. When monocytes were pretreated
with 200 nM calphostin C, the inhibition of MIP-1
chemotaxis by opioids was largely blocked, indicating the involvement
of PKCs in heterologous desensitization (Fig. 6A). MCP-1-mediated
heterologous desensitization was also blocked by calphostin C treatment
(Fig. 6A). However, calphostin C did not interfere with
MIP-1
-mediated chemotaxis but only with heterologous desensitization. To determine which subfamily of PKCs were involved, we
used Go6976, a specific inhibitor for cPKC but not nPKC isozymes. This
drug only partially impaired the inhibition by MCP-1 pretreatment and
had no effect on the inhibition induced by opioids (Fig.
6B). These data suggest that only
Ca2+-independent PKC isotypes are involved in
opioid-mediated heterologous desensitization, consistent with a lack of
Ca2+ flux upon opioid treatment.

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Fig. 6.
Calphostin C, not Go6976, blocks
Met-enkephalin-mediated heterologous desensitization.
A, effects of calphostin C on MCP-1 and Met-enkephalin
desensitization of chemotactic response (p < 0.047).
B, effects of Go6976 on desensitization of chemotactic
response to MIP-1 by MCP-1 and Met-enkephalin (p < 0.05). (Each data point is an average of quadruplicate data.)
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Met-enkephalin Elicited Activation of Calcium-independent PKC, not
cPKC--
The translocation of PKCs from cytosol to different membrane
compartments is a hallmark of PKC activation (14). The activation of
each isozyme can be monitored by its redistribution to the membrane. To
confirm the observations obtained with PKC inhibitors, we investigated
the translocation of different PKC isozymes in monocytes upon MCP-1 and
Met-enkephalin stimulation. As shown in Western blotting analysis, in
freshly isolated monocytes, PKCs distributed in the cytosol (Fig.
7A). When stimulated with 1000 ng/ml MCP-1, monocytes responded with a membrane enrichment of both
Ca2+-dependent and -independent PKCs, such as
,
I,
II,
, and
isozymes (Fig. 7A). The
translocation occurred in as little as 30 s (data not shown).
Similar activation patterns were also found in MIP-1
-activated cells
(data not shown). However, only Ca2+-independent PKCs, such
as
and
, were activated by 10
7 M
Met-enkephalin. We further confirmed the Western blotting data by
immunofluorescent staining of PKC translocation in monocytes (Fig.
7B). In the basal state, different PKC isotypes seem to exhibit different distribution pattern in cytosol. Consistent with
Western blotting analysis, Met-enkephalin induced translocation of
PKC
,
but not calcium-dependent PKC isotypes, whereas
both cPKC and PKC
,
redistributed to the membrane upon MCP-1
stimulation. These data provided additional evidence that the
activation of classical PKC isozymes is undetectable in the presence of
Met-enkephalin, and only Ca2+-independent PKCs are involved
in this heterologous desensitization. The absence of PKC
,
I,
II, and
participation may account for the lack of chemokine
receptor internalization and the lower potency of opioid-mediated
heterologous desensitization.

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Fig. 7.
PKCs translocate to membrane only upon
chemokine stimulation. A, monocytes were treated with
MCP-1 or Met-enkephalin at 37 °C for 10 min and then rapidly cooled
by ice-cold PBS. The distribution of PKC , I, II, , and between membrane (M) and cytosol (C) was analyzed
by Western blotting. B, immunofluorescent staining of the
translocation of PKC isotypes upon MCP-1 or Met-enkephalin
stimulation.
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DISCUSSION |
Opioids suppress chemokine receptor function through
calcium-independent PKC. Extensive studies have shown the essential
role of PKC in heterologous desensitization (8, 9). However, little is
known about the contribution of different PKC isotypes to this process.
Numerous reports indicate that 12 PKC isotypes do not serve redundant
function, but each isotype has its unique activation process and
function (14-17). By comparing Met-enkephalin, MCP-1, and
MIP-1
-mediated down-regulation of MIP-1
receptors, our data
provided the first insights concerning the role of PKC isotypes in
heterologous desensitization. The fact that calphostin C blocked the
inhibitory effects of both MCP-1 and Met-enkephalin suggests that PKCs
were involved in both opioid-induced and chemokine-induced heterologous
desensitization. However, the absence of Ca2+ mobilization
provided the first clue that cPKCs were not involved in desensitization
by opioids. The failure of Go6976 to reverse opioid-induced inhibition
provided further evidence for the lack of cPKC effects. To confirm
these observations, we directly examined activation of PKC isotypes by
a translocation assay. Both Western blotting analysis and
immunofluorescent staining clearly showed that only
Ca2+-independent PKC
,
were activated by opioids.
Because expression of PKC
is very low in monocytes and PKC
translocates to the Golgi, they are not likely to be involved (17, 18,
32). Thus, we conclude that only
,
, µ, and
may be involved
in Met-enkephalin-mediated heterologous desensitization in monocytes, whereas chemokines use those plus calcium-dependent PKCs.
To further dissect the role of PKC isotypes, we propose that
isozyme-specific peptide inhibitors of PKCs may be used to identify the
participating isozymes (14).
Heterologous desensitization of chemokine receptors seems to follow a
hierarchy. Certain receptors, such as the fMLP receptor, have a
relatively high capacity to desensitize other GPCRs, whereas opioid
receptors have a limited capacity (19, 20). The capacity of a receptor
to cross-desensitize GPCRs seems to correlate with its ability to
induce greater phosphoinositide hydrolysis and sustained
calcium mobilization (8). Our data suggest that
calcium-dependent PKCs are also critical for robust
heterologous desensitization. MCP-1 elicited a marked calcium response,
which is necessary for the internalization of MIP-1
-binding sites
and a marked decrease in the coupling efficiency between receptors and
G protein. Such inhibitory effects could be blocked by the PKC
inhibitor calphostin C. In contrast, Met-enkephalin failed to activate
classic PKC isotypes and consequently had moderate inhibitory effects
and no receptor internalization. Thus, we speculate that the hierarchy of heterologous desensitization partially relies on the capacity of
receptors to activate different isotypes of PKC; opioid receptors that
fail to activate cPKC can only mediate moderate heterologous desensitization.
Chemotaxis requires less signaling input from activated receptors than
Ca2+ flux. Most chemokines induce both chemotaxis and a
Ca2+ flux response in a Gi
protein-dependent fashion. It was even suggested that
chemotaxis requires a Ca2+ signal. Our data clearly show
that chemotaxis by opioids can occur in the absence of Ca2+
mobilization. Furthermore, our data suggest that chemotaxis is more
sensitive to ligand stimulation. We speculate that Met-enkephalin receptors induce a low level of phospholipid metabolism, enough for
chemotaxis, but generate insufficient DAG to elicit a detectable Ca2+ flux. Indeed, other weak chemoattractants, such as
certain autoantigens, fail to elicit Ca2+
responses.2
Opioid receptors may serve as a link between immune and nervous systems
by modulating PKC activity of leukocytes. In neuronal systems, opioids
elicit activation of G protein-coupled inwardly rectifying
K+ channels, inhibition of Ca2+-channel, and
adenylyl cyclase activity, eventually resulting in analgesia (33). By
coupling to Gi, opioid receptors on leukocytes mediate
chemotaxis, suggesting that they may share similar downstream signaling
pathways with chemokine receptors (7). Three mechanisms have been
proposed to explain opioid-mediated immunomodulation. First, continuous
activation of opioid receptors in the central nervous system may
elevate the level of circulating corticosterone, which in turn
suppresses the immune system (34, 35). Second, opioids may activate the
sympathetic nervous system to increase the level of epinephrine or
norepinephrine (36, 37). The discovery of opioid receptors in
peripheral blood mononuclear cells added a third mechanism based on
data showing that these opiate receptors can directly modify the
response of proinflammatory receptors (2-4, 7). Our data suggest that
long term usage of opioids directly interferes with chemokine receptor
function and provide a molecular mechanism for such heterologous
desensitization. The dose-response curve of Met-enkephalin-mediated
inhibition showed that Met-enkephalin inhibited chemotaxis at
concentrations as low as 10
9 M, comparable
with the blood level of opiates in patients or addicts (12). In
addition, prolonged exposure to drugs has been shown to increase the
expression of opioid receptors, which may lead to greater inhibitory
effects. Opioid-induced heterologous desensitization suppresses
chemokine responses in leukocytes when the concentration of chemokine
is in the nanomolar range. For instance, dosage curves of
Ca2+ mobilization showed that opioid inhibition was most
effective when the concentration of MIP-1
was between 1-50 ng/ml,
equivalent to its level in vivo. This leads us to suggest
that such cross-desensitization in leukocytes may directly contribute
to well documented opioid-mediated immunosuppression (1-3). Such
down-regulation of chemokine receptor function is not limited to
monocytes in the resting state. The fact that Met-enkephalin
pretreatment also impaired the chemotaxis response of monocyte-derived
macrophages suggests a potential inhibitory role of opioids during
inflammation. Nevertheless, the inhibitory effects of heterologous
desensitization appear to be limited and constitute only one part of
the complex immunomodulatory effects of opioids.
Overall, our studies have identified Ca2+-independent PKCs
as participants in opioid desensitization of chemokine receptors and
provided additional insights of the molecular mechanism of opioid-mediated immunosuppression. This is a first report on the different roles of nPKCs and cPKCs in heterologous desensitization. Based on our results, specific inhibitors for nPKC isozymes can be used
to develop strategies to prevent opiate-mediated immunosuppressive effects. Inhibitors that do not block cPKC presumably will have fewer
undesirable negative effects on endogenous heterologous desensitization
of chemokine receptors.