J. Exp. Med.,
Volume 188, Number 2, July 20, 1998 317-325
Opiates Transdeactivate Chemokine Receptors:
and µ Opiate Receptor-mediated
Heterologous Desensitization
By
M.C.
Grimm,*
A.
Ben-Baruch,*
D.D.
Taub,
O.M.Z.
Howard,
J.H.
Resau,§
J.M.
Wang,
H.
Ali,¶
R.
Richardson,¶
R.
Snyderman,¶
and
J.J.
Oppenheim*
From the * Laboratory of Molecular Immunoregulation, Division of Basic Sciences, and the Intramural
Research Support Program, Science Applications International Corp. Frederick, Frederick, Maryland
21702; the § ABL-Basic Research Program, National Cancer Institute - Frederick Cancer Research
and Development Center, Frederick, Maryland 21702; the Laboratory of Immunology, National
Institute on Aging, Baltimore, Maryland 21224; and the ¶ Department of Medicine, Duke University
Medical Center, Durham, North Carolina 27710
 |
Abstract |
An intact chemotactic response is vital for leukocyte trafficking and host defense. Opiates are
known to exert a number of immunomodulating effects in vitro and in vivo, and we sought to determine whether they were capable of inhibiting chemokine-induced directional migration
of human leukocytes, and if so, to ascertain the mechanism involved. The endogenous opioid
met-enkephalin induced monocyte chemotaxis in a pertussis toxin-sensitive manner. Met-enkephalin, as well as morphine, inhibited IL-8-induced chemotaxis of human neutrophils and
macrophage inflammatory protein (MIP)-1
, regulated upon activation, normal T expressed
and secreted (RANTES), and monocyte chemoattractant protein 1, but not MIP-1
-induced
chemotaxis of human monocytes. This inhibition of chemotaxis was mediated by 
and µ but
not 
G protein-coupled opiate receptors. Calcium flux induced by chemokines was unaffected by met-enkephalin pretreatment. Unlike other opiate-induced changes in leukocyte
function, the inhibition of chemotaxis was not mediated by nitric oxide. Opiates induced
phosphorylation of the chemokine receptors CXCR1 and CXCR2, but neither induced internalization of chemokine receptors nor perturbed chemokine binding. Thus, inhibition of
chemokine-induced chemotaxis by opiates is due to heterologous desensitization through phosphorylation of chemokine receptors. This may contribute to the defects in host defense
seen with opiate abuse and has important implications for immunomodulation induced by several endogenous neuropeptides which act through G protein-coupled receptors.
Key words:
chemokine;
chemokine receptor;
opioid receptor;
desensitization;
neuropeptide
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Introduction |
Long-term abuse of opiates results in immune defects,
including impaired NK cell activity and altered CD4+
and CD8+ T cell numbers (1), and it has been implicated as
a contributing factor in HIV infection (2). In animal models, parenteral use of opiates has been demonstrated to inhibit mitogenic and effector cell responses in B and T cells
(3, 4), cellular immunity (5), phagocytosis by neutrophils
and macrophages (6, 7), tumor cell suppression (6, 7), and
cytokine production (8). Morphine inhibits monocyte and
neutrophil functions through the physiologically defined
µ3 opiate receptor (9, 10); indeed, binding sites and protein
expression for and subclasses of G protein-coupled opiate receptors (11, 12), in addition to gene expression of ,
, and µ subclasses (13), have been described in leukocytes.
Chemokines are a large family of chemotactic cytokines
that mediate their effects on leukocytes through a number
of G protein-coupled, seven transmembrane-spanning
(STM)1 receptors (16). Although there is apparent redundancy in the ligand binding characteristics of these chemokine
receptors, specificity is provided by patterns of receptor and
G protein expression, ligand potency, and levels of receptor
desensitization. Interactions among STM receptors are complex (17): in a process known as receptor cross-regulation,
or heterologous desensitization, thrombin receptor activation has been shown to cause phosphorylation of several
chemoattractant receptors, including the IL-8 receptor CXCR1, the C5a receptor, and the receptor for platelet-activating factor, but not that for FMLP (18). Such cross-regulation does not occur between all classes of STM receptors; for example, 1-adrenergic receptors are unable to
desensitize chemoattractant receptors (19). We have shown
recently that homologous desensitization through phosphorylation of the IL-8 receptor CXCR2 occurs in response to its native ligands IL-8 and neutrophil-activating
peptide (NAP)-2 (20). Together, these findings led us to
the hypothesis that the impairment of leukocyte functions
mediated by opiates may involve inhibition of chemokine
effects based on cross-regulation, resulting in phosphorylation and hence to heterologous desensitization of chemokine receptors. The studies reported here show that opiates, acting through and µ subclasses of opiate receptors expressed on human monocytes and neutrophils, are capable
of inhibiting subsequent migratory responses to chemokines,
and that this process of heterologous desensitization or transdeactivation is associated with phosphorylation of chemokine receptors.
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Materials and Methods |
Cells.
Human polymorphonuclear neutrophils (21) and monocytes were obtained from healthy donor blood packs. Neutrophils
were isolated from human blood by 3% dextran in PBS sedimentation of Ficoll-Hypaque-generated pellets followed by hypotonic
lysis (0.2% NaCl) of contaminating red cells. Neutrophils were
>95% pure by morphological criteria. Monocytes were purified
from Ficoll-Hypaque-generated mononuclear cells using MACS®
CD14 microbeads (Miltenyi Biotec Inc., Auburn, CA) as per the manufacturer's instructions. The monocytes were >90% pure by
nonspecific esterase staining.
Chemotaxis.
In vitro chemotaxis was performed as described
previously (22). In brief, cells were incubated at 37°C with medium, pertussis toxin (Sigma Chemical Co., St. Louis, MO) 500 ng/ml for 90 min, met-enkephalin (Sigma Chemical Co.) in
varying concentrations for 60 min, naloxone (Sigma Chemical
Co.) 10
8 M for 10 min before met-enkephalin, or naloxone alone.
In other experiments, cells were preincubated with opiate receptor-specific agonists or antagonists, or opiate incubation was preceded by pretreatment with the nitric oxide (NO) synthase inhibitor, L-NMMA (NG-monomethyl-L-arginine monoacetate [Sigma
Chemical Co.]). Chemotaxis was assessed in 48-well microchemotaxis chambers, using polyvinylpyrrolidone-free 5-µm pore size
membranes (Nucleopore; NeuroProbe, Cabin John, MD). Migration in response to met-enkephalin, morphine, chemokines, or FMLP was allowed to continue for 90 min at 37°C in 5% CO2. The membrane was removed, and the upper surface was washed
with PBS, scraped, fixed, and stained. The results are expressed as chemotaxis index (mean number of cells per high power field for chemoattractant dilution/mean number of cells per high power
field for medium) ± SE from at least three separate experiments.
Intracellular Calcium Flux.
Human peripheral blood monocytes and neutrophils were incubated at 107/ml for 30 min at
room temperature in HBSS containing 0.1% BSA and 1 µM
Fura-2 (23). Cells were then washed in PBS and incubated with
met-enkephalin (109 M) or morphine (106 M) for 60 min,
with or without preincubation for 10 min in naloxone (108 M).
The cells were washed and resuspended, and Fura-2 excitation was assessed at 340 and 380 nm with detection at 510 nm in a luminescence spectrometer using FL WinLab software (Perkin-Elmer Corp., Norwalk, CT), after stimulation with opiate or
chemokine.
Assessment of Receptor Internalization Using Confocal Laser Microscopy.
Rat basophil leukemia cells (RBL2H3) stably transfected
to express CXCR1 were derived as described previously (24).
Transfected cells were untreated or incubated with IL-8 1 µg/ml
for 30 min or with met-enkephalin 109 M for 3 h, fixed in 4%
paraformaldehyde, and centrifuged onto gelatin-coated slides.
Cells were permeabilized in 0.15% saponin before incubation for
60 min with an antiepitope mAb (12CA5; Boehringer Mannheim Biochemicals, Indianapolis, IN). After washing and incubation with FITC-labeled goat anti-mouse IgG, the cells were incubated with DAPI (Sigma Chemical Co.) for 10 min. Slides
were examined using a confocal laser scanning microscope
(model 310; Carl Zeiss, Inc., Thornwood, NY). Nomarski, FITC
(488 nm, green), and DAPI (UV 364 nm, blue) images were prepared for each specimen. Blue and green images were superimposed onto the Nomarski image.
Binding Assays.
Binding of chemokines to their receptors was
assessed using 1 ng/ml of 125I-labeled chemokine (NEN Research
Products, Boston, MA) in the presence of various concentrations
of unlabeled ligands or met-enkephalin (from equimolar to
1,000-fold excess), or after preincubation of the cells with met-enkephalin for 60 min with or without pretreatment for 10 min
with naloxone. Cells at 107/ml in RPMI 1640/1% BSA/25 mM
Hepes were incubated with ligands for 45 min at room temperature and pelleted through 10% sucrose; cell pellet-associated radioactivity was then determined in a 
-counter. Binding data
were analyzed using the computer program LIGAND.
Chemokine Receptor Phosphorylation.
Epitope-tagged FMLP receptor-transfected (25) and CXCR1-transfected RBL2H3 cells
were washed, then incubated in phosphate-free media for 3 h.
The cells were then labeled with 150 µCi/7.5 × 106 cells of
[32P]orthophosphate (NEN Research Products) for 90 min at
37°C before stimulation with native ligands for 5 min, met-enkephalin 109 M for 60 min, or naloxone 108 M for 10 min before
met-enkephalin. Equivalent numbers of cells for each treatment
were lysed in RIPA buffer containing protease and phosphatase inhibitors. Epitope-tagged FMLP receptors and CXCR1 were immunoprecipitated as described previously (18) and run in 10%
SDS-polyacrylamide gels. The gels were dried and exposed to radiographic film.
| |
Results |
Opiate-mediated Inhibition of Chemokine Chemotaxis.
We
sought initially to confirm previous reports (26, 27) that
opiate compounds, including met-enkephalin, are chemotactic for monocytes and neutrophils. Preliminary studies
confirmed that phagocytes respond chemotactically, with a
chemotaxis index 2-2.5-fold higher than controls, to met-enkephalin and morphine, with optimal activity at 109
and 106 M, respectively. This chemotaxis was inhibited by
the opiate receptor antagonist naloxone (data not shown).
To investigate the mechanism of chemotaxis, monocytes
were incubated for 90 min with pertussis toxin 500 ng/ml
before exposure to met-enkephalin in microchemotaxis
chambers. Fig. 1 demonstrates that the chemotactic activity
of met-enkephalin for monocytes was abolished completely by pertussis toxin, suggesting association with the Gi
subclass of G proteins.
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Fig. 1.
Chemotactic response of human peripheral blood monocytes
to met-enkephalin and effect of pertussis toxin pretreatment. The results
are expressed as chemotaxis index (mean number of cells per high power field
for met-enkephalin dilution/mean number of cells per high power field for
medium) ± SE from two separate experiments. *P <0.05; **P <0.01.
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The immunosuppressive and antiinflammatory effects of
opiates led us to assess the ability of met-enkephalin to
downregulate chemotaxis of monocytes in response to
macrophage-inflammatory protein (MIP)-1 and monocyte chemoattractant protein (MCP)-1. Preincubation of
freshly isolated human monocytes for 60 min with met-enkephalin at concentrations of 1015-106 M demonstrated that the optimal concentration for inhibition of
MIP-1 and MCP-1-induced chemotaxis was equivalent
to the optimal chemotactic dose, 109 M (Fig. 2). This
concentration, as well as the peak chemotactic concentration of 106 M for morphine, was used in subsequent inhibition experiments.
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Fig. 2.
Inhibition of monocyte chemotaxis in response to MIP-1
100 ng/ml and MCP-1 100 ng/ml, after pretreatment using met-enkephalin.
Results are expressed as percentage of chemotactic response of untreated
cells. Data from two experiments. *P <0.05; **P <0.01.
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We next assessed the effect of preincubation of monocytes with met-enkephalin on chemotaxis in response to a
number of CC chemokines. MIP-1, regulated upon activation, normal T expressed and secreted (RANTES), and
MCP-1 at their optimal concentrations of 50 ng/ml were
potent inducers of monocyte chemotaxis but were inhibited by 82, 58, and 52%, respectively, by met-enkephalin (Fig. 3 A). These inhibitory effects were prevented by preincubation with 108 M naloxone. In contrast, chemotaxis
in response to MIP-1 was not inhibited by met-enkephalin (Fig. 3 A). Although MIP-1 and RANTES act through a
number of CC chemokine receptors, including CCR1,
CCR3, and CCR5, MIP-1 acts principally through CCR5
(28). These data suggest that some but not all CC chemokine receptors can be inhibited by met-enkephalin, and
that the degree of inhibition varies between receptors. Preincubation of monocytes with met-enkephalin also had no
effect on chemotaxis induced by FMLP (Fig. 3 A), indicating that the monocyte FMLP receptor, like its neutrophil
counterpart, is spared.
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Fig. 3.
(A) Effects of met-enkephalin on FMLP (fMLP) and CC chemokine-induced monocyte chemotaxis. (B) Effects of met-enkephalin on
IL-8- and FMLP (fMLP)-induced neutrophil chemotaxis. The results are expressed as chemotaxis index (mean number of cells per high power field for
chemokine dilution/mean number of cells per high power field for medium) ± SE from at least three separate experiments. *P <0.05; **P <0.01.
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Preincubation of human neutrophils with met-enkephalin 109 M resulted in 61% inhibition of chemotaxis induced by 100 ng/ml IL-8 (Fig. 3 B). Inhibition was reversed by the presence of 108 M naloxone during the
incubation period, suggesting that this enkephalin-induced
effect was opiate receptor-mediated. Naloxone alone, although thought to act as a partial agonist for opiate receptors (29), was ineffective at inhibiting chemotaxis in response
to IL-8 (Fig. 3 B). Importantly, FMLP-induced chemotaxis of neutrophils was not inhibited by preincubation with
met-enkephalin (Fig. 3 B), indicating that the opiate effect
does not apply to all chemoattractant STM receptors.
Opiate Receptor Specificity.
To determine the potential
role of morphine in inhibiting chemokine effects and to
dissect opiate receptor specificity, monocytes were preincubated with morphine and certain opiate receptor-specific agonists, then tested for their chemotactic response to MIP-1. Like met-enkephalin, morphine caused significant inhibition (86%) of MIP-1-induced chemotaxis of monocytes
(Fig. 4 A), which was reversed by naloxone. Since morphine is thought to mediate its effects in monocytes primarily through µ3 receptors (30), we examined the response of
monocytes to the µ opiate receptor-selective peptide
DAMGO ([D-ala2,N-me-phe-gly5(ol)]-enkephalin). Pretreatment with this compound at 109 M also resulted in
significant inhibition of MIP-1 chemotaxis (76%; Fig. 4
A). Moreover, the -selective agonist dermenkephalin at 5 × 108 M inhibited MIP-1 chemotaxis by 52%, whereas the
-selective agonist dynorphin A at 108 M caused no significant inhibition (Fig. 4 A). Similar results were observed for
neutrophils: DAMGO and dermenkephalin inhibited IL-8-induced chemotaxis by 50 and 34%, respectively, whereas
dynorphin A had no effect (data not shown). Together,
these results suggest that opiate-mediated inhibition of
chemokine-induced chemotaxis occurs through activation
of µ and but not opiate receptors expressed on monocytes and neutrophils.
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Fig. 4.
Effects of opiate receptor-specific agonists and antagonists on
chemokine-induced monocyte chemotaxis. (A) Effect on MIP-1-induced
chemotaxis of monocytes, of preincubation with the -specific agonist
dynorphin A, the -specific agonist dermenkephalin, the µ-specific agonist DAMGO, and morphine. (B) Effect on MIP-1-induced chemotaxis
of monocytes, of pretreating before met-enkephalin with the -specific
antagonist naltrindole and the µ-specific antagonist CTOP. *P <0.05;
**P <0.01.
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To clarify opiate receptor specificity further, monocytes
and neutrophils were incubated with the µ-specific antagonist CTOP (cys2, tyr3, orn5, pen7 amide) at 107 M or the
-selective antagonist naltrindole at 2 × 108 M before
met-enkephalin. CTOP reversed 67% of the met-enkephalin-induced inhibition of MIP-1-induced monocyte chemotaxis, whereas naltrindole reversed 30% (Fig. 4 B), indicating a greater effect through µ than through opiate receptors
on monocytes. Combining these antagonists before met-enkephalin treatment reversed completely the met-enkephalin
effect. Similarly, preincubation of neutrophils with the µ receptor antagonist CTOP and with the antagonist naltrindole reversed 66 and 46% of the met-enkephalin-mediated inhibition of IL-8-induced chemotaxis, respectively
(data not shown).
Intracellular Calcium Flux.
In addition to their ability to
generate chemotaxis of leukocytes, chemokines induce intracellular Ca2+ fluxes in many responding cells. Met-enkephalin and morphine were assessed for their ability to
induce changes in intracellular Ca2+ concentration, and for
any activity in heterologously reducing chemokine-induced
Ca2+ mobilization. There was no evidence of direct induction of Ca2+ flux by met-enkephalin in monocytes, nor did
preincubation with varying concentrations of met-enkephalin
alter the Ca2+ flux induced by activating doses of MIP-1
(Fig. 5). Analysis of the effect of met-enkephalin on the
EC50 of MIP-1 demonstrated a Ca2+ flux response at low
doses. Peak Ca2+ flux occurred using 10 ng/ml MIP-1,
and the EC50 was ~2 ng/ml, with no significant difference
between untreated and met-enkephalin-pretreated monocytes
(data not shown). The chemokines IL-8, NAP-2, MCP-1, MCP-3, and RANTES, and the chemoattractant FMLP,
were also assessed in neutrophils and monocytes, as appropriate, and their capacity to induce Ca2+ flux was similarly
unaffected by met-enkephalin or morphine pretreatment (data not shown).
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Fig. 5.
Effect on Ca2+ flux induced by MIP-1 1 µg/ml in untreated monocytes or monocytes pretreated with met-enkephalin 1011
M, 109 M, and 107 M. Met-enkephalin 109 M did not induce a Ca2+
flux in these cells. Representative experiment of four similar experiments.
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CXCR1 Internalization.
To determine if the ability of
opiates to inhibit chemokine-induced chemotaxis arises
through chemokine receptor internalization, epitope-tagged CXCR1-transfected RBL2H3 cells (24) were examined by confocal laser microscopy. Initial experiments
demonstrated that these cells responded chemotactically to
IL-8, and that this chemotactic effect was inhibited in a
naloxone-sensitive manner by met-enkephalin pretreatment, confirming the presence of functional opiate receptors (data not shown). After preincubation with IL-8 1 µg/
ml for 30 min, the transfected cells showed prominent internalization of CXCR1, but no internalization was seen in
response to preincubation for 60 min using met-enkephalin
109 M (Fig. 6). Similar observations were made for CXCR2
on human neutrophils and CCR5 on human monocytes
(data not shown).
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Fig. 6.
Absence of CXCR1
internalization in response to
pretreatment of epitope-tagged
CXCR1-transfected RBL2H3
cells with met-enkephalin. Transfected cells were (A) untreated,
(B) incubated with IL-8 1 µg/ml
for 30 min, or with (C) met-
enkephalin 10-9 M for 3 h. Representative micrographs from >10
experiments are shown.
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Chemokine Binding after Opiate Treatment.
To confirm
the lack of receptor internalization in response to opiates,
we examined the ability of met-enkephalin to inhibit binding of CXC and CC chemokines to their specific receptors. Using 125I-labeled chemokines and met-enkephalin on
fresh human neutrophils and monocytes, there was no evidence in direct competition assays of binding of met-enkephalin to receptors for IL-8, NAP-2, MCP-1, MCP-2, MCP-3,
MIP-1, MIP-1, or RANTES. Similarly, after preincubation of cells with met-enkephalin for 60 min, there was
no impairment of chemokine binding (Fig. 7), suggesting
that chemokine receptor affinity and number were not
modulated by this duration of met-enkephalin treatment.
Therefore, the observed met-enkephalin effects on chemotactic responses were not occurring at the level of chemokine receptor binding of ligands.
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Fig. 7.
(A) Scatchard analysis of MIP-1 binding to untreated
monocytes, showing 5,356 binding sites per cell. After pretreatment of
monocytes with met-enkephalin 10-9 M for 60 min (B), the binding characteristics of MIP-1 were unchanged, with 6,080 binding sites per cell
detected. Representative plots of two experiments. B/T, Bound/total. B/F,
Bound/free.
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Involvement of NO.
Recent data suggest that morphine-mediated effects in monocytes and neutrophils may be due
to the generation of NO (30). Monocytes treated with the
NO synthase inhibitor L-NMMA (NG-monomethyl-L-arginine monoacetate) before incubation with opiates showed
no change in the opiate-induced inhibition of MIP-1
chemotaxis, nor did the NO donor diethylamine dinitric
oxide (31) at 500 µM, which generates NO at a concentration ~104-fold higher than the inhibitory concentration
thought to be produced by morphine-stimulated monocytes (30), reproduce the opiate effect (data not shown).
Thus, opiate-induced inhibition of chemokine effects is
not mediated by NO.
Phosphorylation of Chemoattractant Receptors.
Since opiate
molecules, like chemokines, mediate their actions through
STM receptors, the possibility that inhibition of chemokines occurred by a process of heterologous desensitization
was next examined. For technical reasons, analysis of
chemokine receptor phosphorylation in neutrophils and
monocytes was unsuccessful; in view of this, phosphorylation was examined in a receptor transfected cell system.
Epitope-tagged FMLP receptor-expressing and epitope-tagged CXCR1-expressing RBL2H3 cells were incubated
in phosphate-free medium, then with [32P]orthophosphate.
They were then stimulated with FMLP 106 M (for the
FMLP receptor-expressing cells), IL-8 500 ng/ml (for the
CXCR1-expressing cells), or met-enkephalin 109 M. After whole cell lysis, chemoattractant receptors were immunoprecipitated and examined on SDS-polyacrylamide gels
exposed to radiographic film (18). The results demonstrate
that in addition to the expected phosphorylation of the
chemoattractant receptors induced by the native ligands,
CXCR1 was phosphorylated in response to met-enkephalin (Fig. 8). This phosphorylation was inhibited by naloxone (Fig. 8). CXCR2 immunoprecipitated from retinoic
acid-differentiated U937 cells also was found to be phosphorylated after exposure of the cells to met-enkephalin
(data not shown). The FMLP receptor was not phosphorylated in response to met-enkephalin treatment (Fig. 8).
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Fig. 8.
Phosphorylation of
chemoattractant receptors in response
to native ligands, met-enkephalin, or
naloxone and met-enkephalin. Equivalent numbers of cells for each treatment were analyzed. Lane 1, Untreated (unt); lane 2, native ligand
(FMLP [fMLP] 10-7 M or IL-8 1 µg/ml); lane 3, met-enkephalin
(met-enk); lane 4, naloxone plus met-enkephalin (nal + met-enk). Representative of three experiments.
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Discussion |
This paper describes for the first time a plausible and intriguing mechanism by which opiates may mediate the impaired leukocyte function observed in drug abusers and in
animal models of opiate abuse. It also suggests the possibility of an immunomodulatory role for endogenous opiates
in controlling leukocyte recruitment. It confirms earlier
studies (26, 27) showing that opiate molecules can act directly, albeit weakly, as chemoattractants, and demonstrates that this functional effect is pertussis toxin-sensitive, suggesting coupling of opiate receptors in monocytes to the
Gi subclass of G proteins. Although this has been shown
for opiate responses in hippocampal slices (32) and µ receptor-transfected Jurkat cells (33), to our knowledge this is
the first analysis of G protein use by opiate receptors on native leukocytes.
The heterologous desensitization of chemokine responses
observed in these experiments has definite selectivity, since
the FMLP receptor is unaffected and CCR5 may also be
relatively spared, indicating that there is no generalized impairment of cell motility. The intracellular controls that
generate this apparent selectivity remain to be clearly defined,
although sparing of the FMLP receptor in heterologous
phosphorylation processes has been observed previously and may reflect the absence of protein kinase C phosphorylation sites in intracellular portions of the receptor (18). A
further clue to molecular mechanisms of opiate-induced
desensitization is suggested by the lack of inhibition of
chemokine-induced Ca2+ flux and absence of chemokine
receptor internalization by opiates, despite both impairment of
the chemotactic response and enhancement of receptor
phosphorylation. These features imply a lower requirement for G protein activation by chemokine receptors for Ca2+
mobilization than for chemotaxis. Alternatively, by analogy
with studies examining cross-regulation among chemoattractant receptors (34), there may be levels in addition to
receptor phosphorylation at which desensitization occurs.
Studies are planned to identify the roles of various receptor
kinases and intracellular receptor residues involved in the divergent desensitization responses observed here.
The effects of met-enkephalin observed in monocytes
and neutrophils are mediated through µ and but not opiate receptors. All three receptor types are known to be
expressed by leukocytes (11), and their differential ability to interact with chemokine receptors suggests the possibility of using specific receptor agonists as potential antiinflammatory molecules. The lack of efficacy of agonists in
inhibiting chemokine effects contrasts with the ability of receptor activation to inhibit replication of HIV-1 in microglial cells, the central nervous system equivalent of resident macrophages (15), suggesting divergence between blood monocytes and tissue macrophages in opiate receptor
expression or responsiveness. The molecular mechanisms
of opiate receptor selectivity in chemokine receptor desensitization remain to be elucidated. Although some opiate-mediated, and specifically morphine-mediated, effects on
leukocytes appear to involve NO-dependent devices (30), it is clear from our results that desensitization of chemokine responses by µ and opiate receptor activation occurs independently of NO generation.
This study has broad implications for the inhibition of
leukocyte responses by opiate molecules, and indeed by
other apparently nonimmune mediators acting through
STM receptors. The data suggest a mechanism by which
opiates function as antiinflammatory or immunosuppressive agents, by interfering with chemokine-induced directional
migration of inflammatory cells. That inhibition or absence
of chemokines can markedly suppress inflammatory reactions has been demonstrated in several animal models of
disease by administration of neutralizing antibodies (35,
36), as well as in virally infected chemokine deletion mutants (37). Therefore, exposure of leukocytes to opiates in
vivo may result in profoundly impaired host responses. This may have direct clinical relevance, such as in the setting of analgesia given in the presence of infectious or surgical insults or in the intravenous drug user exposed to infectious agents. However, this study may also have relevance
in the potential modulation of immune and inflammatory
responses due to endogenous opiate molecules, which
among other neuropeptides are known to be released by
cells of the immune system (38), and which may act as normal regulators of chemotaxis. These molecules also may
play a role in altering inflammatory responses in pathophysiological states such as septic shock (39). Moreover, the
finding of opiate-induced neuroimmunomodulation of
chemokine function raises the prospect that other neuroendocrine mediators, acting through STM receptors, might
operate through similar mechanisms, a possibility we are currently investigating.
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Footnotes |
Address correspondence to Michael C. Grimm, Laboratory of Molecular Immunoregulation, National Cancer Institute - Frederick Cancer Research and Development Center, Bldg. 560, Rm. 31-19, Frederick, MD
21702-1201. Phone: 301-846-1347; Fax: 301-846-7042; E-mail: grimm{at}mail.ncifcrf.gov
Received for publication 10 July 1997 and in revised form 24 April 1998.
This research is sponsored in part by the National Cancer Institute, Department of Health and Human Services, under contract with ABL. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
The authors are grateful to Dr. S.-B. Su for assisting with calcium mobilization experiments; to Drs. P.M.
Murphy (National Institute of Allergy and Infectious Diseases, Bethesda, MD), A. Richmond (Vanderbilt
University, Nashville, TN), and C. Hébert (Genentech Inc., San Francisco, CA) for supplying antibodies; to
Dr. G. Cox of Science Applications International Corp., Frederick, and Dr. R. Shen of ABL-Basic Research
Program, National Cancer Institute - Frederick Cancer Research and Development Center, for their helpful
advice; and to Drs. R. Neta and S. Durum for reviewing the manuscript.
Abbreviations used in this paper
CCR, CC chemokine receptor;
CXCR, CXC chemokine receptor;
CTOP, cys2, tyr3, orn5, pen7 amide;
DAMGO, [D-ala2,N-me-phe-gly5(ol)]-enkephalin;
MCP, monocyte
chemoattractant protein;
MIP, macrophage-inflammatory protein;
NAP, neutrophil-activating peptide;
NO, nitric oxide;
RANTES, regulated
upon activation, normal T expressed and secreted;
STM, seven transmembrane-spanning.
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