Journal of Neuroimmunology 83 Ž1998.

45–56

Evidence for opioid receptors on cells involved in host defense and the
immune system
a,b,)
Burt M. Sharp , Sabita Roy c , Jean M. Bidlack d

a
Endocrine-Neuroscience and Neuroimmunomodulation Research Labs., Minneapolis Medical Research Foundation, Minneapolis, MN, USA
b
Department of Medicine, Hennepin County Medical Center, UniÕersity of Minnesota, Minneapolis, MN, USA
c
Department of Pharmacology and Surgery, Minneapolis Veterans Administration and UniÕersity of Minnesota, Minneapolis, MN, USA
d
Department of Pharmacology and Physiology, UniÕersity of Rochester, NY, USA

Received 7 March 1997; received in revised form 20 October 1997; accepted 24 October 1997

Abstract

Although the role of opiates and opioids in the physiological and pathological function of the immune system is only beginning to be
unraveled, converging lines of evidence indicate that the opioid receptors expressed by immune cells are often the same or similar to the
neuronal subtypes, particularly d and k . Recent studies also point to the existence of novel opioid receptors andror binding sites on
immune cells that are selective for morphine. Opioids and their receptors, particularly those with high affinity for d agonists, appear to
function in an autocrinerparacrine manner. Thus, opioid peptides generated from immune-derived proenkephalin A act as cytokines,
capable of regulating myriad functions of both granulocytes and mononuclear cells. Further identification and characterization of
receptors and signal transduction pathways that account for some of the unique properties of opiate binding and immunomodulation Že.g.,
dose-dependent effects of morphine that occur at exceptionally low concentrations relative to the K d’s of the neuronal m receptor or the
morphine binding site reported on activated human T-cells. represents one of the major research challenges ahead. Elucidating
mechanisms, such as these, may provide unique therapeutic opportunities through the application of opioid immunopharmacology to
disorders involving immune responses in peripheral organs and the central nervous system. q 1998 Elsevier Science B.V.

Keywords: Mu; Delta; Kappa

1. Introduction its in vitro effects occur at exceptionally low concentra-

tions ŽpM–fM range. relative to the affinity of morphine
Opiate alkyloids and opioid peptides such as morphine for neuronal opiate receptors. In contrast, morphine has
and b-endorphin affect the function of lymphocytes and been reported to bind to activated human T-cells with an
other cells involved in host defense and immunity. Phar- approximate K d of 100 nM and minimal stereospecificity.
macological studies of cellular function and biochemical The immunomodulatory effects of b-endorphin have been
determinations of ligand binding and opioid receptor mR- shown to depend on both naloxone-sensitive and
NAs indicate that both opioid receptors and atypical bind- naloxone-insensitive receptors. Lastly, measurements of
ing sites for morphine, naloxone and b-endorphin are the mRNAs encoding the neuronal types of opiate receptor
expressed by leukocytes. Several examples will suffice to have detected rather low levels in immune cells. Although
illustrate this complexity. In the case of morphine, many of much progress has been made, in general, the structures of
the receptors mediating the immunomodulatory effects of
specific opiate alkyloids and peptides have not been identi-
fied definitively and precise structure–function relation-
)
Corresponding author. MMRF, 914 S. 8th St., D-3, Minneapolis, MN
ships have not been defined. This review will critically
55404, USA. Fax: q1 612 3473915; e-mail: assess the functional, biochemical and molecular evidence
[email protected] for opioid receptors on leukocytes.

0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

PII S 0 1 6 5 - 5 7 2 8 Ž 9 7 . 0 0 2 2 0 - 8
46 B.M. Sharp et al.r Journal of Neuroimmunology 83 (1998) 45–56

2. Discussion Sharp, 1995.. This was blocked by naltrindole. These

inhibitory effects required pre-treatment with the DOR
2.1. d-like opioid receptors and the expression of selective agonists prior to stimulation by anti-CD 3-´ . Sev-
proenkephalin A eral approaches have been used to show that the endoge-
nous enkephalins produced by mixed thymocyte cultures,
2.1.1. Functional eÕidence for d-like opioid receptors largely composed of T-cells, inhibited con A-stimulated
Functional studies demonstrating the activation of T-cell proliferation ŽLinner et al., 1995.. Thus, naltrindole,
leukocytes by opiate agonists selective for the d type of anti-methionine–enkephalin antibodies and an anti-sense
opioid receptor ŽDOR. provided some of the earliest evi- oligonucleotide cDNA all enhanced proliferation. Taken
dence for the expression of DOR by cells involved in host together, these investigations suggest that d-like opioid
defense. Stefano et al., observed that wD-Ala2 , D-Met 5 x-en- receptors are constitutively expressed by normal murine
kephalinamide ŽDAMA. rapidly stimulated immunocytes T-cells and that activating these receptors may enhance or
obtained from hemolymph of the mollusc Mytilus edulis inhibit T-cell proliferation, depending on such factors as
to increase their cellular area and to cluster, with a peak the presence of other cells Že.g., accessory cells. and the
effect at 10 pM concentration and an inverted U-shaped condition of culture Že.g., presence of absence of serum
dose-response relationship ŽStefano et al., 1989.. These containing undefined growth factors..
effects on cellular morphology and locomotion were
2.1.2. EÕidence for d-like opioid receptors from ligand
blocked by naloxone Ž10 nM.. Similarly, DAMA induced
binding studies
human peripheral blood granulocytes to elongate. Mollusc
A limited number of laboratories have successfully
and human cells also responded to the DOR agonists
wD-Pen2 , D-Pen5 x-enkephalin ŽDPDPE. and wD-Ala2 , D- detected d-like opioid receptors on leukocytes by conven-
tional radioligand binding assays. One group reported that
Leu5 x-enkephalin ŽDADLE.; however, maximal effects re-
w 3 Hx-deltorphin bound to a single high affinity site on
quired 100-fold greater concentrations than with DAMA.
membranes from human peripheral blood polymorphonu-
In a subsequent study, Stefano et al. found that wD-Ala2 x-
clear leukocytes ŽStefano et al., 1992.. The K d was 0.45
deltorphin I Ždeltorphin., a selective DOR agonist, had
nM with a Bmax of 2.0–8.0 pmol gy1 protein. The authors
similar effects at 10 pM concentrations ŽStefano et al.,
noted considerable variability in Bmax values between
1992.. The U-shaped dose-effect relationship was again
assays using glass-on-glass homogenization. Moreover,
evident. They also showed that deltorphin enhanced the
homogenization with a Brinkmann Polytron frequently led
con A-induced proliferation of unseparated murine spleno-
to a loss of detectable binding. Based on the differential
cytes. However, little dose-dependency was evident in
effects of pretreatment with wD-Ala2 , Leu5, Cys 6 x-enkepha-
these studies.
linamide ŽDALCE–NH2., an irreversible ligand, on the
Studies of human peripheral blood mononuclear and
binding of w 3 Hx-deltorphin compared to w 3 Hx-DPDPE, these
polymorphonuclear leukocytes have shown that methion-
binding studies suggested that human granulocytes express
ine–enkephalin stimulates chemotaxis ŽVanEpps and Sa-
two types of d-like opioid receptor ŽFowler and Fraser,
land, 1984; Foris et al., 1987.. In addition, studies of
1994.. Unexpectedly, the high affinity deltorphin binding
human T-lymphocyte chemotaxis have shown that both
site had a relatively high affinity for wD-Ala2 , MePhe 4 ,
leucine and methionine enkephalin, and enkephalin analogs
GlyŽol.5x-enkephalin ŽDAMGO., a highly selective m opi-
such as DADLE, DPDPE and wD-Ser 2 , Leu5 x-enkephalin-
oid agonist. This report indicates that cells involved in host
Th6 ŽDSLET., were chemotactic ŽHeagy et al., 1990.. The
defense express d-like opioid receptors; some of these may
effects of the naturally occurring enkephalin pentapeptides
Ž10y1 1 –10y5 M. were inhibited by naloxone Ž10y5 M. differ from those present in the CNS.
Covalent cross-linking with selective d ligands has
and were directly dose-dependent, in contrast to the fore-
provided some evidence for the presence of d-like opioid
going reports of Stefano et al. Ž1989, 1992..
receptors on macrophages and lymphocytes. Specific bind-
Both the endogenous enkephalin-like agonists produced
ing was detected on the macrophage cell line, P388d1, the
by thymic T-cells ŽLinner et al., 1991, 1995. and the
T-cell line, MOLT-4, and the B-cell line, IM-9 ŽCarr et al.,
addition of peptides selective for DOR have been shown to
1989.. Covalent cross-linking with the d selective ligand,
exert complex effects on T-cell proliferation. For example,
cis-Žq.-3-methylfentanylisothiocyanate ŽSUPERFIT. has
within a narrow range of concentrations deltorphin
Ž10y1 3 –10y12 M. enhanced con A-stimulated splenocyte also suggested the presence of d-like opiate receptors on
both B- and T-cell-enriched murine splenocytes and on
proliferation—an effect that was blocked by the highly
human peripheral blood lymphocytes ŽCarr et al., 1988..
selective DOR antagonist, naltrindole ŽPortoghese et al.,
1988; Caroleo et al., 1994.. In contrast, our laboratory 2.1.3. EÕidence for d-like opioid receptors from studies of
reported that 3 enkephalin analogs, including deltorphin, mRNA expression
inhibited the proliferation of highly purified CD4q or CDq 8 Recently, it was reported that simian peripheral blood
murine T-cells that was induced by cross-linking the T-cell mononuclear cells express the DOR mRNA originally
receptor ŽTCR. complex with anti-CD 3-´ ŽShahabi and cloned from hybrid neural cells and present within the
 B.M. Sharp et al.r Journal of Neuroimmunology 83 (1998) 45–56 47

central nervous system ŽEvans et al., 1992; Chuang et al., peak effect was observed between 10y1 1 and 10y9 M and
1994.. The detection and sequencing required reverse tran- an inverted U-shaped dose-response profile was found.
scription followed by polymerase chain reaction ŽPCR.. Naloxone 10y6 M blocked the ME-induced Ž100 nM.
Another laboratory has reported that DOR transcripts were elevation of wCaq2 x i . The efficacy of highly selective m
undetectable in human peripheral blood lymphocyte and and k opioid receptor agonists suggested that ME may be
monocyte populations after PCR, but could be found at interacting with m opioid receptors on both cell lines and
low levels in human T-, B- and monocyte cell lines that both m and k opioid receptors may be coupled to
ŽGaveriaux et al., 1995.. In addition, DOR transcripts were calcium mobilization on B-cells. Since DADLE was less
found in murine splenocytes and in some B- and T-cell effective than DAMGO or U50,488H, the role of d opi-
lines. Most recently, a third group reported that human oid-like receptors was not defined unambiguously in this
peripheral blood lymphocytes and several human lymphoid study.
cell lines expressed DOR transcripts that were nearly Recently, our laboratory reported that pre-incubation
identical to the known sequence from human brain ŽWick with DADLE or b-endorphin enhanced the con A-induced
et al., 1996.. Northern blots with riboprobes were used to mobilization of intracellular free calcium by murine splenic
detect these low levels. T-cells ŽShahabi et al., 1996.. The peak effect of b-en-
Our group ŽSharp et al., 1997. has reported that the dorphin required 10y1 0 –10y9 M concentrations and an
sequence of a PCR transcript amplified from enriched inverted U-shaped dose–response relationship was appar-
Ž90%. murine splenic and lymph node T-cells had 98% ent. In contrast, DADLE showed a direct dose–response
identity with the murine brain DOR ŽEvans et al., 1992.. curve with a maximal effect at 10y7 M concentration.
Using RT–PCR, fresh splenocytes from female Balbrc N-Ac-b-endorphin, which shows minimal potency at neu-
mice were found to express very low levels of this DOR ronal opiate receptors, was unable to substitute for b-en-
mRNA which was enhanced by culturing splenocytes in dorphin; whereas, naltrindole, a highly selective d opiate
the presence of serum-containing media. This increase was receptor ŽDOR. antagonist, inhibited the effect of b-en-
evident by 24 h in culture and persisted at 48 and 72 h; dorphin. In addition, a selective m opiate receptor antago-
con A attenuated this at 24 h and by 72 h the transcript nist was ineffective. Therefore, concentrations of b-en-
was undetectable ŽSharp et al., 1997.. dorphin within the physiological range found in the sys-
temic circulation modulated the increase in T-cell wCaq2 x i
2.1.4. Signal transduction by d-like opioid receptors on induced by mitogenic signals. The efficacy of DADLE
mononuclear cells alone and antagonism by naltrindole suggests that d-type
A limited number of reports provide insight into the opiate receptors may mediate the effects of b-endorphin.
effects that activating d opioid-like receptors have on To further study signal transduction through d opioid
intracellular signal transduction pathways. In one of the receptors, our laboratory has developed a T cell line
earliest studies, rat peritoneal macrophages were incubated ŽDOR-Ju1.1. that stably expresses the neuronal d opioid
with methionine–enkephalin ŽME. at 10y9 –10y7 M con- receptor ŽSharp et al., 1996.. The DOR agonists, deltor-
centrations ŽForis et al., 1986.. These investigators ob- phin and DADLE, elevated wCa2q x i , at concentrations from
served an early influx of Naq that was followed by the 10y1 1 to 10y7 M; both agonists dose-dependently in-
efflux of Caq2 . Intracellular cGMP levels were also in- creased wCa2q x i from 60 nM to peak concentrations of 400
creased by ME 10y8 M and this was inhibited by a rather nM within 30 s ŽED50 of approximately 5 = 10y9 M..
higher concentration of naloxone, 10y5 M. Since highly Naltrindole, a selective DOR antagonist, abolished the
selective DOR agonists and antagonists were unavailable increase in wCa2q x i and pre-treatment with pertussis toxin
at the time of these studies, these findings only suggest was also effective. To assess the role of extracellular
that d opioid-like receptors may be involved in these calcium, cells were pre-treated with EGTA which reduced
signaling events. the initial deltorphin-induced elevation of wCa2q x i by more
More recently, a biphasic effect of ME on intracellular than 50% and eliminated the second phase of calcium
cAMP levels was reported in human peripheral blood mobilization. Additionally, the effect of DADLE on
lymphocytes ŽMartin-Kleiner et al., 1991.. Low concentra- forskolin-stimulated cAMP production was determined.
tions of ME Že.g., 10y1 2 M. induced a three-fold elevation DADLE reduced cAMP production by 70% ŽIC50 of
of cAMP when measured 15 min after treatment, whereas approximately 10y1 1 M. and pertussis toxin inhibited the
higher concentrations Ž10y9 M. reduced cAMP levels 2 h action of DADLE. Thus, the DOR expressed by a trans-
after exposure. No antagonists were used to determine fected Jurkat T-cell line is positively coupled to pathways
whether opioid receptors mediated these effects on cAMP. leading to calcium mobilization and negatively coupled to
ME has been shown to rapidly elevate intracellular free adenylate cyclase.
Caq2 concentrations ŽwCaq2 x i . in human B-cell lines im-
mortalized at early pre-B ŽNalm 6. and mature stages ŽJY. 2.1.5. Expression of proenkephalin A by lymphocytes
of B-cell differentiation ŽHeagy et al., 1992. ME was The regulated expression of proenkephalin A ŽPEA.
effective at concentrations between 10y1 5 and 10y7 M; its mRNA and enkephalin-related peptides has been demon-
48 B.M. Sharp et al.r Journal of Neuroimmunology 83 (1998) 45–56

strated in human, rat and mouse lymphocytes. Zurawski et opioid receptors. In splenocytes obtained from BALBrc
al. Ž1986. first showed that con A-stimulated murine T-cell mice, T-cell-dependent antibody production was inhibited
clones expressed relatively high levels of PEA mRNA by in vitro exposure of the isolated splenocytes to the k
Ž0.1–0.5% of mRNA. that was undetectable in resting agonists U50,488 and U69,593, which was blocked by
cells. In addition, ME-immunoreactivity ŽME-IR. was nor-BNI ŽTaub et al., 1991.. This inhibition was observed
found in culture supernatants. by pre-treating either mouse T-cells or macrophages with
Other laboratories have reported that ME-IR was pre- U50,488 prior to measuring antibody production by the in
sent in supernatants from phytohemaglutinin ŽPHA.- vitro plaque forming assay, suggesting that both cell popu-
stimulated human peripheral blood lymphocytes and in cell lations are targets for k agonists ŽGuan et al., 1994..
extracts from rat mononuclear cells ŽPardros et al., 1989; Similarly, the administration of the k agonist MR2034 to
Rosen et al., 1989; Saravia et al., 1993.. Supernatants from mice in vivo suppressed the antibody response to sheep red
the human lymphocytes contained high, intermediate and blood cells in vitro ŽJankovic and Radulovic, 1994;
low molecular weight ME-IR that was detected 18–24 h Radulovic et al., 1995.. In addition to the high affinity
after PHA-stimulation ŽPardros et al., 1989.. Extracts of rat k-selective agonist U50,488, dynorphin, the endogenous
splenic mononuclear cells contained ME-IR that co-eluted opioid peptide for the k receptor, has been shown to have
with ME on reverse phase HPLC ŽSaravia et al., 1993.. direct effects on immune cells. Dynorphin A Ž1–13. at
This co-eluting material constituted a large fraction of the concentrations as low as 10y1 4 M enhanced superoxide
total splenic ME-IR. Furthermore, rat lymphocytes from production in human polymorphonuclear leukocytes and
spleen, lymph nodes and bone marrow expressed PEA peritoneal macrophages ŽSharp et al., 1985.. This enhance-
mRNA ŽRosen et al., 1989.. In the spleen, the B-cell ment was blocked by the opioid antagonist naloxone,
population was positive for PEA mRNA; this increased suggesting that the k opioid receptor mediated the dynor-
significantly after 3 h of incubation with lipopolysaccha- phin-induced increase in superoxide production. Dynor-
ride, but not with con A. phin and related peptides were also found to enhance
Our laboratory has been involved in collaborative stud- tumoricidal activity of pre-activated murine macrophage
ies showing that PEA mRNA is induced by con A-stimula- cultures, without affecting activation of the macrophages
tion of normal murine thymocytes from 4–5-week-old ŽFoster and Moore, 1987.. Collectively, these studies
CD-1 mice. This occurs in the CD4q population of T-cells strongly suggest the presence of k opioid receptors on
ŽLinner et al., 1991.. In addition, PEA mRNA was consti- cells from the immune system.
tutively expressed in a subpopulation of thymic T-cells on
gestational days 15–16, but not at birth ŽLinner et al., 2.2.2. EÕidence for k opioid receptors from ligand binding
1996.. In thymic T-cells from 4–5 week old mice, the con studies
A-stimulated expression of PEA mRNA was modulated by Demonstrating k opioid binding to a mixed population
IL-1b in a bi-phasic manner ŽLinner et al., 1993.. Subpico- of lymphocytes has been difficult, probably due to the low
molar concentrations of IL-1b enhanced PEA mRNA ex- density of opioid receptors on lymphocytes and the fact
pression by 1.5–2.5-fold, whereas subnanomolar concen- that only subpopulations of lymphocytes may express the
trations were inhibitory. Con A-induced PEA mRNA ex- receptor. Consequently, cell lines have been used to char-
pression was also modulated by deltorphin I; picomolar acterize the presence of k opioid receptors on immune
concentrations enhanced expression and nanomolar con- cells. Fiorica and Spector Ž1988. identified Žy .
centrations inhibited ŽLinner et al., 1995.. These effects of w 3 Hxbremazocine binding sites on the EL-4 thymoma cell
deltorphin I were abolished by the d-2 opioid receptor line; however, this binding site was not stereoselective,
antagonists, naltrindole and naltriben, but not by the d-1 and concentrations of greater than 1 mM U50,488 were
antagonist, BNTX Ž7-benzylidenenaltrexone.. needed to observe complete inhibition of binding. The
macrophage cell line P388d1 expressed binding sites for
2.2. k opioid receptors on mononuclear cells and lym- the k agonist w 3 HxU69,593, but neither dynorphin nor
phoma cell lines naltrexone completely displaced binding ŽCarr et al., 1989..
However, the mouse R1.1 thymoma cell line, derived from
2.2.1. Functional eÕidence for k opioid receptors a thymoma from a C58rJ mouse, expressed a single high
Evidence for the presence of k opioid receptors on affinity binding site for w 3 Hxnaloxone and w 3 HxU69,593
cells from the immune system is derived from studies ŽBidlack et al., 1992.. The order of potency of competing
demonstrating that k agonists exert direct effects on im- ligands, including dynorphin peptides, was consistent with
mune function. For example, the k-selective agonist the presence of a k opioid receptor. This binding site was
U50,488 suppressed peritoneal macrophage phagocytosis further characterized using Žy.w 3 Hxbremazocine, which
of Candida albicans in mice ŽSzabo et al., 1993.. This also bound with high affinity to a single binding site on
inhibition was reversed by the k-selective antagonist nor- R1.1 membranes ŽLawrence and Bidlack, 1992.. Competi-
binaltorphimine Žnor-BNI., suggesting that the suppression tion experiments showed that the site was stereoselective,
of phagocytosis induced by U50,488 was mediated by k and displayed a binding profile consistent with that of the
 B.M. Sharp et al.r Journal of Neuroimmunology 83 (1998) 45–56 49

brain k 1 receptor, described by Clark et al. Ž1989., partic- was deduced from cDNA sequences from human and
ularly, because the site had high affinity for both U50,488 monkey lymphocytes ŽChuang et al., 1995a,b.. Recently,
and -neo-endorphin. In addition, Žy.w 3 Hxbremazocine the full-length nucleotide sequence for the k receptor
binding to R1.1 membranes was potently inhibited by both expressed on the R1.1 thymoma cell line was reported
mono- and divalent cations ŽLawrence and Bidlack, 1992., ŽBelkowski et al., 1995c.. The nucleotide sequence shares
similar to results reported for k opioid binding in brain 99.8% sequence homology and the deduced amino acid
membranes ŽPaterson et al., 1986.. The nucleotides GTP, sequence shares 100% sequence homology with the re-
˜
GDP and the non-hydrolyzable analog guanylyl-5O-imido- ported murine brain k opioid receptor ŽYasuda et al.,
diphosphate further reduced Žy.w 3 Hxbremazocine binding 1993.. Another mRNA population obtained from the R1.1
in the presence of NaCl, whereas other nucleotides were cells possesses a 30-base pair insertion 15 base pairs
ineffective ŽLawrence and Bidlack, 1992.. This study sug- upstream of the initiation codon. This 30-base pair inser-
gested that the k opioid binding site on R1.1 membranes tion is also present in the cDNA of the rat brain k opioid
was coupled to a G protein, as has been reported for brain receptor ŽLi et al., 1993.. These results suggest that multi-
k opioid receptors ŽMack et al., 1985.. The R1.G1 and the ple k opioid receptor mRNA species are present in the
R1EGO cell lines, two derivative cell lines obtained from R1.1 cell line. Splice variants of the k receptor may exist
the mouse R1.1 thymoma, express the k opioid receptor at on cells from the immune system and may provide a
densities that are three- and six-fold, respectively, greater source for protein heterogeneity. The R1.1 cell line is
than the parent R1.1 cell line ŽLawrence et al., 1995b.. negative for the cell surface phenotypic markers CD4 and
Using the R1.1 and related cell lines, radioligand binding CD 8 , characteristics of thymocytes in one of the early
studies have demonstrated that a cell derived from the stages of differentiation ŽBelkowski et al., 1995b.. Using
immune system can express a brain-type k opioid binding cell fractionation techniques, CD4yrCDy 8 thymocytes were
site. isolated and analysis by RT–PCR showed that these pri-
In order to detect the expression of k opioid receptors mary immature thymocytes also expressed mRNA for the
on a mixed lymphocyte population, such as thymocytes k receptor ŽBelkowski et al., 1995b.. The full-length
and splenocytes, an indirect immunofluorescence method sequence for the k opioid receptor has also been detected
that is more sensitive than radioligand binding assays was on human fetal microglia, the resident macrophages of the
developed ŽLawrence et al., 1995a.. Using FITC-AA, a brain ŽChao et al., 1996.. There was 100% identity be-
high affinity k agonist conjugated to fluorescein, followed tween microglial cell cDNA and the human brain k opioid
by an amplification of FITC-AA binding using biotinyl- receptor gene ŽZhu et al., 1995.. Human microglia were
ated anti-fluorescein IgG, followed by extravidin-R-phyco- also shown to express the k opioid receptor protein, as
erythrin, specific labeling of k opioid receptors on thymo- detected by flow cytometry using FITC-AA ŽChao et al.,
cytes from C57BLr6ByJ mice was detected ŽLawrence et 1996.. These studies demonstrate that cDNA for the
al., 1995a; Bidlack et al., in press.. The k-selective antago- brain-type k opioid receptor is present on cells from the
nist nor-BNI inhibited greater than 50% of the phyco- immune system and these cells express the k receptor
erythrin fluorescence measured by flow cytometry. Double protein.
labeling experiments using fluorescent antibodies directed
against the cell surface markers CD4 and CD 8 and the 2.2.4. Signal transduction by k opioid receptors on
labeling of the k receptor were performed on thymocytes mononuclear cells
from 6–8-week-old C57BLr6ByJ mice. Greater than 80% Signal transduction studies have been difficult to per-
of the thymocytes were positive for both CD4 and CD8 form on mixed lymphocyte populations because it has
cell surface markers ŽIgnatowski and Bidlack, 1996.. The been very difficult to detect the presence of the receptor on
k opioid receptor was expressed on greater than 50% of these cells. However, the R1.1 and related cell lines have
the CD4qrCDq 8 thymocytes. This study demonstrates that been useful model systems to address the signal transduc-
the majority of mouse thymocytes express the k opioid tion pathways induced by the binding of k agonists to the
receptor, but at a density that is too low to be detected by receptor. Unlike mixed cell populations, such as thymo-
radioligand binding. Incubating nylon–wool purified mouse cytes, splenocytes, and peripheral mononuclear cells, cell
splenocytes with FITC-AA, followed by the amplification lines are a homogenous population of cells that can be
protocol, showed that the k receptor was presented on readily used in second messenger studies. The k receptor
splenocytes from 6–8-week-old C57BLr6ByJ mice ŽBi- on the R1.1 thymoma cell line was shown to be negatively
dlack et al., in press.. coupled to adenylyl cyclase through a pertussis toxin
ŽPTX.-sensitive G protein ŽLawrence and Bidlack, 1993..
2.2.3. Expression of k opioid receptor transcripts by There was strong correlation between the rank order of the
immune cells affinity of k agonists for the receptor, as measured in
The k opioid receptor is a member of the family of radioligand binding assays, and their ability to inhibit both
seven transmembrane receptors that are coupled to G basal and forskolin-stimulated adenylyl cyclase activity
proteins. A partial k opioid receptor amino acid sequence ŽLawrence and Bidlack, 1993.. Incubation of R1.1 cell
50 B.M. Sharp et al.r Journal of Neuroimmunology 83 (1998) 45–56

membranes with PTX and wadenylate- 32 PxNAD q resulted has been shown to increase macrophage superoxide pro-
in the exclusive labeling of a 41-kDa protein, as deter- duction ŽSharp et al., 1985., modulate macrophage oxida-
mined by separating the membrane proteins under reduc- tive burst ŽTosk et al., 1993., enhance macrophage tumori-
ing conditions on a sodium dodecyl sulfate polyacrylamide cidal activity ŽFoster and Moore, 1987; Hagi et al., 1994.
gel, followed by autoradiography. The size of the labeled and increase production of the cytokine interleukin-1 ŽIL-1.
protein is consistent with the molecular weights reported from bone marrow macrophages ŽApte et al., 1990.. In the
for the a subunits of other Gi proteins ŽKatada and Ui, macrophage cell line P388D1, the k agonist U50,488
1982; Law et al., 1985.. Therefore, the k receptor ex- inhibited the synthesis of IL-1 and tumor necrosis factor-a
pressed in the R1.1 cell line is coupled through a Gi ŽTNF-a . ŽBelkowski et al., 1995a.. U50,488 failed to
protein to adenylyl cyclase, similar to brain k receptors modulate IL-6 production in these cells. Both T-cells and
ŽKonkoy and Childers, 1989, 1993.. macrophages are targets for k agonists and the interaction
The cell lines R1.G1 and R1EGO, obtained from the of these cells will orchestrate how k opioids alter immuno-
R1.1 parent cell line, also expressed k receptors that were competence and produce inhibition of T-cell-mediated an-
negatively coupled to adenylyl cyclase ŽLawrence et al., tibody production ŽGuan et al., 1994.. These studies sug-
1995a.. However, there was variation in the amount of gest that k receptors on T-cells and macrophages are
opioid-induced inhibition of adenylyl cyclase activity that involved in maintaining homeostasis of the cells. Over-
was observed among the cell lines. For example, the stimulation of the k receptor on T cells and macrophages
maximal inhibition of adenylyl cyclase activity by by exogenous opioids or endogenous opioid peptides may
Žy.U50,488 was 66%, 49% and 37% for the R1.G1, alter the levels of many cytokines. Changes in cytokine
R1EGO and R1.1 cells, respectively ŽLawrence et al., levels may lead to the suppression of antibody production
1995b.. While the maximal inhibition of adenylyl cyclase ŽTaub et al., 1991; Radulovic et al., 1995..
activity by Žy.U50,488 did not correlate with receptor Using co-cultures of human fetal brain cells and a
number, it did correlate with the % stimulation of low-K m chronically human immunodeficiency virus-1 ŽHIV-1.-in-
GTPase activity by Žy.U50,488. fected promonocytic line U1, dynorphin A Ž1–13. and
The k opioid receptor expressed on the R1.1 cells U50,488 promoted HIV-1 expression ŽChao et al., 1995..
downregulated when the cells were cultured in the pres- Pretreatment with the antagonist nor-BNI completely
ence of U50,488 for 3 h or longer ŽJoseph and Bidlack, blocked this enhancement. The stimulation of HIV-1 ex-
1995.. Culturing cells in the presence of 100 nM U50,488 pression was largely blocked by antibodies to the cy-
for 24 h resulted in a 50% decrease in the number of tokines TNF-a and IL-6, but not IL-10. In addition, dynor-
receptors as measured by the binding of w 3 HxU69,593 and phin stimulated TNF-a and IL-6 expression in the brain
the antagonist w 3 Hxnaloxone to membranes prepared from cell cultures at both mRNA and protein levels, suggesting
the cells. These results are similar to the results obtained that k agonists enhanced HIV-1 expression by increasing
with the neuroblastoma= glioma hybrid NG108-15 cells, the levels of TNF-a and IL-6. In contrast to the chroni-
which expresses the d opioid receptor ŽLaw et al., 1985.. cally infected U1 cells, U50,488, dynorphin A Ž1–13. and
In contrast to studies using neuroblastoma cell lines, the k dynorphin A Ž1–17. inhibited HIV-1 expression in acutely
receptor on R1.1 cells did not desensitize upon chronic infected human microglial cell cultures, in a nor-BNI-sen-
exposure to Žy.U50,488, suggesting that a component sitive manner ŽChao et al., 1996..
required for receptor desensitization may be lacking in Collectively, these findings suggest that k opioids act
these cells ŽJoseph and Bidlack, 1995.. The b-adrenergic directly on k receptors on immune cells. This interaction
receptor kinase Ž b-ARK. has been shown to be necessary probably leads to a number of second messenger events,
for the desensitization of the k receptor expressed in including the inhibition of cyclic AMP levels. These signal
COS-7 cells ŽRaynor et al., 1994.. It is possible that the transduction events may lead to alterations in cytokine
R1.1 cell line expresses b-ARK at levels lower than most levels, which in turn, may alter antibody production and
cells. These cell lines provide an excellent model for susceptibility to viral and bacterial infections. The immune
examining signal transduction pathways mediated by k system expresses many different cell types at varying
agonists in cells from the immune system. By understand- stages of development. The effects of k opioids on each of
ing the signal transduction pathways coupled to k opioid these multiple cell types and the interaction of these cells
receptors on immune cells, the mechanisms responsible for will orchestrate how k opioids alter immunocompetence.
the changes in immune function induced by k agonists and
drugs of abuse will be understood, ultimately leading to 2.3. m-opioid-like receptors (MORS) and atypical m opi-
potential therapeutic interventions. oid binding sites on mononuclear cells and cell lines

2.2.5. Modulation of normal lymphocyte and mononuclear The exact mechanisms responsible for morphine Ža
cell function by k opioid receptors m-type ligand. induced changes in the immune system are
k opioids modulate both cellular and humoral immune not fully defined; they may be directly mediated through
responses. The endogenous k-selective peptide dynorphin opiate receptors present on lymphocytes ŽSibinga and
 B.M. Sharp et al.r Journal of Neuroimmunology 83 (1998) 45–56 51

Goldstein, 1988; Carr et al., 1988., indirectly through creased binding resulted from both an increase in affinity
opiate receptors in the central nervous system, or by Ž K d s 30 nM. as well as increase in the number of recep-
activating the hypothalamic–pituitary–adrenal axis ŽGeo- tors Ž Bmax ..
rge and Way, 1955. to release immunosuppressive gluco- The existence for a similar low affinity, naloxone-insen-
corticoid ŽBryant et al., 1991.. sitive morphine binding site, designated m 3 , has also been
Morphine increases mortality rates in experimentally reported by Makman et al. Ž1995. in human peripheral
infected mice ŽTubaro et al., 1985; Chao et al., 1990.; it blood macrophages. More recently, we have shown that
also inhibits, when administered centrally or peripherally, morphine, in a dose-dependent manner, inhibited the pro-
the cytolytic activity of NK cells as well as mitogen- liferation of a murine macrophagermonocyte cell line, Bac
stimulated lymphocyte proliferation ŽShavit et al., 1986; 1.2F5 ŽRoy et al., 1996.. Characterization of binding sites
Weber and Pert, 1989; Bayer et al., 1990, 1992; Fecho et using w N-methyl- 3 Hx-morphine revealed two populations
al., 1993.. The acute effect of a single subcutaneous of binding sites on these cells. The physiological role of
injection of morphine was found to be biphasic. There was these binding sites is not known. Nevertheless, the exis-
an initial Žfirst 40 min. increase in the phagocytic response tence of these sites lends support to the idea that morphine
followed by suppression after 24 h ŽPacifici et al., 1994.. acts directly on these cells. In contrast to the inhibition of
Additional studies have shown that subcutaneous im- proliferation by morphine, treatment of Bac 1.2F5 cells
plantation of time-release morphine pellets induced rapid with DADLE resulted in a significant increase in prolifera-
and profound thymic atrophy ŽBryant et al., 1987, 1988; tion. These results suggest that this macrophage cell line
Fuchs and Pruett, 1993.. This atrophy reflected an in- expresses two types of opioid receptors—a classical high
creased rate of thymocyte apoptosis. When flow cytomet- affinity d opioid receptor and a low affinity naloxone-in-
ric analysis of thymocytes stained with monoclonal anti- sensitive morphine binding site.
bodies to CD4 and CD 8 was performed, it was found that
the CD4qrCDq 8 thymocytes were depleted to a greater ex- 2.3.2. Expression of m opioid receptor transcripts by
tent than the other subpopulations ŽFuchs and Pruett, 1993; immune cells
Sei et al., 1991; Arora et al., 1990.. With the recent cloning and sequencing of the neuronal
Morphine, after chronic exposure in vivo, attenuates m , d and k opioid receptors ŽEvans et al., 1992., several
lymphocyte proliferative responses ŽBryant et al., 1987., laboratories have sought to detect m-opioid receptors in
NK cell cytotoxicity ŽLefkowiz and Chiang, 1975; Shavit immune cells using RT–PCR. Sedqi et al. Ž1995. were
et al., 1986., antibody and serum hemolysin formation, among the first to report the existence of a m-opioid
ŽGungor et al., 1980. and the phagocytic and killing receptor on rat peritoneal macrophages. Subsequently,
properties of PMN leukocytes ŽTubaro et al., 1985.. Chuang et al. Ž1995a,b., using a similar technique, re-
ported on the presence of m opioid receptors in human
2.3.1. EÕidence for m-opioid receptor(s) on immune cells T-and B-cell lines, CD4q T-cells, monocytesrmacrophages
from ligand binding studies and granulocytes. In addition, transcripts were found in
While the specific mechanisms responsible for the ef- simian peripheral blood mononuclear cells and granulo-
fects of morphine on the immune system are undefined, cytes.
they are thought to be mediated through classical m opioid
receptors Žnaloxone blockade implies an opioid-receptor 2.3.3. m opioid receptor signal transduction in neurons
mediated effect. or through opioid binding sites that are and immune cells
quite different from the classical receptors ŽSibinga and Several studies indicate that neuronal opioid receptors
Goldstein, 1988.. Studies suggest that immune cells con- interact with different G proteins. In reconstitution studies,
tain opioid receptors ŽMadden and Donahue, 1990; it has been shown that both Gi and Go restore the binding
Mehrishi and Mills, 1983; Mendelsohn et al., 1985. and characteristics of the m opioid receptor. Morphine treat-
opioid peptides ŽLolait et al., 1986; Petrov et al., 1986.. In ment of neuronal cells has been shown to stimulate Gi
vitro binding assays, performed to characterize these recep- protein-coupled opioid receptors ŽLaugwitz et al., 1993.
tors, have produced controversial results. Several studies and to modify intracellular levels of potassium, calcium
suggest that these receptors are different from the classical and cyclic adenosine 3,5-monophosphate ŽcAMP. ŽLaw et
opioid receptors present in mammalian brain which are al., 1981; Cooper et al., 1982.. Activation of m receptors
known to mediate analgesia and other in vivo pharmaco- has also been shown to open potassium channels, causing
logical effects ŽSibinga and Goldstein, 1988; Roy et al., hyperpolarization and inhibition of neuronal activity in the
1996.. For example, the morphine receptors expressed on locus ceruleus ŽNorth et al., 1987., and to close calcium
resting thymocytes have relatively low affinity for mor- channels ŽNorth and Williams, 1983.. All three types of
phine Ž K d s 100 nM. ŽRoy et al., 1991a,b.. Interestingly, opioid receptor are known to inhibit adenylyl cyclase, and
interleukin ŽIL-1. activation of thymocytes results in a the subsequent reduction in cAMP levels alters the phos-
dramatic increase in the specific binding of 3 H-morphine phorylation state of several protein substrates of protein
on the activated T-cells ŽRoy et al., 1991a,b.. This in- kinase A ŽOsugi et al., 1991.. To our knowledge, no
52 B.M. Sharp et al.r Journal of Neuroimmunology 83 (1998) 45–56

investigations have been reported showing that similar lation Že.g., b-endorphin and enkephalins. appear to func-
pathways mediate the effects of m opioid ligands on tion as endocrine hormones, mediating the influence of the
immune cells. CNS on the peripheral cells responsible for host defense
and immunity.
2.3.4. Modulation of immune cell function by direct effects Identification of the cell surface molecules that are
of morphine recognized by morphine is a critical area of investigation.
There is considerable evidence for the involvement of Direct binding studies have shown the existence of a novel
cytokines and growth factors in the mechanism of mor- class of low affinity morphine sites on immune cells that
phine-induced immunomodulation. Studies by Peterson et are insensitive to naloxone. Molecular cloning and further
al. Ž1987, 1993. indicated that exposure of con A-stimu- characterization of this binding site will likely yield addi-
lated human peripheral blood mononuclear cells ŽPBMC. tional information on the mechanism of morphine action
to morphine suppressed the production of the lymphokine on immune cells. Sensitive methods such as RT–PCR also
interferon-g ŽIFN.. We have shown that treatment with suggest the existence of neuronal m opioid receptor-related
morphine in vivo decreased the responsiveness to transcripts in immune cells. However, ligand binding stud-
macrophage-colony stimulating factor, but not to granulo- ies have not provided supporting evidence for neuronal m
cytermacrophage stimulating factor ŽRoy et al., 1991a.. opioid receptors on immune cells.
Similarly, it has been shown that cytokines which are Exposure to morphine alters a multitude of physio-
either produced by macrophages or activate them Že.g., logical functions of the immune system. Many of these
IL-6, IL-1b, and interferon-g. will reverse the suppression functional defects involve mature immunocompetent cells
of primary antibody responses by morphine ŽBussiere et that mediate both cell mediated and humoral immune
al., 1993.. In thymocytes, we have shown that morphine in responses. In addition to affecting mature cells, morphine
vivo and in vitro severely inhibits T-cell proliferation in modulates the differentiation of pluripotent stem cells into
response to IL-1. Furthermore, morphine significantly re- the myeloid and lymphoid cell lineages. Specifically,
duced IL-2 synthesis. This was associated with a signifi- chronic morphine affected the M-CSF-dependent genera-
cant reduction in the level of IL-2 mRNA which was due, tion of monocytermacrophage colony forming cells while
at least in part, to transcriptional effects on the IL-2 gene the penultimate step in the differentiation pathway of
ŽRoy et al., 1995.. macrophages was not modified Že.g., GM–CSF induced
colony formation of bone marrow cells..
Morphine also arrests thymocyte proliferation in re-
3. Final comments sponse to mitogens. This anti-proliferative action may
involve the entire population of T-cell lineage—both com-
Converging lines of evidence from numerous laborato- mitted and immature T-cells. Recent work on the effects of
ries show that a variety of cells involved in host defense morphine on thymocytes has shown that opioids interfere
express opioid receptors that are often the same or similar with the expression of the autocrine growth factor, IL-2. It
to the neuronal d , m and k subtypes. Both mononuclear appears that morphine inhibits transcription of the IL-2
and polymorphonuclear cells involved in host defense gene. Since mitogen stimulation of T-cells leads to the
express d-like opioid receptors. Activation of these recep- concomitant expression of both IL-2 and its high affinity
tors results in intracellular signaling that involves the receptor Žheterotrimeric complex., the potential effects of
mobilization of calcium and the regulation of cAMP lev- morphine on IL-2 receptor expression is an important
els. Other signal transduction pathways may also be af- subject for further study.
fected, although these have not been characterized at this Both functional and structural evidence indicates that
time. neuronal k opioid receptors are expressed by cells in-
The expression of d-like opioid receptors and of PEA volved in host defense. k opioid binding to both T-cell
mRNA and methionine–enkephalin-related peptides by lines and normal murine thymocytes has recently been
cells involved in host defense and immunity implies the demonstrated. Recently, the full-length nucleotide se-
existence of an autocrinerparacrine regulatory system. quence for the k receptor expressed on the R1.1 thymoma
These locally generated opioid peptides function as cy- cell line was reported. The nucleotide sequence shares
tokines, regulating myriad functions of both granulocytes 99.8% sequence homology and the deduced amino acid
and mononuclear cells. From an evolutionary point of sequence shares 100% sequence homology with the re-
view, at least with respect to the presence of d opioid-like ported murine brain k opioid receptor ŽYasuda et al.,
receptors, this host defense opioid system appears to be 1993.. Additional findings suggest that multiple k opioid
highly conserved. Thus, opioid signaling by cells involved receptor mRNA transcripts are present in the R1.1 cell
in host defense has probably been adapted for various line. Thus, splice variants of the k receptor may exist on
functions by most species. In addition to the cells from the immune system and may provide a source
autocrinerparacrine significance of this opioid system, for protein heterogeneity. Analysis of normal
opioid peptides which are secreted into the systemic circu- CD4yrCDy 8 thymocytes by RT–PCR has also shown that
 B.M. Sharp et al.r Journal of Neuroimmunology 83 (1998) 45–56 53

these primary immature thymocytes expressed mRNA for Acknowledgements

the k receptor.
k opioids modulate both cellular and humoral immune This work was supported by NIDA DA-04196 ŽB.
responses. The endogenous k-selective peptide dynorphin Sharp., DA-08188 ŽS. Roy., DA-04355 ŽJ. Bidlack. and
has been shown to modulate a number of the functions of DA-09676 ŽJ. Bidlack..
normal macrophages including superoxide and cytokine
production. Furthermore, in the macrophage cell line
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