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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
JPET 286:931–937, 1998
Vol. 286, No. 2
Printed in U.S.A.
Rat Natural Killer Cell, T Cell and Macrophage Functions after
Intracerebroventricular Injection of SNC 801
JASON E. NOWAK, RICARDO GOMEZ-FLORES, SILVIA N. CALDERON, KENNER C. RICE and RICHARD J. WEBER
Section of Medical Sciences, Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria,
Illinois (J.E.N., R.G.F., R.J.W.) and Laboratory of Medicinal Chemistry, NIDDK, NIH, Bethesda, Maryland (S.N.C., K.C.R.)
Accepted for publication March 24, 1998
This paper is available online at http://www.jpet.org
Mu, delta and kappa opioid receptor classes have been
identified in neural tissue (Pert and Snyder, 1973; Simon et
al., 1973; Terenius, 1973), and their stimulation has the
potential to relieve pain. However, in addition to analgesic
properties, mu opioid receptor agonists have been associated
with the alteration of immune responses through central or
peripheral pathways (Shavit et al., 1986; Weber and Pert,
1989; Bayer et al., 1992; Carr et al., 1993; Lysle et al., 1993;
Flores et al., 1995). ICV injection of b-endorphin and
DAMGO has been reported to enhance rat splenic NK cell
activity (Jonsdottir et al., 1996) and nitric oxide production
(Iuvone et al., 1995), respectively. In contrast, ICV injection
of morphine and b-endorphin was shown to suppress Con A-,
PHA- or LPS-induced rat splenic lymphocyte proliferative
responses (Lysle et al., 1996; Panerai et al., 1994). In addition, morphine action in the periaqueductal gray matter of
Received for publication October 3, 1997.
1
This work was supported by National Institutes of Health R01 Grant
DA/AI08988. Animals used in these studies were maintained in accordance
within the Guide for the Care and Use of Laboratory Animals.
splenic lymphocyte proliferation compared to controls. Additionally, SNC 80 did not significantly affect splenic T cell or
natural killer cell populations as measured by the expression of
T cell receptorab, and T helper (CD4), T suppressor/cytotoxic
(CD8) and natural killer cell surface markers. Finally, SNC 80 did
not affect interferon-g- or lipopolysaccharide (LPS)-induced
splenic nitric oxide, and LPS-induced tumor necrosis factor-a
production by splenic macrophages. These results suggest that
SNC 80 could be useful in the treatment of pain without suppressing immune function. However, the potential immunoenhancing properties of SNC 80 may be also valuable in immunocompromised individuals.
the mesencephalon has been linked to immunosuppression
(Weber and Pert, 1989; Lysle et al., 1996), through central
mu receptors (Band et al., 1992). Because of their effects on
immune function, mu opioid agonists are not optimal for pain
management in many clinical situations when suppression of
immune function is undesired, such as AIDS patients, burn
victims or cancer patients with intractable pain who opt for
immunotherapy. We previously reported lack of immunosuppression following administration of buprenorphine, a partial
agonist at mu opioid receptors (Williams et al., 1991).
Early studies with delta opioid agonists included naturally
occurring peptidic ligands such as enkephalins and deltorphin, or exogenous analogues such as D-Pen2,D-Pen5enkephalin (DPDPE). These peptidic compounds are rapidly
degraded by the human body thus limiting their clinical
application. In contrast, nonpeptide opioids are more stable,
and their usefulness as analgesics without affecting immune
function has been proven (Williams et al., 1991). Burroughs
Wellcome synthesized the nonpeptide molecule BW373U86
which was shown to produce analgesia via the delta opioid
receptor (Chang et al., 1993). This compound, however, had
ABBREVIATIONS: SNC 80, (1)-4-[(a R)-a-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; Con A, concanavalin A; FACS, fluorescent antibody cell sorter analysis; FITC, flourescein isothiocyanate; HPLC, high-performance liquid chromatography;
IL-2, interleukin-2; ICV, intracerebroventricular; NK, natural killer; antiTCR, IgG1 monoclonal antibody to rat T cell receptorab; CD4, IgG1
monoclonal antibody to rat T helper cells; CD8, IgG1 monoclonal antibody to rat T suppressor/cytotoxic cells; NKCR, IgG1k monoclonal antibody
to rat NK cells; LPS, lipopolysaccharide; DPDPE, D-Pen2, D-Pen5-enkephalin; DAMGO, H-Tyr-d-Ala-Gly-Phe(N-Me)-Gly-ol.
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ABSTRACT
We investigated the effects of (1)-4-[(a R)-a-((2S,5R)-4-allyl2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC 80), a nonpeptidic delta-opioid receptor-selective
agonist, on rat leukocyte functions. Intracerebroventricular injection of SNC 80 (20 nmol) in Fischer 344N male rats did not
affect splenic natural killer cell activity compared with intracerebroventricular saline-injected controls. SNC 80 also had no
effect on concanavalin A-, anti-T cell receptor-, interleukin-2and anti-T cell receptor 1 interleukin-2-induced splenic and
thymic lymphocyte proliferation in most experiments. In some
experiments, however, SNC 80 significantly (P , .01) caused a
41 to 93% increase of concanavalin A-, anti-T cell receptor-,
interleukin-2- and anti-T cell receptor 1 interleukin-2-induced
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Nowak et al.
several adverse effects, such as convulsions and barrel rolling
(Comer et al., 1993). Calderon et al. (1994) were able to use
BW373U86 to derive its optically pure methyl ether enantiomer SNC 80, a compound proven to be a potent d-selective
analgesic (Bilsky et al., 1995).
ICV therapy has been reported to be as effective as other
neuraxial treatments to control pain (Ballantyne et al.,
1996). ICV opioid treatment has been successfully used to
control refractory pain due to cancer when systemic treatments have failed (Ballantyne et al., 1996). Our study was
conducted to investigate the effects of acute ICV injection of
SNC 80 on NK cell and T cell, and macrophage functions.
Materials and Methods
suspensions were centrifuged for 7 min at 1400 rpm and supernatants were discarded. Pellets were then resuspended in 2 ml of red
blood cell lysing buffer; after homogenizing, 2 ml AIM-V medium was
added, and suspensions centrifuged for 7 min at 1400 rpm. After this,
supernatants were discarded, and pellets were resuspended in 3 ml
AIM-V medium. Splenic cells were then counted visually, adjusted to
a density of 4.5 3 106 cells/ml in this medium, and incubated for 2 hr
in flat-bottomed 96-well plates (Becton Dickinson). Nonadherent
cells were removed, and adherent cells were then incubated overnight in 100 ml AIM-V in the presence or absence of IFN-g (50 U/ml)
(higher doses of IFN-g resulted in more than 30% reduction of macrophage viability, data not shown); the final monolayer consisted of
.95% macrophages as judged by morphology and phagocytic activity.
NK cell assay. NK cell cytotoxic activity was assessed by the
chromium release assay using [51Cr]-labeled YAC-1 murine lymphoma cell line as reported previously (Weber and Pert, 1989).
YAC-1 cells were labeled by incubating 107 cells with 200 mCi sodium
51
chromate (NEN Research Products, Boston, MA) for 2 hr at 37°C,
and then washed three times with RPMI 1640 medium and resuspended in this medium to a density of 5 3 104 cell/ml. YAC-1 cells
were added to round-bottomed 96-well plates (Becton Dickinson,
Lincoln Park, NJ) containing splenic cells at various concentrations
to give effector/target ratios ranging from 25:1 to 400:1. Spontaneous
and maximal 51chromium release were obtained by incubating
[51Cr]-labeled YAC-1 cells in AIM-V medium alone or medium containing 2% sodium dodecyl sulfate plus 0.1N HCl, respectively. After
4 hr of incubation, supernatants were harvested and 51Cr release
was measured in a gamma counter (Packard, Downers Grove, IL).
Four separate wells per animal per effector:target were analyzed; the
mean of the four wells was used for final data analysis.
T cell proliferation assay. T cell proliferation was determined
by [3H]-thymidine uptake as previously reported (Lysle et al., 1993).
Thymic and splenic cells were adjusted to 5 3 106 cells/ml and
cultured in round-bottomed 96-well plates (Becton Dickinson). Cell
cultures were then incubated in the presence or absence of Con A,
aTCR (5 mg/ml), IL-2 (5% of a 24-hr conditioned medium from Con
A-stimulated splenic cells) and aTCR 1 IL-2 for 48 hr. [3H]-thymidine (1 mCi/well, ICN Pharmaceuticals Inc., Costa Mesa, CA) was
added 4 hr before the end of the incubation period. Cell cultures were
then harvested with a semiautomatic cell harvester (Tomtec, Orange, CT) and cell-incorporated radioactivity determined using a
Microbeta Plus liquid scintillation counter (Wallac Oy, Turku, Finland). Three wells were analyzed for each animal with each stimulant studied; the mean of the three wells was used for final data
analysis.
FACS. Spleen and thymus cell suspensions containing 1 3 107
cells/ml were incubated in ice for 20 min with 10 ml of FITC-labeled
IgG1 monoclonal antibodies to TCR (rat T cell receptorab), CD4 (rat
T helper cells), CD8 (rat T suppressor cells) and NKCR (rat NK cell
receptor) surface markers. Cells were washed two times with Hanks’
balanced salt solution containing 5% fetal bovine serum and 0.02%
sodium azide, then washed one time with Hanks’ balanced salt
solution alone. The cells were then fixed with 2% paraformaldehyde,
and percent of cells with TCR, CD4, CD8 and NKCR surface markers
was determined by FACS analysis.
Nitrite determination. Accumulation of nitrite in the supernatants of macrophage cultures was used as an indicator of nitric oxide
production by resident or activated cells. Resident macrophages and
macrophages activated with IFN-g (50 U/ml) or LPS (25 ng/ml,
higher doses do not significantly increase nitric oxide production,
data not shown) were incubated at 37°C in an atmosphere of 5%
CO2-95% air for 3 days in a total volume of 200 ml AIM-V medium per
well. After incubation, supernatants were obtained and nitrite levels
were determined with the Griess reagent as reported elsewhere
(Gomez-Flores et al., 1997a), using NaNO2 as standard. Optical
densities at 540 nm were then determined in a microplate reader
(Molecular Devices Corporation, Palo Alto, CA).
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Reagents and culture media. Rat rIFN-g (specific activity of
4 3 106 U/mg), penicillin-streptomycin solution, and DMEM/F12,
RPMI 1640 and AIM-V media were obtained from Life Technologies
(Grand Island, NY). SNC 80 was synthesized by Dr. Silvia N. Calderon at National Institutes of Health (NIH), and generously donated by Dr. Kenner Rice from the NIH. LPS from Escherichia coli
serotype 0128:B12, methanol, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, sodium dodecyl sulfate, HCl, Con A, red
blood cell lysing buffer and fetal bovine serum were purchased from
Sigma Chemical Co. (St. Louis, MO). FITC-labeled monoclonal antibodies (IgG1) to aTCR, T helper cell (CD4) and T suppressor cell
(CD8) surface markers were obtained from Harlan Bioproducts for
Science (Indianapolis, IN). FITC-labeled monoclonal antibodies to
NKR-P1 (anti NK cells) surface marker (Chambers et al., 1989) were
kindly provided by Dr. William Chambers of the Pittsburgh Cancer
Institute (Pittsburgh, PA). Cell sorter analysis was performed on a
Becton Dickson FACScan (San Jose, CA). Actinomycin D was obtained from ICN Pharmaceuticals (Aurora, OH). The murine fibrosarcoma cell line L929 was purchased from American Type Culture
Collection (Bethesda, MD) (clone CCL 1).
Animals. Fischer 344N male rats (150 –250 g), purchased from
Harlan Sprague Dawley (Indianapolis, IN), were housed three to
four per cage with water and rat food available ad libitum. Measures
were taken to reduce micro-organism infestation of our colony by
housing our animals in a room separate from the general University
vivarium. Animals were anesthetized by i.m. injection of 100 mg/kg
ketamine (Fort Dodge Laboratories, Inc., Fort Dodge, IA) and 20
mg/kg xylazine (Bayer Corporation, Shawnee Mission, KS) after
which a double 23-gauge stainless steel guide cannula (outer diameter, 0.02 inches; inner diameter, 0.01 inches) was stereotaxically
implanted 0.75 mm above the right lateral ventricle using the following coordinates: anterior-posterior, 21.0 mm; mediolateral, 11.5
mm; dorsoventral, 23.5 mm in reference to the bregma. Rats were
allowed 10 days for recovery from cannulation surgery before morphine injection and were adapted to handling daily by being picked
up and held in the identical manner used during injections.
Drug preparation and administration. SNC 80 was dissolved
in pyrogen-free saline to a concentration of 2 nmol/ml. Ten microliters
of SNC 80 or vehicle (saline) was then administered ICV (total dose
of SNC 80 was 20 nmol) at the speed of 10 ml/min with a microinjection pump (Harvard Apparatus, Southnatick, MA). Three hours
after morphine injection, rats were killed by asphyxia with CO2.
Cell preparation and culture. Spleen and thymus were removed immediately after the rat was killed. Single-cell suspensions
were prepared by disrupting the organs in RPMI 1640 medium
supplemented with 0.5% penicillin-streptomycin solution. Lymphocyte suspensions were washed three times in this medium, and the
final pellets were resuspended and adjusted at appropriate densities
with AIM-V medium containing 0.5% penicillin-streptomycin solution. The culture medium was changed at this step to the serum-free
medium AIM-V which has been observed to support cell culture
(Kaldjian et al., 1992). For the macrophage assays, 2 ml spleen
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Rat Immune Status after ICV Injection of SNC 80
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TNF-a assay. TNF-a production by macrophages was determined
by the L929 bioassay. In brief, macrophage monolayers were incubated in the presence or absence of 25 ng/ml LPS, in a total volume
of 200 ml of AIM-V medium, for 4 hr after which supernatants were
collected and kept at 280°C until use. TNF-a levels in the supernatants were then quantified by the L929 bioassay as described elsewhere (Gomez-Flores et al., 1997b). The bioassay was performed in
D-MEM/F12 medium using 1⁄2 serial dilutions of the supernatants.
Recombinant murine TNF-a (a gift from NCI Biological Resources
Branch, Rockville, MD, lot 88/532) was used as standard. After 24 hr
of incubation, cell viability of the L929 cells was determined by a
colorimetric technique using methanol,3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide to a final concentration of 0.5 mg/
ml, and incubating the cells for 1.5 hr at 37°C (Belkowski et al.,
1995). Formazan crystals were dissolved in DMSO and optical densities at 540 nm were determined in a microplate reader (Molecular
Devices Corporation). TNF-a levels represented the inversed of the
dilution causing 50% cytotoxicity, and were expressed in U/ml (Klostegaard, 1985).
Histology. The brains were removed, fixed in isopentane on dry
ice (240°C) and kept at 280°C. Sequential 40-mm coronal sections
through the injection site were obtained using a freezing-stage cryostat (222°C) and the slides were Nissl-stained. The lateral-ventral
placement of microinjection (Paxinos and Watson, 1986) was confirmed by light microscopy.
Statistical analysis. Each data point for animals within the
same experimental group were pooled and expressed as the mean 6
S.E.M. for each experiment. Four rats were used per experimental
group in every experiment. Level of significance was assessed by
Student’s t test, and by one-way analysis of variance, comparing the
experimental group to the control group at each effector:target ratio
for NK-cell analysis and each level of Con A, aTCR or IL-2 for T cell
analysis.
Results
Effect of SNC 80 on NK-cell activity. As shown in figure
1 (experiments 1–3), ICV microinjection of SNC 80 did not
affect splenic NK cell activity. However, in one experiment
(experiment 3) SNC 80 caused a significant (P , .05) 12 6
0.2% increase of NK cell activity at an effector:target ratio of
200:1 compared with cell response of ICV-injected saline
control.
Effect of SNC 80 on splenic and thymic lymphocyte
proliferation. ICV injection of SNC 80 was associated with
either a significant increase or no effect on splenic and thymic lymphocyte proliferative response to Con A, antiTCR-,
IL-2-, antiTCR 1 IL-2 as compared with ICV-injected saline
control. As observed in figure 2 (experiment 1), SNC 80
caused significant (P , .01) 41 6 1, 42 6 1 and 55 6 3%
increase of splenic lymphocyte proliferation induced by Con
A at doses of 1.25, 2.5 and 5 mg/ml, respectively, compared
with cell response of ICV-injected saline control. Similarly,
SNC 80 caused significant (P , .001) 93 6 11, 106 6 23 and
69 6 16 percent increase of splenic lymphocyte proliferation
in the presence of Con A at doses of 1.25, 2.5 and 5 mg/ml,
respectively (figure 2, experiment 2), compared with cell response of ICV-injected saline control. In addition, significant
(P , .001) 80 6 10, 62 6 7 and 75 6 10% increase in splenic
lymphocyte proliferation in the presence of antiTCR, IL-2
and antiTCR 1 IL-2 respectively, compared with cell response of ICV-injected saline control, were also observed. In
some experiments, ICV injection of SNC 80 did not affect
splenic lymphocyte proliferation induced by Con A (experiment 3, figure 2) or antiTCR, IL-2, and antiTCR 1 IL-2
Fig. 1. Effect of SNC 80 on NK-cell activity. NK-cell activity was measured 3 hr after ICV acute microinjection of SNC 80 or saline, by the
specific lysis of [51Cr]-labeled YAC-1 cells as explained in the text. Data
represent mean 6 S.E.M. of percent cytotoxicity (test cpm-spontaneous
cpm/maximal cpm-spontaneous cpm) of four replicate determinations per
treatment (four rats per treatment). * P , .05 as compared with control.
combination (experiments 1 and 3, figure 2). Similarly, SNC
80 did not affect thymic lymphocyte proliferation induced by
Con A, antiTCR, IL-2 and antiTCR 1 IL-2 combination (experiments 1–3, figure 3).
Effect of SNC 80 on splenic T cell and NK cell populations. As observed in figure 4 (experiments 1–3), ICV
injection of SNC 80 did not affect splenic T cell or NK cell
populations as measured by the expression of TCR, CD4,
CD8 and NKCR surface markers.
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Fig. 2. Effects of SNC 80 on splenic lymphocyte proliferation. Con A-, antiTCR- and IL-2-induced splenic lymphocyte proliferation was measured 3
hr after ICV acute microinjection of SNC 80 or saline, by [3H]-thymidine incorporation as explained in the text. Data represent mean 6 S.E.M. of three
replicate determinations per treatment (4 rats per treatment).
Effect of SNC 80 on nitric oxide and TNF-a production by splenic macrophages. As observed in figure 5, ICV
injection of SNC 80 did not affect IFN-g- or LPS-induced
splenic nitrite and LPS-induced TNF-a production by splenic
macrophages.
Discussion
Opioid agonists selective for mu, delta and kappa opioid
receptors have been shown to possess the ability to alleviate pain (Zaki et al., 1996). However, in vivo injection of
mu opioid receptor selective agonists has been reported to
suppress rat splenic B (ICV injection, Lysle et al., 1996)
and T cell proliferation (s.c. injection, Lysle et al., 1993;
Flores et al., 1996; ICV injection, Lysle et al., 1996). They
have also been shown to suppress a variety of functions
including murine T cell-mediated cytotoxicity (s.c. injection, Carr et al., 1995), production of rat (s.c. injection,
Fecho et al., 1996) or murine (s.c. injection, Scott and Carr,
1996) interferon-g, NK cell cytotoxic activity in rats
(s.c. injection, Lysle et al., 1996; Fecho et al., 1996; PAG injection, Weber and Pert, 1989; Lysle et al., 1996; ICV injection,
Lysle et al., 1996; Band et al., 1992), mice (s.c. injection, Scott
and Carr, 1996; Carr et al., 1994), Rhesus monkeys (s.c.
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Rat Immune Status after ICV Injection of SNC 80
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Fig. 3. Effects of SNC 80 on thymic lymphocyte proliferation. Con A-, antiTCR- and IL-2-induced thymic lymphocyte proliferation was measured 3 hr
after ICV acute microinjection of SNC 80 or saline, by [3H]-thymidine incorporation as explained in the text. Data represent mean 6 S.E.M. of three
replicate determinations per treatment (four rats per treatment).
injection, Carr et al., 1993) and humans (Provinciali et al.,
1996), and phagocytosis of Candida albicans by murine (s.c.
injection, Rojavin et al., 1993) or human (s.c. injection,
Tubaro et al., 1987) macrophages.
Delta selective opioid compounds are devoid of many of the
adverse effects seen with mu selective opioid agonists, including, in most cases, immunosuppression. They also have
been shown to have diminished abuse potential. For these
reasons, it is essential to develop analgesics which are selective for delta rather than mu opioid receptors (Rapaka and
Porreca, 1991). Early studies were performed with deriva-
tives of the naturally synthesized delta opioid receptor-selective enkephalin peptides, such as DPDPE, in search of new
analgesics. It was found that certain delta selective agonists
enhance lymphocyte proliferation even in the absence of mitogen (Hucklebridge et al., 1990). Band et al. (1992) reported
that ICV injection of DPDPE did not significantly alter NK
cell function. However, DPDPE and other peptidic opioids
were shown to be very unstable in animal models and had a
low potential to cross the blood brain barrier, thus limiting
its use (Hambrook et al., 1976).
SNC 80 has been tested and proven to be a potent analge-
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Nowak et al.
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Fig. 4. Effects of SNC 80 on splenic cell populations. Expression of splenic
lymphocyte and NK-cell surface markers was determined 3 hr after ICV
acute microinjection of SNC 80 or saline, by using FITC-labeled IgG1
monoclonal antibodies to TCR (rat T cell receptorab), CD4 (rat T helper
cells), CD8 (rat T suppressor cells) and NKCR (rat NK cell receptor)
surface markers, as explained in the text. Data represent mean 6 S.E.M.
of three replicate determinations per treatment (four rats per treatment).
sic, acting at the delta opioid receptor, in both rats and
Rhesus monkeys (Calderon et al., 1994; Bilsky et al., 1995).
In the results reported in this study, we generally observed
that ICV injection of SNC 80 did not affect NK cell, T cell and
macrophage functions (figures 1–5). In some experiments we
showed that this opioid increased T cell proliferative responses to various stimulus (figures 1 and 2). Therefore, the
delta opioid receptor selective agonist SNC 80 could potentially be used in many different clinical situations where
immunosuppression is undesirable as shown for mu selective
ligands such as morphine (Weber and Pert, 1989; Bayer et
al., 1992; Carr et al., 1993; Lysle et al., 1993; Flores et al.,
1995). Furthermore, SNC 80 was observed to enhance T cell
proliferative responses in some instances thus making this
compound potentially suitable in treating not only pain, but
also ameliorating the immune status of immunocompromised
individuals. SNC 80 could be used as a reference to further
develop analgesics with minimal or no impact, even with
enhancing properties, on immune functions.
Acknowledgments
The authors thank Mary E. Riley, Amod Sureka and Aiqin Wang
for technical assistance.
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