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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 288:635–642, 1999
Vol. 288, No. 2
Printed in U.S.A.
The Effects of Morphine, Nicotine and Epibatidine on
Lymphocyte Activity and Hypothalamic-Pituitary-Adrenal Axis
Responses
R. DANIEL MELLON and BARBARA M. BAYER
Department of Pharmacology, Georgetown University Medical Center, Washington, DC
Accepted for publication September 1, 1998
This paper is available online at http://www.jpet.org
Evidence implicating the central nervous system (CNS) as
a key regulator of immune function has been presented by
many laboratories (Madden and Felten, 1995). Both the autonomic nervous system (ANS) and the neuroendocrine system have been suggested to be the primary mechanisms
mediating CNS-induced immunomodulation (Madden and
Felten, 1995). Recent studies have demonstrated that selective drugs of abuse modulate both immune cell activities and
the hypothalamic-pituitary-adrenal (HPA) axis. In particular, this and other laboratories have demonstrated that opioid drugs such as morphine suppress the immune system via
activation of central opioid receptors (Mellon and Bayer,
1998a). Similar immunomodulatory effects have also been
reported after nicotine treatment (Caggiula et al., 1992;
McAllister et al., 1994); however, the exact site of action and
mechanism remains to be determined. The similarity of the
Received for publication May 26, 1998.
1
This work was supported by National Institute on Drug Abuse Grants R01
DA04358 (B.M.B.) and F31 DA05779 (R.D.M.). Some preliminary observations
in this paper have been reported previously (Mellon et al., 1996).
glionic antagonist chlorisondamine (0.5 mg/kg, i.p.), completely
blocked the effect of epibatidine on blood lymphocytes without
altering the elevation of corticosterone levels. Although naltrexone (10 mg/kg, s.c.) blocked all effects of morphine, the effects
of epibatidine were not blocked by the opioid receptor antagonist. Furthermore, in contrast to morphine (Hernandez et al.,
1993), central injection of neither nicotine (30 or 240 nmol) nor
epibatidine (5, 50, or 500 ng) altered blood lymphocyte responses. These results suggest that, like morphine, nicotinic
agonists decrease blood lymphocyte proliferation responses,
apparently independent of elevated corticosterone. However,
unlike morphine, nicotinic agonists appear to act predominantly
at peripheral receptors, suggesting that nicotinic receptors are
downstream of opioid receptors in a centrally mediated opioidinduced immunomodulatory pathway.
findings reported with nicotine to those produced by morphine suggest a possible relationship between nicotinic and
opioid receptor-mediated immunomodulation.
To examine the potential overlap between opioid and nicotinic receptors in a neuroimmunomodulatory pathway, we
compared the effects of morphine with those of nicotine, and
also examined the effects of epibatidine, a highly selective
and potent nicotinic receptor agonist. Epibatidine, an alkaloid isolated from skin extracts of the Ecuadoran poison frog,
Epipedobates tricolor, was first described by Daly and colleagues in 1992 (Spande et al., 1992). Initial interest in this
compound was elicited due to its ability to produce both a
Straub tail reaction (typical of opioid receptor activation) and
potent antinociception at doses 200- to 500-fold lower than
morphine. In subsequent studies, epibatidine was shown to
bind with high affinity to the nicotinic receptor and demonstrated little, if any, activity at any other receptor (Badio and
Daly, 1994; Houghtling et al., 1994; Houghtling et al., 1995).
Recent studies suggest that this compound may bind with
high affinity to the subtype of neuronal nicotinic receptor
ABBREVIATIONS: CNS, central nervous system; ANS, autonomic nervous system; DMPP, 1,1-dimethyl-4-phenylpiperazinium; HPA, hypothalamic-pituitary-adrenal; ConA, concanavalin A.
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ABSTRACT
Acute administration of morphine alters various neuroendocrine
and immune parameters via opioid receptors located within the
central nervous system. Similar effects have been reported
after systemic nicotine treatment. To examine the possible
relationship between opioid and nicotinic receptor activation on
the immune system, we compared the effects of morphine with
both nicotine and the highly selective nicotinic agonist, epibatidine. Male Sprague-Dawley rats were treated with either morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. 5 1 mg/kg
freebase), or epibatidine (5 mg/kg, s.c.) and sacrificed 2 hours
later. Each drug increased plasma corticosterone levels and
decreased the magnitude of the peripheral blood lymphocyte
proliferation response to the T cell mitogen concanavalin A.
None of the treatments had a significant effect on splenic or
thymic lymphocyte responses. The effects of nicotine treatment
were dose-dependent. Pretreatment with the quaternary gan-
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Mellon and Bayer
predominating at the autonomic ganglia (Houghtling et al.,
1995; Flores CM et al., 1996) and thus promises to be a
powerful pharmacological tool for the study of autonomic
functions.
The present studies compare the effects of morphine with
those of both nicotine and epibatidine on the proliferation
responses of T lymphocytes and activation of the HPA axis.
To determine the site of action of nicotinic drugs, the effects
of systemic and central administration of nicotine and epibatidine were measured. In addition, the quaternary ganglionic
antagonist chlorisondamine was used to determine the relative contribution of peripheral and central nicotinic receptors
in the modulation of lymphocyte activity.
A preliminary report of some of the observations in this
paper has been presented previously (Mellon et al., 1996).
Materials and Methods
drug dispersal into the ventricle. Flow of the drug solution was
monitored via tracking a small air bubble created in the polyethylene
tubing that separated the drug solution and distilled water. The
animal was returned to the home cage for the duration of the treatment period. Nociception was measured as described 30 min after
either morphine administration or 3 min after either nicotine or
epibatidine treatment. Animals were sacrificed either 1 or 2 h later,
as indicated in the figure legends.
Whole-Blood Lymphocyte Proliferation Assays. Animals
were sacrificed via decapitation and trunk blood was collected in
50-ml polypropylene tubes containing 0.2 ml of heparin (1000 U/ml)
and immediately placed on ice until processed. Whole blood was
diluted 1:5 with cold RPMI 1640 cell culture media (Gibco, Grand
Island, NY) containing 1% fetal bovine serum and gentamicin (20
mg/ml). Triplicate samples of blood (0.1 ml) were plated into 96-well
microtiter plates containing 0.1 ml/well of the T cell-specific mitogen
concanavalin A (ConA; 0, 2, 4, and 6 mg/well) and incubated for 72 h
at 37°C with 8% CO2. Cells were pulsed with 0.5 mCi of [methyl3
H]thymidine (6.7 Ci/mmol; New England Nuclear, Boston, MA) in a
20 ml volume and incubated for an additional 24 h. Samples were
lysed with distilled water and harvested onto glass fiber filters using
a 96-well cell harvester (Brandel, Gaithersburg, MD). The amount of
labeled DNA was determined via liquid scintillation spectrophotometry (Beta Plate; L.K.B. Pharmacia).
Spleen and Thymus Lymphocyte Proliferation Assays. After
decapitation, spleen and thymus were removed via sterile forceps
and each placed in cold RPMI 1640 cell culture media (10 ml) containing 1% fetal bovine serum and gentamicin (20 mg/ml). The tissues were teased apart with sterile forceps, and the resulting cell
suspension was washed twice in cold RPMI 1640 media and adjusted
to a concentration of 2 3 106 cells/ml for spleen and 5 3 106 cells/ml
for thymus. Cell preparations (100 ml) were then cultured with
increasing concentrations of ConA (0, 0.0625, 0.125, 0.25, and/or 0.5
mg/well) as described for the blood proliferation assay to provide
both optimal and suboptimal concentrations of mitogen.
Antinociception. Antinociception was measured by the radiant
heat tail-flick method (D’Amour and Smith, 1941), as described previously (Hernandez et al., 1993). All animals were acclimated to
handling and the tail-flick device for 2 to 3 days before experimental
manipulations. Light intensity was controlled to provide a predrug
latency between 2 and 3 s. A cut-off of 8 s was used to prevent
damage to tail tissue. Data were expressed as the percent maximum
possible effect (% MPE) as defined below:
% MPE 5
Postdrug latency (s) 2 Predrug latency (s)
Cutoff (8 s) 2 Predrug latency (s)
3 100
Plasma Corticosterone Assay. Plasma samples were maintained
at 220°C until assayed via solid phase 125I radioimmunoassay kits
purchased from ICN Biochemicals, Inc. (Costa Mesa, CA). Because
baseline levels of plasma corticosterone were found to vary depending
on the nature of the experimental manipulations involved in the assays,
matched treatment controls were included in all studies.
Histological Analysis. Brains of centrally treated animals were
removed postmortem and stored in 10% formaldehyde followed by
20% sucrose solution. All brains from drug-treated animals were cut
into 40-mm sections on a freezing microtome (HistoSTAT Cryostat
Microtome, model 975c; Reichert, Buffalo, NY) and placed on gelatincoated slides for microscopic verification of cannula placement by
comparison to the atlas of Paxinos and Watson (1997).
Statistical Analysis. Only animals whose cannula were determined to be accurately placed in the ventricle were used for data
analysis. For proliferation assays, the median of triplicate samples
was determined for each concentration of mitogen and the resulting
dose-response curves generated. To compare the ConA dose-response
curves, nonlinear regression analysis was completed to generate the
best-fit curve using GraphPad Prism software (San Diego, CA). For
blood proliferation data, regression analysis indicated that the
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Animals. Pathogen-free male Sprague-Dawley rats (200 –225 g
upon receipt) were obtained from Taconic Laboratories (Germantown, NY). Animals were group-housed three per cage with microisolator tops and were provided food (Purina rat chow, Ralston Purina
Co., St. Louis, MO) and water ad libitum. The light cycle was regulated automatically (lights on at 6 AM, off at 6 PM) and temperature
was maintained at 23 6 1°C. All animals were allowed to acclimate
to this environment for 1 week before use in an experiment or
surgical implantation of a guide cannula into the ventricular system.
All experiments were conducted between 7 and 11 AM.
Drugs. Morphine sulfate was generously provided by the National
Institute on Drug Abuse (Research Triangle Park, NC). Epibatidine
dihydrochloride was purchased from Research Biochemicals International (Natick, MA). Nicotine hydrogen tartrate and naltrexone
hydrochloride were purchased from Sigma Chemical Co. (St. Louis,
MO). Chlorisondamine chloride (Ecolid) was kindly provided by
Ciba-Geigy Corporation (Summit, NJ). All drugs were dissolved in
sterile isotonic saline, which also served as the control treatment in
these studies. The injection volume for all systemic studies was 1
ml/kg and the route of administration was as indicated in the figure
legends. The doses of all drugs reported in these studies are based on
the molecular weight of the salt and not that of the freebase; however, the freebase doses of nicotine are also provided in parentheses
following that of the salt.
Cannulation and Microinjection Procedures. Animals were
anaesthetized with Equithesin (3 ml/kg, i.p.). Equithesin was prepared by dissolving chloral hydrate (4.25 g) and magnesium sulfate
(2.13 g) in a solution consisting of deionized water (43 ml), propylene
glycol (26.6 ml), ethyl alcohol (12.45 ml), and pentobarbital (18 ml, 50
mg/ml). Surgical implantation of a guide cannula into either the
right lateral ventricle (final coordinates relative to bregma: anteriorposterior 5 23.6; medial-lateral 5 24.6, dorsal-ventral 5 27.5) or
the dorsal third ventricle (final coordinates relative to bregma: anterior-posterior 5 24.2; medial-lateral 5 0; dorsal-ventral 5 23.4)
was completed based on the atlas of Paxinos and Watson (1997).
After surgery, a stylet was inserted into the guide cannula to maintain sterility and patency. All animals were given gentamicin sulfate
(4 mg, s.c.) and allowed to recover for approximately 1 week before
experimentation.
Microinjection of drugs was accomplished in freely moving awake
animals as described previously (Hernandez et al., 1993). Briefly,
test animals were removed from the home cage, the protective stylet
was removed, and the internal cannula, which extended 1 mm beyond the end of the guide cannula, was inserted. The animal was
placed into a separate cage for the injection procedure. The injection
volume for all centrally administered drugs was 2 ml, administered
over 45 s using a Sage Infusion pump set to dispense 2.7 ml/min. The
internal cannula remained in place for an additional 75 s to ensure
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1999
Opioid/Nicotinic Immunomodulation
Results
Comparison of the Effects of Morphine with Nicotinic Agonists. The antinociception, lymphocyte suppression, and activation of the HPA axis induced by morphine
were compared to those of the prototypical ganglionic agonist
nicotine and the potent nicotinic agonist epibatidine. The
rationale for the dose of each drug was based on the results
of previously published studies in our and other investigators’ laboratories (Tripathi et al., 1982; Bayer et al., 1992;
Caggiula et al., 1992; Qian et al., 1993; McAllister et al.,
1994). A representative study comparing the antinociceptive
effects of these compounds as measured by tail-flick latency
is shown in Fig. 1. Both nicotine (2.85 mg/kg, s.c. 5 1.0 mg/kg
freebase) and epibatidine (5 mg/kg, s.c.) treatment produced
approximately 60% of the maximum possible effect 3 min
after administration, whereas the response to morphine (10
mg/kg, s.c.) was not significant at this time point. However,
when tested 30 min after drug treatment, the antinociceptive
Fig. 1. Effect of morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. 5 1.0
mg/kg freebase), and epibatidine (5 mg/kg, s.c.) on tail-flick latency to
radiant heat. Animals (n 5 7 per group) were treated with the indicated
drug and tail-flick latency was measured 3 min later. Morphine-treated
animals were again tested 30 min after the injection. Data are expressed
as the maximum possible effect as described in Materials and Methods.
**P , .01 compared with saline and morphine treatment at 3 min, ***P ,
.001 compared with saline and morphine treatment at 3 min, and P , .05
compared with both nicotine and epibatidine treatment (one-way
ANOVA, Newman-Keuls).
effect of morphine was maximal (Fig. 1), whereas by this
time, nicotine and epibatidine were only marginally effective
(data not shown).
In addition to their antinociceptive effects, treatment with
morphine, nicotine or epibatidine significantly decreased the
magnitude (Emax) of the peripheral blood lymphocyte proliferation response to the T cell mitogen ConA when compared
with responses of saline-treated control animals (Fig. 2). No
significant alteration in the sensitivity (EC50) of lymphocytes
to ConA was noted after any drug treatment. In contrast to
the effect of these agents on peripheral blood lymphocytes, no
significant effects were detected in lymphocytes isolated from
either the spleen or the thymus under these conditions (data
not shown).
Since activation of the HPA axis has been reported to
produce altered immune parameters (Parrillo and Fauci,
1979), plasma levels of corticosterone were also measured in
these studies. Figure 3 represents the combined results from
two studies where animals were sacrificed at either 1 or 2 h
after drug administration. As depicted, all drug treatments
produced significantly elevated plasma levels of corticosterone compared with saline treatment when measured at 1 h.
However, only morphine treatment significantly continued to
sustain elevated corticosterone levels for 2 h (Fig. 3).
Dose Dependence of Nicotine-Induced Alterations in
Peripheral Blood Lymphocyte Proliferation. Unlike
epibatidine or morphine, the dose of nicotine used in these
studies (2.85 mg/kg, s.c. 5 1.0 mg/kg freebase) produced some
seizure activity. Because this could complicate the interpretation of the results, experiments were carried out to determine whether the effects of nicotine on lymphocyte responses
could be observed at lower doses. Figure 4 represents the
combined results from two studies testing nicotine at four
Fig. 2. Effect of morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. 5 1.0
mg/kg freebase), and epibatidine (5 mg/kg, s.c.) on blood lymphocyte
proliferation responses to ConA. Animals (n 5 6 –7 per group) were
sacrificed 2 h after drug treatment. All drug treatments significantly
decreased the magnitude (Emax) of the blood lymphocyte proliferation
responses compared with saline-treated controls (***P , .001, one-way
ANOVA, Newman-Keuls). The EC50 for ConA treatment was unaltered
by any drug treatment (P . .05, one-way ANOVA, Newman-Keuls).
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curves best fit a sigmoidal dose-response curve with the following
equation: Y 5 Emin 1 (Emax 2 Emin)/(1 1 10LogEC50 2 X), where Y is
the proliferation response measured in cpm, Emin is the calculated
minimum proliferation response (i.e., absence of mitogen), Emax is
the calculated maximal proliferation response, EC50 is the concentration of mitogen producing 50% of the maximal response, and X is
the logarithm of concentration. From the resulting equations the
values for maximal response (Emax) and EC50 were obtained and
compared. Significant differences in Emax were interpreted as alterations in the magnitude of the response of lymphocytes to ConA
treatment, whereas alterations in EC50 values were interpreted as
alterations in the sensitivity of the lymphocytes to ConA treatment.
For all analyses, Student’s t test was used to compare results from
experiments involving only two treatment groups, whereas a oneway analysis of variance (ANOVA) with Newman-Keuls post hoc
analysis was conducted for comparison of three or more groups.
When no significant difference between basal proliferation in the
control groups occurred, the results obtained in individual experiments were combined directly and proliferation data were expressed
as the mean cpm 6 S.E.M. For all parameters, any value greater
than 2 SDs from the mean of the treatment group was omitted.
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Mellon and Bayer
Fig. 3. Effect of morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. 5 1.0
mg/kg freebase), and epibatidine (5 mg/kg, s.c.) on plasma levels of corticosterone. Animals (6 –7 per group) were treated with the indicated drug
and sacrificed either 1 or 2 h later. Plasma corticosterone was determined
as described in Materials and Methods. *P , .05 compared with salinetreated control value for indicated time (one-way ANOVA, NewmanKeuls).
different doses. ANOVA indicated that nicotine at a dose of
either 2.0 mg/kg (0.7 mg/kg freebase) or 2.85 mg/kg (1.0
mg/kg freebase) significantly decreased the magnitude of
blood lymphocyte proliferation responses compared with saline-treated controls and doses of nicotine at either 0.5 mg/kg
(0.175 mg/kg freebase) or 1 mg/kg (0.35 mg/kg freebase).
Although not significant, a dose of 1 mg/kg (0.35 mg/kg freebase) slightly decreased peripheral blood lymphocyte responses, whereas the lowest dose of nicotine (0.5 mg/kg, s.c.
5 0.175 mg/kg freebase) slightly elevated the response. The
sensitivity of lymphocytes to ConA as measured by EC50
values was not significantly altered. In these studies, nicotine at doses of 2 mg/kg (0.7 mg/kg freebase) or 2.85 mg/kg,
s.c. (1.0 mg/kg freebase) also significantly increased latency
to tail-flick, whereas lower doses were without any detectable
effect (data not shown).
The Role of Central Nicotinic Receptor Activation
on Peripheral Lymphocyte Activity. As nicotine and epibatidine readily enter the CNS, the role of central nicotinic
receptors in the effect of these drugs on peripheral blood
lymphocyte proliferation was examined by administering
these compounds directly into the ventricular system of
freely moving conscious rats. Figure 5 shows the effects of
administration of nicotine at doses of 30 and 240 nmol into
the third ventricle. Neither of these doses of nicotine significantly altered either the magnitude (Emax) or sensitivity
(EC50) of blood lymphocytes to ConA treatment. To maximize
the central tissues potentially exposed to injected drug, the
higher dose of nicotine (240 nmol) was administered into the
lateral ventricle. As shown in the inset in Fig. 5, nicotine
treatment was still without effect on blood lymphocyte proliferation. No significant alterations in spleen lymphocyte
proliferation were noted after microinjection into either the
third or the lateral ventricle under these conditions (data not
shown).
To evaluate whether epibatidine may have centrally mediated effects on lymphocyte activity, the drug was also administered directly into the lateral ventricle of the rat. The doses
of epibatidine used (5, 50 and 500 ng) were based on our
previous experience with central administration of morphine
(Hernandez et al., 1993), which detected significant effects on
blood lymphocyte proliferation and antinociceptive responses
after microinjection of doses 1000-fold lower than those used
systemically. After the central administration of epibatidine,
the highest dose tested (500 ng) produced a slight but insignificant decrease in the magnitude of the peripheral blood
lymphocyte proliferation response (Fig. 6).
Nicotinic Antagonist Studies. To further examine
whether the effects of epibatidine were mediated predominantly by peripheral nicotinic receptors, chlorisondamine
(0.5 mg/kg, i.p.), the quaternary nicotinic antagonist, was
administered 30 min before epibatidine (5 mg/kg, s.c.) treatment. Animals were sacrificed 1 h later, at a time when
epibatidine significantly elevates corticosterone levels (Fig.
3). The rationale for the dose of chlorisondamine was based
on a review of the published literature. Although higher
doses of chlorisondamine have been reported in the literature
(Irwin et al., 1988; Britton and Indyk, 1989; Nikolarakis et
al., 1989; Saito et al., 1991), we chose a lower dose to decrease
Fig. 5. Effect of central (3V) nicotine on the magnitude of the peripheral
blood lymphocyte proliferation response. Animals (n 5 5–7) were treated
with the indicated dose of nicotine directly into the third ventricle and
sacrificed 2 h later. Blood lymphocyte proliferation responses were measured as described in Materials and Methods. No significant differences
in either Emax or EC50 between treatment groups were detected (P . .05,
one-way ANOVA, Newman-Keuls). Inset, nicotine (240 nmol, n 5 11–14)
was administered directly into the lateral ventricle and animals were
sacrificed as above. No significant differences in either Emax or EC50
between treatment groups were detected (P . .05, Student’s t test).
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Fig. 4. Dose-dependent effects of nicotine on the magnitude of blood
lymphocyte proliferation response. Animals were treated with either
saline (n 5 13) or the indicated dose of nicotine (n 5 6 –7 per group) and
sacrificed 2 h later. The proliferation response of blood lymphocytes was
determined as described in Materials and Methods. Calculated Emax
values (cpm): saline, 40,770 6 2,506; 0.5 mg/kg, 47,780 6 5,574; 1.0
mg/kg, 33,040 6 3,738; 2.0 mg/kg, 23,650 6 2,890**; 2.85 mg/kg, 17,900 6
1751***. **P , .01 compared with saline and nicotine 0.5 mg/kg (0.175
mg/kg freebase), ***P , .001 compared with saline, nicotine 0.5 mg/kg
(0.175 mg/kg freebase), and P , .05 compared with nicotine 1 mg/kg, s.c.
(0.351 mg/kg freebase) (one-way ANOVA, Newman-Keuls). The EC50 for
ConA treatment was unaltered by any drug treatment (P . .05, one-way
ANOVA, Newman-Keuls).
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1999
the potential for this quaternary drug to gain access to the
CNS (Clarke, 1984). As previously shown at 2 h (Fig. 2),
epibatidine treatment also significantly suppressed the magnitude of the blood lymphocyte proliferation response within
1 h (Fig. 7). Furthermore, pretreatment with chlorisondamine completely antagonized the suppressive effects of systemic epibatidine on the blood lymphocyte proliferation response.
To examine the possibility that this dose of chlorisondamine may be blocking central nicotinic receptors, plasma levels
of corticosterone were measured. As shown in Fig. 8, epibatidine significantly elevated corticosterone 1 h after treatment; however, pretreatment with chlorisondamine did not
antagonize this effect. Thus, in contrast to the effects of
chlorisondamine on blood lymphocyte proliferation, chlorisondamine did not antagonize the effects of epibatidine on
plasma levels of corticosterone (Fig. 8).
Naltrexone Antagonist Studies. To determine whether
the effects of epibatidine were due to interaction with opioid
Fig. 7. Effect of chlorisondamine pretreatment on magnitude of epibatidine-induced suppression of peripheral blood lymphocyte proliferation
response. Animals (n 5 5– 6 per group) were pretreated with either saline
or chlorisondamine (0.5 mg/kg, i.p.) 30 min before a 1-h systemic treatment of epibatidine (5 mg/kg, s.c.). Blood lymphocyte proliferation response was measured as described in Materials and Methods. Calculated
Emax values (cpm): saline saline, 49,240 6 4,452; saline epibatidine,
21,240 6 3,042 ***; chlorisondamine saline, 55,810 6 5,153; chlorisondamine epibatidine, 45,580 6 3,148. *** P , .001 compared with all other
treatment groups (one-way ANOVA, Newman-Keuls). No significant differences in EC50 were detected (P . .05, one-way ANOVA, NewmanKeuls).
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Fig. 8. Effect of chlorisondamine pretreatment on epibatidine-induced
elevation of plasma corticosterone. Animals were treated as described in
the legend to Fig. 7. Plasma corticosterone was determined as described
in Materials and Methods. **P , .01 compared with saline saline- and
chlorisondamine saline-treated groups (one-way ANOVA, NewmanKeuls).
receptors, animals were pretreated with the opioid receptor
antagonist naltrexone (10 mg/kg, s.c.) 30 min before epibatidine treatment (5 mg/kg, s.c.). Animals were sacrificed 1 h
after epibatidine treatment. As shown in Fig. 9, epibatidine
treatment significantly suppressed the blood lymphocyte proliferation response. In contrast to the effects of the nicotinic
antagonist chlorisondamine, the opioid antagonist naltrexone did not antagonize the effect of epibatidine. Likewise,
epibatidine treatment significantly elevated plasma levels of
corticosterone in an opioid-independent manner (Fig. 10).
Additional experiments were carried out under identical
experimental conditions to confirm that the effects of morphine were sensitive to antagonism by naltrexone. Morphine
treatment (10 mg/kg, s.c.) significantly decreased blood lymphocyte proliferation compared with all other treatment
groups (Fig. 11). However, in contrast to the lack of an effect
of naltrexone in epibatidine-treated animals, naltrexone (10
mg/kg, s.c.) pretreatment completely antagonized the effect
of morphine on the blood lymphocyte proliferation response
Fig. 9. Effect of naltrexone pretreatment on epibatidine-induced suppression of peripheral blood lymphocyte proliferation response. Animals (n 5
6 per group) were pretreated for 30 min with either naltrexone (10 mg/kg,
s.c.) or saline, and then treated with either saline or epibatidine (5 mg/kg,
s.c.) and sacrificed 1 h later. Blood lymphocyte proliferation response to
ConA was determined as described in Materials and Methods. Calculated
Emax values (cpm): saline saline, 38,710 6 5,197; saline epibatidine,
14,000 6 1,235 ***; naltrexone saline, 44,290 6 2,370; naltrexone epibatidine, 17,710 6 2,306 **. **P , .01 and ***P , .001 compared with both
saline saline- and naltrexone saline-treated animals (one-way ANOVA,
Newman-Keuls). No significant differences in EC50 were detected (P .
.05, one-way ANOVA, Newman-Keuls).
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Fig. 6. Effect of central epibatidine (LV) on peripheral blood lymphocyte
proliferation response. Animals were treated with either saline (n 5 14)
or epibatidine at a dose of 5 ng (n 5 9), 50 ng (n 5 4), or 500 ng (n 5 6)
directly into the lateral ventricle and sacrificed 2 h later. Blood lymphocyte proliferation responses were measured as described in Materials and
Methods. No significant differences in either Emax or EC50 were detected
(P . .05, one-way ANOVA, Newman-Keuls).
Opioid/Nicotinic Immunomodulation
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Mellon and Bayer
Fig. 10. Effect of naltrexone pretreatment on epibatidine-induced elevation of plasma corticosterone. Animals were treated as described in the
legend to Fig. 9. Plasma corticosterone was determined as described in
Materials and Methods. **P , .01 compared with saline saline- and
naltrexone saline-treated groups (one-way ANOVA, Newman-Keuls).
(Fig. 11). Likewise, naltrexone antagonized the effect of morphine on the HPA axis (Fig. 12).
Discussion
The close similarity of the reported findings with nicotine on
immune function (Caggiula et al., 1992; McAllister et al., 1994)
with the results of the studies conducted in our laboratory with
morphine led us to further explore the relationship between
nicotinic and opioid-induced alterations in immune function.
The results (Figs. 1–3) with acute systemic morphine, nicotine
and epibatidine treatment presented here demonstrated that
each of these compounds produce: 1) antinociception, 2) decreased magnitude of peripheral blood lymphocyte proliferation
responses to mitogen without altering the sensitivity of the
lymphocytes, 3) no alteration of either splenic or thymic proliferation responses, and 4) an elevation of circulating corticosterone levels. Collectively, these results indicate that the effects of
systemic morphine are largely mimicked by both nicotine and
epibatidine treatment.
Although considerable evidence supports the involvement
of a central site of action for systemic morphine on blood
Fig. 12. Effect of naltrexone pretreatment on morphine-induced elevation
of plasma corticosterone. Animals were treated as described in the legend
to Fig. 11. Plasma corticosterone was determined as described in Materials and Methods. ** P , .01 compared with all other groups (one-way
ANOVA, Newman-Keuls).
lymphocyte responses (Hernandez et al., 1993; Mellon and
Bayer, 1998), the site(s) of action for nicotine-induced immunomodulatory effects has not been well characterized. Based
on several observations in this report, the CNS does not
appear to be the predominant site of action of nicotine- and
epibatidine-induced alterations in peripheral blood lymphocyte responses. First, central administration of either nicotine or epibatidine failed to significantly alter blood lymphocyte proliferation (Figs. 5 and 6). Although the highest dose
of epibatidine tested did produced a slight trend toward a
decrease in blood lymphocyte proliferation, this dose was
only 10-fold lower than the dose used for systemic administration. Second, chlorisondamine, a quaternary nicotinic antagonist, completely antagonized the effects of systemic epibatidine on blood lymphocyte proliferation responses. High
doses of chlorisondamine (10 mg/kg, s.c.) have been reported
to produce prolonged blockade of central nicotinic receptors
(Clarke, 1984; Clarke et al., 1994), suggesting that some of
the drug gained access to the central compartment under
these conditions. However, the lower doses of chlorisondamine used in the present study (0.5 mg/kg, i.p.) had no effect on
the stimulation of the HPA axis by epibatidine (Fig. 8). Because nicotinic receptor-mediated activation of the HPA axis
appears to be due to central nicotinic receptors (Cam et al.,
1979; Matta et al., 1987), these data suggest that it is unlikely that chlorisondamine gained access to the CNS. The
conclusion that this dose of chlorisondamine does not gain
access to the CNS is further supported by studies examining
nicotine-induced antinociception (Caggiula et al., 1995).
These observations also suggest that systemic administration of epibatidine decreases peripheral blood lymphocyte
responses by stimulating peripheral rather than central nicotinic receptors.
Similar to the findings reported here, Caggiula et al. (1992)
observed that systemic nicotine administration to the
Sprague-Dawley rat decreased peripheral blood lymphocyte
proliferation responses in a dose-dependent manner. Additional studies by this group also demonstrated that acute
systemic nicotine treatment in the Sprague-Dawley rat had
no effect on splenic lymphocyte proliferation responses to
ConA, suggesting that the blood lymphocytes were specifically targeted by nicotine (McAllister et al., 1994). However,
in contrast to these observations, in the Lewis rat, Fecho et
al. (1993) found that systemic administration of low doses of
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 11. Effect of naltrexone pretreatment on morphine-induced suppression of peripheral blood lymphocyte proliferation response. Animals (n 5
6 per group) were pretreated for 30 min with either naltrexone (10 mg/kg,
s.c.) or saline, and then treated with either saline or morphine (10 mg/kg,
s.c.) and sacrificed 1 h later. Blood lymphocyte proliferation responses to
ConA were determined as described in Materials and Methods. Calculated Emax values (cpm): saline saline, 76,740 6 6,758; saline morphine,
41,870 6 5,918 ***; naltrexone saline, 95,870 6 7,589; naltrexone morphine, 94,970 6 6,706. *** P , .001 compared with all other groups
(one-way ANOVA, Newman-Keuls). No significant differences in EC50
were detected (P . .05, one-way ANOVA, Newman-Keuls).
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1999
641
munosuppressive (Keller et al., 1983; Keller et al., 1988;
Bayer et al., 1990; Flores et al., 1990).
Overall, the results are consistent with the conclusion that
activation of the autonomic ganglia appear to lead to decreased peripheral blood lymphocyte proliferation. However,
the possibility for a direct effect of nicotinic agonists on
lymphocyte proliferation response has not been eliminated
by these studies. Although there are a few published reports
which provide evidence for nicotinic receptors on lymphocytes (Richman and Arnason, 1979; Maslinski et al., 1980;
Konno et al., 1986; Menard and Rola-Pleszczynski, 1987;
Toyabe et al., 1997), the pharmacology and functional significance of these receptors are not entirely clear.
In summary, these studies provide the first comparison of
the immune effects of nicotine with the potent nicotinic agonist epibatidine and suggest that nicotine and epibatidine
treatment appear to mimic the effects of morphine on peripheral lymphocyte responses. These treatments appear to decrease the overall magnitude of the response to mitogen
treatment without clearly altering the sensitivity of the cells
to this potent stimuli. Although the physiological significance
of this altered cellular responsiveness is not known, this
profile resembles that which would be produced in the presence of a noncompetitive antagonist to immunological stimuli. Given this potential immunomodulatory role, activation
of opioid or nicotinic receptors may lead to a functional,
although attenuated, immune response. Furthermore, these
studies provide evidence that nicotinic agonists produce their
immunomodulatory effects on blood lymphocyte proliferation
by acting predominantly at peripheral nicotinic receptors. In
addition, nicotinic receptor mediated immunomodulation,
like that produced by opioids, appear to be independent of
their ability to activate the HPA axis. Collectively, the studies discussed here suggest that peripheral nicotinic receptors
appear to be located downstream of central opioid receptors
in an neuroimmunomodulatory pathway.
Acknowledgments
We thank Valerie Lewis-Morris and Monica C. Hernandez for
expert technical assistance in these studies, Dr. Kenneth J. Kellar
(Georgetown University) for generously providing epibatidine and
nicotine for the initial studies, and Dr. John C. Pezzullo (Georgetown
University, Washington, DC) and Dr. Harvey J. Morulsky (GraphPad Software, Inc.) for assistance with statistical analyses.
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