Journal of Neuroimmunology 127 (2002) 88 – 95

www.elsevier.com/locate/jneuroim

Abrupt or precipitated withdrawal from morphine induces

immunosuppression
Rahil T. Rahim a, Martin W. Adler b,c, Joseph J. Meissler Jr. a,c, Alan Cowan b,c,
Thomas J. Rogers a,c, Ellen B. Geller b,c, Toby K. Eisenstein a,c,*
a
Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA
b
Department of Pharmacology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA
c
Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, PA 19140, USA
Received 11 January 2002; received in revised form 6 March 2002; accepted 25 March 2002

Abstract

The present studies tested the effect of withdrawal from morphine by two different paradigms, abrupt withdrawal (AW) or precipitated
withdrawal (PW), on the capacity of murine spleen cells to mount an in vitro antibody response. Mice were made dependent by chronic
treatment using s.c. implanted morphine slow-release pellets. Splenocytes were harvested at various time points after withdrawal and the
number of antibody-forming cells determined using a plaque-forming cell (PFC) assay. The results indicate that induction of abstinence from
morphine in dependent mice by either paradigm caused marked immunosuppression between 24 and 48 h post-withdrawal. However, the
kinetics of onset and recovery from immunosuppression were different in AW and PW. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Morphine; Abrupt withdrawal; Precipitated withdrawal; Immunosuppression

1. Introduction to normal responses by 120 h after pellet implantation (Bus-

It is well established that morphine given acutely or
subacutely is immunosuppressive as assayed by several
different parameters including natural killer (NK) cell activ-
ity (Weber and Pert, 1989; Band et al., 1992), T-cell mitogen
responses (Bayer et al., 1990, 1992), and antibody responses
(Bussiere et al., 1992; Eisenstein et al., 1990, 1995). There
are few studies examining the effects of chronic morphine
exposure on immune responses. Bryant et al. (1988a,b)
followed the kinetics of murine splenocyte proliferative
responses to concanavalin A in mice implanted with a 75-
mg slow-release morphine pellet. They observed profound
immunosuppression, which reached a maximum at 48 h but
returned to normal by 96 h after pellet implantation, although
morphine levels in the sera at 48 and 96 h were equivalent.
Previous studies from our laboratory examined murine
splenic plaque-forming cell (PFC) responses to sheep red
blood cells (SRBCs) over time after implantation of a 75-mg
slow-release morphine pellet and showed comparable re-
sults, with maximal immune suppression at 48 h, and a return
*
Corresponding author. Tel.: +1-215-707-3585; fax: +1-215-707-7920.
E-mail address: tke@astro.ocis.temple.edu (T.K. Eisenstein).
siere et al., 1993). Recently, West et al. (1997) have shown
that continuous administration of morphine to rats for 20
days by osmotic minipump led to tolerance in some immune
assays but not others. These studies support the hypothesis
that tolerance to many of the immunomodulatory effects of
morphine develops after chronic exposure to the opioid.
The phenomenon of opioid tolerance is well established
by standard pharmacological tests measuring behavioral,
biochemical, and physiological parameters (Way et al.,
1969; Cicero and Meyer, 1973; Patrick et al., 1978; Berg-
ström et al., 1984; Martinez et al., 1990; Paronis and
Holtzman, 1992). The majority of these studies show that
morphine is a potent analgesic, but upon repeated admin-
istration, its effects diminish. It is also well established that
once tolerance develops, termination of the drug by abrupt
(drug cessation) or precipitated withdrawal (with or without
drug cessation plus administration of an opioid antagonist)
can lead to an abstinence syndrome indicating a state of
physical dependence. There are only two studies in mice
and one in humans on the effects of abrupt withdrawal (AW)
from morphine on immune responses (Bhargava et al.,
1994; Govitrapong et al., 1998; West et al., 1999), and none
that we are aware of on precipitated withdrawal.

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 R.T. Rahim et al. / Journal of Neuroimmunology 127 (2002) 88–95
The present study examines and compares the effects of
precipitated and abrupt withdrawal from morphine on
splenic antibody responses in mice. It was found that abrupt
withdrawal, by removal of morphine pellets from dependent
animals, resulted in profound immunosuppression that was
maximal at 48 h post-pellet removal and was still present at
144 h. In contrast, precipitated withdrawal resulted in a
short period of immunopotentiation at 3 h post-pellet
removal, followed by profound immunosuppression at 24
h post-withdrawal, with a rapid return to normal immune
responses by 72 h. The results are novel, as both paradigms
led to immunosuppression at 24 h post-withdrawal, but
there were marked differences in the kinetics of the effects
on immune responses.
2. Materials and methods
2.1. Mice
Six-week-old C3HeB/FeJ female mice were purchased
from Jackson Laboratories (Bar Harbor, ME) and housed in
sterilized cages with mouse chow and water provided ad
libitum. All mice were acclimated for a minimum of 1 week
prior to being used in experiments.
2.2. Surgery and experimental design
Mice were anesthetized with methoxyflurane and areas
of the back were shaved. A 1-cm incision was made in the
skin and mice were implanted s.c. with either a 75-mg
morphine pellet or a cellulose placebo pellet (The National
Institute on Drug Abuse (NIDA), Rockville, MD) encased
in a 1  1-cm square nylon mesh bag to permit easy
removal. Control mice had no surgery and received no
drugs. Based on published results (Bussiere et al., 1993)
and preliminary studies (Eisenstein and Rahim, 2001), it
was established that mice were tolerant to the immunosup-
pressive effects of morphine by 96 to 120 h post-morphine
pellet implantation. In all experiments, pellets were left in
place for 96 h before removal of the pellet to examine the
effects of withdrawal. The group designated ‘‘morphine-
dependent’’ received a pellet for 96 h and were sacrificed
without removal of their pellets. Withdrawal was initiated
by two paradigms, abrupt and precipitated withdrawal. For
abrupt withdrawal (AW), morphine pellets were surgically
removed from dependent mice 96 hr after implantation, and
at various times from 2 to 144 h after pellet removal, the
mice were sacrificed and the number of antibody-forming
cells determined using the MD-PFC assay (see below). In
the second paradigm, precipitated withdrawal (PW), mor-
phine pellets were removed, and upon recovery from
anesthesia, mice were given two subcutaneous injections
(1 mg/kg) of the opioid antagonist, naloxone, 30 min apart
to measure physical dependence and to assess immunosup-
pression at early time points. After each injection of the
89
antagonist, physical dependence was measured by quantita-
tion of naloxone-precipitated jumping of individual animals
using a PlexiglasR withdrawal chamber. The average num-
ber of jumps per group was determined for each injection.
Physical dependence was also evaluated by measuring
mouse body weight at various times after either abrupt or
precipitated withdrawal, and other signs and symptoms such
as writhing and diarrhea were also noted. For immune
studies, at 3 h after initiation of precipitated withdrawal,
splenocytes were harvested and the number of PFCs deter-
mined. To assess immune responses at time points between
6 and 72 h in mice undergoing precipitated withdrawal,
dependent mice had their pellets removed and they were
implanted s.c. with an AlzetR osmotic minipump adminis-
tering naloxone hydrochloride (1 mg/kg/day at a rate of 1 Al/
h) (The National Institute on Drug Abuse (NIDA), Rock-
ville, MD). Use of the naloxone pump was necessitated due
to the short half-life of naloxone in rodents (Weinstein et al.,
1974). For immune studies, mouse spleen weights were also
obtained at the time of sacrifice.
2.3. Primary in vitro antibody response
Morphine-dependent mice and animals undergoing with-
drawal were sacrificed and the number of antibody-forming
cells was determined using a Mishell – Dutton (MD) plaque-
forming cell (PFC) assay (Mishell and Dutton, 1967). Four
mice per group were sacrificed and their spleens removed.
Spleen cells from two mice in each group were pooled to
obtain two cohorts per treatment. Immune function was
assessed using an in vitro plaque-forming cell (PFC) assay
described previously, which measures the capacity of spleen
cells to mount a primary antibody response to sheep red
blood cells (SRBCs). A single cell suspension of spleen cells
was obtained by pushing the spleen through nylon mesh bags
in RPMI supplemented with 5% fetal calf serum. Red blood
cells were lysed by hypotonic shock with sterile water. Cells
were washed twice and resuspended in tissue culture
medium consisting of RPMI supplemented with 10% FCS,
2 mM L-glutamine, 50 mg/ml gentamicin, 1 mM nonessen-
tial amino acids, 1 mM sodium pyruvate, 10 mg/ml of
adenosine, uridine, cytosine, and guanosine, and 0.05 mM
of 2-mercaptoethanol. The cells were counted and resus-
pended to 1.0  107 cells/ml and dispensed into flat-bottom
24-well tissue culture plates. Sheep red blood cells (Rock-
land, Gilbertsville, PA) were washed three times in sterile
saline and 3.5  106 cells were added to each well in 50 Al.
For each cohort, a minimum of triplicate cultures was tested,
each with its own negative control (i.e., splenocytes without
SRBCs). Cultures were incubated in an atmosphere consist-
ing of 7% O2, 10% CO2, and 83% N2 at 37 jC. Twenty-four
hours later, cultures were fed with nutrient cocktail consist-
ing of RPMI supplemented with approximately 30% FCS,
5.5 mM L-glutamine, 3 mM each of essential amino acids
and nonessential amino acids, 5.5 mg/ml dextrose, 0.61%
sodium bicarbonate, and 40 mg/ml each of adenosine,
90 R.T. Rahim et al. / Journal of Neuroimmunology 127 (2002) 88–95

Fig. 1. Suppression of splenic antibody responses following abrupt withdrawal. Mice were implanted with either a placebo or morphine pellet (encased in
nylon) for 96 h. Mice were then anesthetized and the pellet was surgically removed. Control mice did not undergo surgery or pellet implantation. Values at T = 0
for morphine-pelleted mice represent responses of animals that received a sham operation, leaving morphine pellets in place at the time of sacrifice. Placebo
and morphine groups had their pellets removed at various time points between 2 and 144 h prior to sacrifice and harvest of their spleens for the PFC assay. For
clarity, the early time points up to 6 h are plotted separately in Panel A. Panel B shows all time points up to 144 h post-pellet removal. Antibody responses are
expressed as the mean number of PFC/107 cells F S.E. Each point except the one at 144 h is the average of data from two separate experiments, using two
cohorts per experiment, and each cohort having been done in triplicate. Thus, all points but the one at 144 h are the average of 12 data points. The 144-h time
points were carried out on one cohort in triplicate. p < 0.0001 placebo vs. morphine at 4 to 6 h. p < 0.001 placebo vs. morphine at 24 to 96 h.

uridine, cytosine, and guanosine. On day 5, cells were
harvested, washed in RPMI, and the number of direct PFCs
(cells producing IgM antibodies against SRBCs) quantitated
using the Cunningham modification of the Jerne hemolytic
plaque assay (Cunningham and Szenberg, 1968). Data from
the PFC assay were calculated as the mean number of PFC/
107 cells. Each point represents a minimum of two pooled
cohorts, with each of these cohorts tested in at least triplicate.
Many points are the average F S.E. of more than two cohorts
obtained from multiple experiments.

Fig. 2. Potentiation of the PFC response following precipitated withdrawal. Mice were implanted with either morphine or placebo pellets for 96 h. One group of
placebo and one group of morphine-treated mice had pellets left in place but received a sham operation. The sham-operated group was morphine-dependent.
Other groups of mice underwent precipitated withdrawal by removal of their pellets prior to receiving either two s.c. naloxone (mg/kg) injections (30 min apart)
or two s.c. saline injections. Control mice had no surgery and received no drugs. Spleens were harvested from all experimental groups for the PFC assay at 3 h
post-pellet removal. Antibody responses are expressed as the mean number of PFC/107 cells F S.E. Each point represents data from two experiments, with two
cohorts per experiment, each cohort being done in triplicate. p < 0.0001 morphine + naloxone vs. all other groups.
 R.T. Rahim et al. / Journal of Neuroimmunology 127 (2002) 88–95
2.4. Statistical analysis
Plaque counts, body weights, and spleen weights were
analyzed using a mixed model analysis of variance followed
by pairwise group comparisons using the Dunn – Bonferoni
method or the Scheffe procedure adjusting for multiple
comparisons and random experimental effects. Differences
were considered significant for p values V 0.05.
3. Results
3.1. Abrupt withdrawal (AW) results in suppression of
splenic antibody responses
To examine the effect of abrupt withdrawal on immune
responses, the PFC assay was carried out at various time
points after pellet removal (Fig. 1). At 2 h post-withdrawal,
mice that were implanted with a placebo or a morphine
pellet showed approximately a 50% decrease in the number
of PFCs. By 4 h, the immune response of the placebo
animals had returned to normal, suggesting that the transient
immunosuppression observed at 2 h was a result of the
surgical procedure. In contrast, PFC responses of the mice,
which had received morphine, continued to decline (Fig.
1A). Suppression reached a maximum at 24 to 48 h after
pellet removal. By 48 h post-pellet removal, antibody
responses of mice that had received morphine were sup-
pressed over 75% compared with placebo mice (Fig. 1B).
Suppression was still evident at 96 h (55%). At 144 h, PFC
responses of morphine-abstinent mice were not statistically
different from those at the 96-h time point. However, at 144
h, the placebo value was decreased, but this point represents
data from only one cohort in one experiment. Decreased
PFC responses of morphine-abstinent mice are not due to
changes in body or spleen weight (Fig. 6).
3.2. Measurement of splenic antibody responses following
precipitated withdrawal
To assess immune status shortly after initiation of pre-
cipitated withdrawal, spleens were harvested for the PFC
assay after removal of morphine pellets and administration of
two doses of naloxone. Animals were sacrificed 3 h after
pellet removal. The results show a significant (40%) increase
in PFC responses of animals undergoing withdrawal above
levels of dependent mice which did not have their pellets
removed and did not receive naloxone (Fig. 2). Mice that had
morphine pellets removed and received saline had PFC
responses that did not differ from those of control mice or
dependent, sham-operated mice not undergoing withdrawal.
To examine the effect of precipitated withdrawal on
immune responses at time points beyond 3 h, naloxone (1
mg/kg/day) was administered by s.c. implantation of an
AlzetR osmotic minipump at the time of morphine pellet
removal (Fig. 3). Thus, mice were implanted with either
91
Fig. 3. Modulation of the PFC response following precipitated withdrawal.
Values at T = 0 represent responses of mice that were implanted with either
a morphine or placebo pellet for 96 h and received a sham operation,
leaving pellets in place at the time of sacrifice. Groups at later time points
had their pellets removed and received naloxone. PFC responses were
measured at 3 h after induction of withdrawal by administration of naloxone
injections (as described in Fig. 2). Precipitated withdrawal groups at other
time points were administered naloxone (1 mg/kg/day) using AlzetR
osmotic minipumps for 6 to 72 h. Control mice had no surgery and did not
receive any drug. Antibody responses are expressed as the mean number of
PFC/107 cells F S.E. Each point represents data from at least two pooled
cohorts done in triplicate. The 24-h time point represents three cohorts from
two experiments. Comparison of placebo vs. control values at 24 and 48 h
after withdrawal was made using 95% confidence limits. Placebo at 24 h
vs. control is significant. Placebo at 48 h vs. control is not significantly
different.
morphine or placebo pellet for 96 h. The pellets were then
surgically removed and replaced with an osmotic minipump
administering naloxone. Induction of precipitated with-
drawal by implantation of a naloxone minipump resulted
in normal PFC responses at 6 h post-withdrawal but
profound immunosuppression (80%) by 24 h post-with-
drawal. PFC responses were returning to normal levels
(20% suppression) by 48 h and had returned to control
levels by 72 h post-withdrawal. Some immunopotentiation
(compared with control values) was observed in placebo-
pelleted animals at 24 h (by comparison of 95% confidence
limits) and at 48 h (not statistically different). Comparison
of PFC values of abstinent mice at these time points shows
highly significant immunosuppression whether compared
with values of control or placebo animals.
3.3. Comparison of PFC responses following abrupt or
precipitated withdrawal
A comparison of the kinetics of alterations in the PFC
responses of mice undergoing abrupt or precipitated with-
drawal is shown in Fig. 4. First, at 3 h after precipitated
92 R.T. Rahim et al. / Journal of Neuroimmunology 127 (2002) 88–95

post-withdrawal. It is evident that the shape of the recovery

curve is different for the two withdrawal paradigms. For
abrupt withdrawal, suppression was sustained at 144 h post-
withdrawal, in contrast to the results obtained following
precipitated withdrawal where PFC responses returned to
normal by 72 h.

3.4. Withdrawal from morphine pellets results in behavioral

signs of physical dependence

In order to demonstrate that 96 h after pellet implantation

Fig. 4. Comparison of kinetics of abrupt vs. precipitated withdrawal from
morphine on PFC responses. All mice received morphine in vivo by s.c.
implantation of a 75-mg pellet for 96 h. In the first paradigm, abrupt
withdrawal was induced by removal of the pellet. In the second paradigm,
naloxone was used to induce precipitated withdrawal in mice which had
their pellets removed prior to receiving naloxone s.c. (1 mg/kg injections in
the 3-h time point or 1 mg/kg/day by osmotic minipumps for the other time
points).
withdrawal, PFC responses were potentiated, whereas those
of mice undergoing abrupt withdrawal showed a steady
decline with no window of potentiation. Time points at 2, 4,
5, and 6 h were done for abrupt withdrawal (Fig. 1A) to
assure that if potentiation occurred, it would be detected.
PFC responses of mice subjected to either withdrawal
paradigm showed profound immunosuppression by 24 h
mice were physically dependent on morphine, signs of an
abstinence syndrome were measured when animals were
subjected to precipitated withdrawal (PW). Jumping activity
was quantitated following removal of the morphine pellet
and administration of naloxone or saline (Fig. 5). Morphine-
abstinent mice receiving naloxone had an average of 14
jumps after the first injection, whereas morphine-abstinent
mice receiving saline showed no significant jumping activ-
ity. Mice that had a placebo pellet removed and received
either naloxone or saline also showed no jumping activity.
Mice subjected to abrupt withdrawal did not show increased
jumping.
Physical dependence was also verified by the loss in
body weight following either abrupt or precipitated with-
drawal (Fig. 6A and B). In both withdrawal paradigms,
morphine-abstinent mice had a significantly greater loss in
body weight between 6 to 48 h than mice that had a
placebo pellet removed at the same time points. For abrupt
withdrawal mice, body weights returned to normal by 96 h
post-initiation of abstinence, a time when immune res-
ponses were still suppressed. In contrast, in precipitated
withdrawal mice, body weights were still significantly sup-

Fig. 5. Demonstration of physical dependence by naloxone-precipitated jumping activity. Mice (four to eight per group) in each experiment were implanted
with either a placebo or morphine pellet for 96 h. The mice were then anesthetized and the pellet surgically removed. Upon recovery, the mice were placed in
withdrawal chambers and then given either an s.c. naloxone injection (1 mg/kg) in a volume of 0.2 ml or an injection of saline (s.c.). The mice were then
allowed to rest for another 30 min before administration of a second injection of either naloxone or saline. Jumping activity of each mouse was scored within a
15-min period after each injection. Data are expressed as a mean F S.E. and are the average of three individual experiments. p < 0.01 morphine plus saline vs.
morphine plus naloxone after the first naloxone injection.
 R.T. Rahim et al. / Journal of Neuroimmunology 127 (2002) 88–95 93

Fig. 6. Effect of withdrawal on mouse body and spleen weights. Mice were implanted with either morphine or placebo pellets for 96 h prior to induction of
either abrupt or precipitated withdrawal. Data are mean F S.E. of body weights (Panels A and B) and spleen weights (Panels C and D) in grams. For abrupt
withdrawal, graphs are pooled results of three experiments with data points representing from 4 to 13 individual treated mice per group. For precipitated
withdrawal, graphs are pooled results of five experiments with data points representing from 4 to 20 individual treated mice per group. Not all time points were
done in each experiment. p < 0.01 placebo vs. morphine at 0 and 4 to 48 h (Panel A). p < 0.01 placebo vs. morphine at 3 to 72 h (Panel B). p < 0.001 placebo vs.
morphine at 0, 2, and 24 to 96 h (Panel C). p < 0.001 placebo vs. morphine at 24 to 72 h (Panel D).

pressed at 72 h, a time when immune responses had returned 4. Discussion

to normal.
Spleen weights of morphine-abstinent mice were also
significantly decreased by 6 h post-withdrawal with a nadir
at 24 to 48 h (Fig. 6C and D). For abrupt withdrawal mice,
spleen weights returned to normal by 144 h post-withdrawal.
As was observed for body weight, there was a lack of
correlation between levels of immunosuppression and spleen
weight. In precipitated withdrawal, immune responses were
normal at 72 h but spleen weights were still suppressed.
There was a parallel between depressed body weights and
spleen weights. Other symptoms of the abstinence syndrome,
such as writhing and diarrhea, were observed in many
animals subjected to either withdrawal paradigm, but were
not quantified for these studies.
This study is the first to compare the effects of both
abrupt and precipitated withdrawal from morphine on
immune responses in mice and shows that withdrawal, by
either paradigm, results in profound immunosuppression.
The present study is also the first to examine and compare
the kinetics of abrupt and precipitated withdrawal on
immune responses. Mice were made dependent by admin-
istration of a morphine pellet for 96 h and a morphine-
abstinence syndrome was initiated by two different para-
digms. In the first paradigm, mice undergoing abrupt
withdrawal (surgical removal of the morphine pellet)
showed a steady decline in their PFC responses starting
at 4 h post-pellet removal. Maximal (80%) suppression of
94 R.T. Rahim et al. / Journal of Neuroimmunology 127 (2002) 88–95
PFC responses occurred at 24 and 48 h post-withdrawal
with significant suppression (50%) still evident at 144 h. In
the second paradigm, mice undergoing precipitated with-
drawal had a morphine pellet removed and received nalox-
one. In contrast to abrupt withdrawal, at 3 h following
precipitated withdrawal, PFC responses were potentiated
by 40% above normal levels. However, PFC responses
returned to normal levels by 6 h post-withdrawal, followed
by 80% suppression by 24 h, and a return to normal PFC
levels by 72 h. Thus, while withdrawal by either paradigm
suppressed the capacity of spleen cells to make antibody,
the kinetics of each are different. Early immunopotentiation
was observed in precipitated withdrawal but not in abrupt
withdrawal, and abrupt withdrawal induced a longer lasting
immunosuppression. Evidence presented in this study
shows that mice implanted with a slow-release morphine
pellet for 96 h developed drug dependency, as evidenced
by abstinence-induced jumping activity following precipi-
tated withdrawal, and a significant loss in body and spleen
weights following either withdrawal paradigm. In addition,
results from the present study support data from West et al.
(1999), which showed that abrupt withdrawal led to sig-
nificant decreases in body weight with a return to normal
by 96 h post-withdrawal.
A few general observations can be made about the
parameters that influence the PFC response and how it
might be modified by abstinence. First, although there were
alterations in spleen weights as a result of withdrawal, all
results are expressed as PFC/107 cells, which normalizes
the data accounting for alterations in spleen size. Also,
spleen size was a poor correlation of immunosuppression,
as mice undergoing abrupt withdrawal had normal spleen
weights by 72 h, but immune responses were still sup-
pressed at 144 h. Alternatively, mice undergoing precipi-
tated withdrawal had normal immune responses by 72 h
but spleen weights were still suppressed at that time point.
Immunosuppression of the PFC response could occur if
there was an alteration of the ratio of B-cells, T-cells, and
macrophages, as all three cell types are needed for develop-
ment of cells secreting antibody to SRBCs. Preliminary
data show that ratios of B-cells, T-cells, and macrophages
are not significantly altered at 24 h after withdrawal, nor is
there evidence of apoptosis at various time points up to 24
h (unpublished data).
The present study shows that precipitated withdrawal has a
time-dependent effect on immune responses. PFC responses
were elevated above control levels immediately after the
induction of precipitated withdrawal. This result may be
comparable to data from previous studies with more tradi-
tional pharmacological endpoints showing that precipitated
withdrawal results in a rebound effect that is opposite to the
acute effects of the drug (Sharma et al., 1975; Nestler, 1992).
However, the period of immunoenhancement observed in the
present studies is short, and suppression of immune responses
is observed by 24 h post-withdrawal, with a return to normal
PFC responses by 72 h.
In regard to abrupt withdrawal in the present study,
maximal immunosuppression occurred at 24 to 48 h follow-
ing pellet removal with suppression still evident by 144 h.
No immunopotentiation was observed. In the literature,
Bhargava et al. (1994) reported that 8 h after abrupt with-
drawal from slow-release pellets in mice, cytotoxic T-cell
activity, B-cell proliferation to mitogen stimulation, and
production of IL-2 by splenocytes were significantly
inhibited, but not NK cell activity. In contrast, West et al.
(1999) reported that rats exposed to morphine in their
drinking water for 20 days and then subjected to abrupt
withdrawal had depressed splenic NK cell activity that was
maximal at 12 to 48 h post-withdrawal. Responses to
mitogens and IFN-g levels were also suppressed, but the
kinetics were different, with maximal suppression at 12 h
post-withdrawal and a return to normal levels by 24 h.
These studies and the results presented in the current paper
support the conclusion that abrupt withdrawal suppresses a
number of parameters in the immune system, although the
kinetics for each may vary.
The value of studies on the kinetics of the effects of
withdrawal is evident. The protracted immunosuppression
observed in the present study is comparable to the only paper
in the literature examining the effects of abrupt withdrawal
on immune responses in humans (Govitrapong et al., 1998).
Chronic heroin addicts in Thailand had depressed responses
to a T-cell mitogen, PHA. In addition, alterations in the total
number and percentage of T-cells, B-cells, and NK cells in
the peripheral blood of heroin addicts following abrupt
withdrawal showed that there were increases in T-cells and
B-cells, but a decrease in the CD4/CD8 ratio and in NK cell
levels. Perturbations in the percentages of B-cells, NK cells,
and the CD4/CD8 ratio did not return to normal until 3 years
post-withdrawal. Comparable studies on the immunomodu-
latory effects of precipitated withdrawal in humans have not
been done. The overall duration of abnormal effects
observed in mice in the present studies is comparable to
clinical studies on the kinetics of abstinence signs in pre-
cipitated withdrawal. Opiate-dependent patients undergoing
rapid opiate detoxification by repeated injections of nalox-
one experienced withdrawal symptoms which dissipated by
72 h post-withdrawal (Resnick et al., 1976).
The present experiments show that both abrupt and
precipitated withdrawal from morphine can lead to windows
of profound immunosuppression. The results imply that
intravenous drug abusers experiencing periods of drug
taking followed by periods of withdrawal may be immuno-
compromised. Modulation of immune responses by mor-
phine-abstinence could have both practical and theoretical
implications, as alterations in immune function could lead to
an enhanced susceptibility to infection or reactivation of
latent infections. In support of this concept, Donahoe (1993)
reported that when SIV-infected monkeys (Macaca mulatta)
were subjected to precipitated withdrawal, their viral load
increased. Thus, withdrawal in HIV-infected individuals
may have adverse consequences for disease progression.
 R.T. Rahim et al. / Journal of Neuroimmunology 127 (2002) 88–95
Further, maintenance of constant drug levels, for example,
by methadone, may be beneficial once tolerance develops
(Novick et al., 1989; Kreek et al., 1990).
Acknowledgements
This work was supported by National Institute on Drug
Abuse grants DA06650, DA11134, and DA13429-01. We
also thank Dr. John P. Gaughan for assistance with statistical
analyses.
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