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Departments of Microbiology and Immunology,
* Pharmacology, and Center for Substance Abuse Research,
Temple University School of Medicine, Philadelphia, Pennsylvania
Correspondence: Toby K. Eisenstein, Ph.D., Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. E-mail: tke{at}astro.ocis.temple.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-: 100
units/mL) to induce cytokine production. After 24 h RNA was
extracted for analysis of cytokine mRNA levels by reverse
transcriptase-polymerase chain reaction, or supernatants were collected
after 48 h for determination of cytokine production by
enzyme-linked immunosorbent assay (ELISA). Morphine enhanced mRNA
expression of interleukin (IL)-12 p40 and tumor necrosis factor 
(TNF-) compared with controls, whereas IL-10 levels were unchanged
by drug treatment. ELISA data showed that both IL-12 p40 and p70 were
increased by morphine. The enhancement of IL-12 at both the mRNA and
protein levels was antagonized by naltrexone, indicating that the
modulation of this cytokine by morphine is via a classic opioid
receptor. These results are particularly interesting in light of our
previous observation that 48 h after morphine pellet implantation,
the peritoneal cavity is colonized with gram-negative and other enteric
bacteria. The enhancement of IL-12 by morphine might be related to
morphine-induced sepsis.
Key Words: interleukin-10 • interferon- • naltrexone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Proinflammatory cytokines such as interleukin (IL)-1, IL-6, tumor
necrosis factor (TNF-), and IL-12 produced by macrophages are
major mediators of inflammatory responses and also play a prominent
role in the development of sepsis [3
4
5
6
]. It has been
shown that injection of IL-1 or TNF- mimics the symptoms of sepsis
and endotoxic shock in experimental animals [3
,
4
]. Furthermore, antibodies to these cytokines block the
toxic effects of injected LPS [7
, 8
]. Other
cytokines, including IFN-, and mediators such as nitric oxide, also
contribute to the shock syndrome [9
, 10
].
In this study, we investigated the capacity of morphine to modulate
expression of cytokines in peritoneal macrophages after in
vivo treatment. The data show that morphine sensitized the
macrophages to the stimulating effects of LPS and interferon-
(IFN-) resulting in enhanced production of IL-12 and other
proinflammatory cytokine mRNAs and proteins 48 h post opioid
administration. The opioid receptor antagonist, naltrexone, blocked the
effect of morphine on cytokine responses.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drug treatment
Mice were anesthetized with Metofane® and given a
subcutaneous, intrascapular implant of either a single 75-mg
slow-release morphine pellet, a 30-mg naltrexone pellet (morphine
antagonist), a placebo pellet, or a morphine pellet plus a naltrexone
pellet (all pellets were obtained from the National Institute on Drug
Abuse, Rockville, MD). The incision through which the pellet was
inserted was closed with a 9-mm surgical clip (Autoclip; Clay Adams,
Sparks, MD). Mice were observed until they recovered from anesthesia
and were housed in groups of 5 for 48 h. Seventy-five milligrams
slow-release morphine pellets result in initial blood levels of 2
µg/mL of morphine and by 48 h in constant blood levels of 0.6
µg/mL for 2–3 days [11
]. This is a standard method
for continuously administering morphine to prevent cycles of
withdrawal, and drug levels are considered physiological. The 30-mg
naltrexone pellets have been shown to effectively antagonize morphine
released from the 75-mg pellet [12
, 13
].
Cell isolations
Mice were killed at 48 h after pellet implantation.
Resident peritoneal macrophages were harvested from mice by peritoneal
lavage using ice-cold RPMI 1640 medium. As controls, cells were also
harvested from normal mice with no pellet implantation. The lavage
fluid from four to five mice in each group was pooled, and cells were
washed once in RPMI 1640 with 10% fetal calf serum (FCS). The cells
were counted in a Coulter counter (Coulter, Hialeah, FL), adjusted to a
density of 5 x 106 cells/mL, and 10-mL samples were
added to 100 x 15-mm plastic petri dishes. After a 2-h incubation
at 37°C in 5% CO2, nonadherent cells were removed by
washing with warm medium, and the remaining adherent cells were
collected by scraping with a rubber policeman. After washing, the
adherent cells were adjusted to 0.5 x 106/mL, and
were placed in 24-well plates. Cells were stimulated with LPS (10
µg/mL) plus IFN- (100 units/mL) for 24 h for RNA isolation or
for 48 h for harvest of supernatants for ELISA.
Reverse transcriptase-polymerase chain reaction (RT-PCR) and
QC-RT-PCR
Macrophage cell cultures were harvested at 24 h after
initial stimulation to analyze mRNA levels. Total RNA was extracted
using RNAzolB according to the manufacturer’s instructions. One to
three micrograms of RNA was reverse transcribed using Superscript II RT
(GIBCO BRL) and random hexamer primers (Promega, Madison, WI). The cDNA
samples were then subjected to PCR analysis. Primers used were: HPRT,
5’-GTTGGATACAGGCCAGACTTTGTTG-3’ (forward) and
5’-GAGGGTAGGCTGGCCTATAGGCT-3’ (reverse); IL-1ß,
5’-GCAACTGTTCCTGAACTCA-3’ (forward) and 5’-CTCGGAGCCTGTAGTGCAG-3’
(reverse); IL-6, 5’-TTCCTCTCTGCAAGAGACT-3’ (forward) and
5’-TGTATCTCTCTGAAGGACT-3’ (reverse); IL-10,
5’-CCAGTTTTACCTGGTAGAAGTGATG-3’ (forward) and
5’-TGTCTAGGTCCTGGAGTCCAGCAGACTCAA-3’ (reverse); IL-12,
5’-ATGGCCATGTGGGAGCTGGAGAAAG-3’ (forward) and
5’-GTGGAGCAGCAGATGTGAGTGGCT-3’ (reverse); TNF-,
5’-GTTCTATGGCCCAGACCCTCACA-3’ (forward) and
5’-TACCAGGGTTTGAGCTCAGC-3’ (reverse); IFN-,
5’-AACGCTACACACTGCATCT-3’ (forward) and 5’-TGCTCATTGTAATGCTTGG-3’
(reverse); inducible nitric oxide synthase (iNOS),
5’-TGGGAATGGAGACTGTCCCAG-3’ (forward) and
5’-GGGATCTGAATGTGATGTTTG-3’ (reverse). The sequencing primers
were obtained from Great American Gene Company (Ramona, CA). cDNA was
amplified for 35 cycles (94°C for 40 s, 60°C for 20 s,
72°C for 40 s, and a final extension at 72°C for 10 min),
using Taq polymerase (Boehringer Mannheim, Indianapolis, IN). PCR
products were analyzed by electrophoresis on 2% agarose gels and
visualized by ethidium bromide staining. Blots were densitometrically
scanned and quantified using NIH Image software. Data are the average
of three separate experiments.
Semiquantitative, competitive RT-PCR was performed using a plasmid, PQRS, containing multiple cytokine competitors including IL-12 [14 ]. Constant volumes of normalized cDNAs were then amplified in the presence of different concentrations of competitor. The concentration of the cDNA from morphine-treated cells or placebo-treated cells was determined as the concentration where the intensity of the competitor and wild-type bands were equivalent.
Cytokine ELISA
Macrophage cell cultures were harvested at 48 h after
initial stimulation as described above. Cell-free supernatants were
collected and frozen at -70°C until determination of cytokine levels
by sandwich ELISA. All monoclonal antibodies used in cytokine ELISAs
were from PharMingen, San Diego, CA (the following list indicates the
description of the coating antibody followed by the biotinylated second
antibody IL-12p40 C15.6, C17.8; IL-12p70: C18.2, C17.15; TNF-:
G281–2626, MP6-XT3; IL-10: JESS-2A5, JESS-16E). Briefly, samples were
incubated overnight at 4°C on plates coated with monoclonal Ab
specific for the cytokine. The plates were washed with PBS containing
Tween 20 before the addition of biotinylated second antibody for 1 h. The plates were washed, and a 1:1000 dilution of
streptavidin-alkaline phosphatase was added for 30 min. After a
thorough washing, p-nitrophenyl phosphate was added, and the
plates were incubated at room temperature for approximately 60 min.
Optical density at 405 nm was determined by using an automated
microplate reader. Cytokine concentrations were calculated from
standard curves developed for each cytokine.
Statistics
Statistical analysis was done with analysis of variance.
P values of < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(100 units/mL). After
24 h RNA was extracted, and RT-PCR was carried out using specific
primers for IL-12 p40, TNF-, IL-1, IL-6, and IL-10. No significant
difference in cytokine mRNA levels of stimulated or unstimulated cells
was found between placebo-treated mice and control mice (data not
shown). As shown in Figure 1
, mRNA was detected for IL-1, IL-6, and TNF- in unstimulated
macrophages taken from animals in all experimental groups (lanes 1, 3,
5, 7). When densitometry values were averaged for three experiments, it
was found that morphine did not significantly potentiate expression of
mRNA for these cytokines above that observed in the placebo group
(Fig. 2
). No message was detected in unstimulated cells for IL-12, IL-10,
IFN-, or iNOS. Activation of cells with LPS plus IFN- strongly
induced IL-10, IL-12, and iNOS gene expression in all groups. Message
levels for IL-1, IL-6, and TNF- were also elevated in all groups
compared with unstimulated cells. When comparisons were made among the
stimulated cells from treatment groups, morphine significantly
increased IL-12 p40 mRNA levels compared with the placebo group.
Morphine also significantly elevated TNF- and IL-1 levels compared
with the placebo group. However, IL-10 mRNA levels were unchanged by
morphine treatment. The specificity of the morphine effect was tested
using naltrexone. Naltrexone blocked the enhancement of mRNA for IL-12
and TNF-, indicating that these effects were mediated by classical
opioid receptors. On the other hand, the antagonist did not completely
block the increase in mRNA for IL-1 and IL-6.
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(Fig. 3
). Constant volumes of normalized cDNAs were amplified in the
presence of different concentrations of the competitor plasmid PQRS
(0.00016–0.005 ng/µL), using primers for the p40 subunit of IL-12.
As shown in Figure 3 , the upper band represents the competitor DNA
products, and the lower band represents the wild-type DNA products. The
lane showing equivalent transcription of competitor and wild-type cDNA
in the morphine-treated group was 12 x 10-4 ng/µL
and that in the placebo group was 1.6 x 10-4 ng/µL.
These results indicate that morphine resulted in an approximately
eightfold increase in IL-12 p40 mRNA when compared with the placebo
group (Fig. 3)
.
|
Morphine increases IL-12 and TNF- production, but decreases
production of IL-10
To confirm the results obtained by molecular analysis for cytokine
gene expression, cytokine protein levels were measured by ELISA.
Peritoneal macrophages were incubated with medium alone or with LPS
plus IFN- for 48 h. The cell-free supernatants were collected
and assayed by sandwich ELISA. As shown in Figure 4
, resting macrophages did not produce cytokines. However, upon
stimulation with LPS plus IFN- significant levels of both IL-12 p40
and p70 were produced by cells taken from all groups. It is important
to note that both IL-12 p40 (Fig. 4A)
and IL-12 p70 (Fig. 4B)
were
significantly increased by morphine compared with levels in the placebo
group, and naltrexone blocked this enhancement. Morphine also resulted
in a significant enhancement of TNF- levels that was antagonized by
naltrexone (Fig. 4C)
. When tested by ELISA, morphine was found to
suppress IL-10 production, and naltrexone blocked this inhibition (Fig. 4D)
.
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were added into the cultures. After
incubation for 48 h, supernatants were harvested and assayed by
ELISA. No significant increase in IL-12 levels were found between
control and morphine-treated macrophage cultures (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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both at the mRNA level and at the
protein level, when cells were harvested and stimulated in
vitro 48 h post opioid administration. Furthermore,
naltrexone blocked these increases. Elevation of IL-1 mRNA levels was
also observed, but increases above the placebo group were smaller, and
antagonism by naltrexone was less robust. IL-6 mRNA was not
significantly elevated above placebo levels. Morphine inhibited
production of IL-10. These results indicate that morphine given
continuously in vivo for 48 h sensitizes peritoneal
macrophages to respond to LPS plus IFN- with production of
pro-inflammatory cytokines, particularly IL-12. The results are
particularly interesting in light of our previous observation that
48 h after morphine pellet implantation, the peritoneal cavity is
colonized with gram-negative and other enteric bacteria
[1
]. The bacteria translocating from the
gastrointestinal tract may act as a stimulus in vivo to
prime macrophages to increase production of inflammatory cytokines.
IL-12, a heterodimeric cytokine with potent IFN--inducing ability
for T cells and NK cells, has been shown to contribute to the sepsis
syndrome [6
]. IL-10, an anti-inflammatory cytokine,
inhibits the production of IL-12, TNF-, and IFN-, and prevents
endotoxin shock in mice [15
]. In humans, initial
clinical trials have demonstrated that IL-10 administration ameliorates
inflammatory symptoms associated with endotoxemia, inflammatory bowel
disease, and rheumatoid arthritis [16
17
18
].
There are few studies in the literature reporting effects of morphine
administered in vivo on elaboration of cytokines. Pacifici
et al. [19
] found that chronic injection of morphine for
8 days depressed IL-1 and TNF- levels, but animals were not given an
opioid antagonist to prove specificity of the effect. Other
investigators have measured T cell cytokines after morphine. Carr et
al. [20
] found that monkeys chronically treated for
months with morphine had elevated levels of IL-2 production from
stimulated peripheral blood mononuclear cells. In contrast, Bhargava et
al. [21
] found decreased levels of IL-2 and IL-4 in
splenocytes taken from mice given morphine chronically for 5 days and
stimulated in vitro with anti-CD3. Lysle et al.
[22
] reported that rats had depressed levels of IL-2 and
IFN- in supernatants of spleen cell cultures harvested 90 min after
a single subcutaneous injection of morphine and stimulated in
vitro with concanavalin A. Bencsics et al. [23
]
found that a single subcutaneous injection of morphine 30 min before
injection of LPS decreased plasma TNF- levels measured at various
times up to 240 min after the LPS injection. Recently, Roy et al.
[2
] reported that morphine-treated mice injected with
LPS had increased IL-6 and TNF- production, as compared with giving
LPS alone in vivo, in peritoneal macrophages harvested after
24 h. This work supports our findings reported previously and in
this study, that morphine sensitizes to LPS. There may be different
mechanisms impinging on cytokine levels in acute versus subacute or
chronic administration of morphine, which may account for differences
noted in the literature.
A larger literature exists examining the effects of opioids added to
normal cells in vitro on modulation of cytokines. In most of
these studies, the alkaloids were suppressive and the endogenous
peptides were stimulatory. Thus, Peterson et al. [24
]
and Chao et al. [25
] reported that morphine inhibited
the release of IFN- and TNF-, respectively, by human peripheral
blood mononuclear cells stimulated with concanavalin A, and Nair et al.
[26
] found that morphine inhibited IFN- and IFN-ß
production by peripheral blood mononuclear cells. Roy et al.
[27
] showed that morphine inhibited concanavalin
A-stimulated IL-2 production and mRNA transcription in murine
thymocytes. Our laboratories have reported that a kappa opioid agonist,
U50,488H inhibits LPS-induced release of TNF- and IL-1 by murine
peritoneal macrophages and macrophage cell lines [28
,
29
]. In contrast, endorphins and enkephalins have been
shown to increase IL-1 and/or IL-6 production by bone-marrow
macrophages [30
], mouse peritoneal macrophages
[31
], and splenic adherent cells [32
].
Studies by Brown et al. [33
] and van den Bergh et al.
[32
, 34
] showed that endorphin and
enkephalin increased IFN- and/or IL-2 and IL-4 production by
concanavalin A-stimulated human mononuclear cells or mouse
CD4+ T cells.
In our experiments, we did not find an effect on IL-12 levels of peritoneal cells treated with morphine in vitro in doses of 10-6 to 10-10 M. Thus, the modulation of this cytokine by the drug does not seem to be a direct effect on the macrophages. We propose that sensitization of macrophages for up-regulation of IL-12 and other proinflammatory cytokines during subacute continuous treatment with morphine for 48 h is due to occult sepsis [1 ]. The septic state is transitory and the up-regulation of cytokines at this time point is consonant with the time when microbes can be cultured from the organs. Sepsis could not be due to morphine contamination with LPS because naltrexone blocked the effect, showing that it is opioid mediated. Also, morphine was inactive in the Limulus lysate assay for endotoxin.
In conclusion, morphine given in vivo was shown to prime
macrophages for enhanced production of the pro-inflammatory cytokines
IL-12 and TNF-, and to suppress the anti-inflammatory cytokine IL-10
in mouse peritoneal macrophages harvested 48 h after morphine
treatment.
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ACKNOWLEDGEMENTS |
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Received April 30, 2000; revised July 9, 2000; accepted July 11, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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challenge in mice J. Immunol. 145,4185-4191[Abstract]
production and lethality in lipopolysaccharide-induced shock in mice Eur. J. Immunol. 25,672-676[Medline]
) Circ. Shock 30,279-292[Medline]
as a mediator of the lethality of endotoxin and tumor necrosis factor- J. Immunol. 149,1666-1670[Abstract]
production by cultured peripheral blood mononuclear cells J. Clin. Invest. 80,824-831
production following treatment of macrophages with the kappa opioid agonist U50,488H J. Pharmacol. Exp. Ther. 273,1491-1496
-opioid agonist U50,488H J. Neuroimmunol. 64,83-90[Medline]
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