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Department of Medicine, Long Island Jewish Medical Center, New Hyde Park, NY 11040. The Long Island Campus for Albert Einstein College of Medicine, Bronx, NY 10461.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) and compromises their
phagocytic capability for immune complexes and bacteria. We hypothesize
that morphine decreases the number of, as well as compromises the
phagocytic capability of, M by programming their death. We studied
the effect of morphine on M apoptosis in vivo as well as in vitro.
Peritoneal M harvested from morphine-treated rats showed DNA
fragmentation. Morphine enhanced murine M (J 774.16) apoptosis in a
dose-dependent manner. Human monocytes treated with morphine showed a
classic ladder pattern in gel electrophoretic and end-labeling studies.
Morphine promoted nitric oxide (NO) production both under basal and
LPS-activated states.
NG-nitro-L-arginine methyl ester
(L-NAME) and
NG-monomethyl-L-arginine
monoacetate (L-NMMA), inhibitors of NO synthase, attenuated
the morphine-induced generation of NO by M. Morphine also enhanced
M mRNA expression of inducible NO synthase (iNOS). Since
morphine-induced M apoptosis was inhibited by L-NAME and L-NMMA, it
appears that morphine-induced M apoptosis may be mediated through
the generation of NO. Morphine promoted the synthesis of Bax and p53
proteins by M. Moreover, IL-converting enzyme (ICE)-1 inhibitor
attenuated morphine-induced M apoptosis. These studies suggest that
morphine activates the induction phase of the apoptotic pathway through
accumulation of p53. The effector phase of morphine-induced apoptosis
appears to proceed through the accumulation of Bax and activation of
ICE-1. The present study provides a basis for a hypothesis that
morphine may be directly compromising immune function by promoting M
apoptosis in patients with opiate addiction. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production from these cells (6).
Opiates suppress primary and secondary Ab response at the cellular
level and in the whole animal (7). Polymorphonuclear cells and
monocytes from patients subjected to morphine treatment show decreased
phagocytic and bacterial killing properties, as well as modulation of
the generation of reactive oxygen species (8, 9).
The mononuclear phagocyte system plays an important role in the host
defense against microorganisms (10, 11). Previously, we and other
investigators demonstrated that morphine attenuated macrophage
(M)4 phagocytosis of
microbial organisms and immune complexes (12, 13, 14). Morphine has been
reported to decrease the number of murine peritoneal and rabbit
alveolar M (12). However, the mechanism of morphine-induced
decreased M was not examined in these studies (12). We hypothesize
that morphine may be promoting M apoptosis. This will provide an
explanation for morphine-induced decreased peritoneal and alveolar M
and decreased phagocytosis of immune complexes by M (in vitro
studies).
In the present study, we evaluated the effect of morphine on apoptosis
of M. We also evaluated the effect of morphine on the apoptotic
pathway of M. Recently, Meßmer et al. reported that endogenously
generated or exogenously supplied nitric oxide (NO) promoted apoptosis
in the mouse M cell line RAW 264.7 (15). In these studies, apoptotic
signaling caused an early accumulation of p53 before DNA fragmentation.
Therefore, we hypothesized that morphine-induced M apoptosis may be
mediated through the generation of NO and may also be associated with
the accumulation of p53. To study whether the effect of morphine is
species specific, experiments were performed on M harvested from
different species (i.e., rats, mice and humans). To determine the
direct effect of morphine on M apoptosis, we conducted the majority
of studies in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were harvested (16).
Four mice weighing 50 g were used for the isolation of mouse
peritoneal M.
Murine M culture
To determine whether morphine has any species-specific effect,
we also studied the effect of morphine on murine M (M cell line J
774.16, American Type Culture Collection, Rockville, MD). M were
grown in DMEM (Life Technologies, Grand Island, NY) containing 10% FCS
(Life Technologies), 50 U/ml of penicillin, and 50 µg/ml of
streptomycin (Life Technologies).
Isolation of human monocytes
One hundred milliliters of blood was collected in 25-ml tubes containing EDTA from healthy volunteers. Monocytes were isolated with the use of a monocyte-separating solution (Accurate Chemical and Scientific, Westbury, NY).
Confirmation of peritoneal M by anti-CD14 Abs
Isolated mouse peritoneal M were labeled with mouse
anti-CD14 Abs (Becton Dickinson, San Jose, CA) and examined under
immunofluorescence microscope.
Apoptosis studies
Hoechst (H)-33342 (Molecular Probes, Eugene, OR) stains the nuclei of live cells and identifies apoptotic cells by increased fluorescence whereas propidium iodide (Sigma) costains the necrosed cells. Double staining by these two agents enables us to obtain the percentage of live, apoptotic, and necrosed cells (17).
Equal numbers (10,000 cells/well) of J774.16 M were seeded in
24-well plates containing DMEM + 10% FCS. Forty-eight hours
later, cells were washed twice with PBS and incubated in media
(DMEM + 1% FCS) containing either buffer or variable
concentrations of morphine (10-10 to
10-4 M, National Institute on Drug Abuse (NIDA),
Rockville, MD) for 48 h. Three sets of experiments were conducted,
each in triplicate.
To determine the dose response effect of morphine on murine peritoneal
M, equal numbers of subconfluent M were incubated in media
(DMEM + 1% FCS) containing either buffer (control) or variable
concentrations of morphine (10-12 to 10-6 M)
for 24 h. Three sets of experiments were performed.
To determine the time course effect of morphine on M apoptosis,
equal numbers (10,000 cells/well) of M were plated in 24-well plates
containing DMEM and 10% FCS for 48 h. Subsequently, cells were
washed twice with PBS and incubated in media (DMEM + 1% FCS)
containing either buffer or morphine (10-8 M) for variable
periods (12, 24, and 48 h) at 37°C. Three sets of experiments
were performed, each in triplicate.
To determine whether morphine-induced M apoptosis may be mediated
through opiate receptors, we evaluated the effect of morphine on M
apoptosis with or without naloxone (NIDA), an opiate receptor
antagonist. In brief, equal numbers of M (10,000 cells/well) were
seeded in 24-well plates containing DMEM + 10% FCS for 24 h.
Subsequently, cells were washed twice with PBS and incubated in media
(DMEM + 1% FCS) containing either buffer (control), morphine
(10-8 M), naloxone (10-6 M), or morphine
(10-8 M) + naloxone (10-6 M) for 48 h.
Three sets of experiments were conducted, each in triplicate.
To determine the effect of various opiate receptor agonists (µ
receptor: morphine, [D-Ala2,
N-Me-Phe4, Gly5-ol]enkephalin
(DAGO), ß-endorphin; 
receptor: [D-Pen2,
D-Pen5]enkephalin (DPDPE), and
[DAla2,
DLeu5]enkephalin (DADLE)) and antagonists
(naloxone, naltrexone), subconfluent M (J774.16) were incubated in
media (DMEM + 1% FCS) containing either buffer or variable
concentrations (10-8 M and 10-6 M) of
agonists (morphine, DAGO, ß-endorphin, DPDPE and DADLE, NIDA) and
antagonists (naloxone, naltrexone, NIDA) for 24 h. At the end of
the incubation period, cells were stained with H-33342 and propidium
iodide and examined under ultraviolet light. Three sets of experiments
were performed, each in triplicate.
To determine whether morphine-induced M apoptosis may be mediated
through the generation of NO by M, we studied the effect of
NG-nitro-L-arginine methyl ester
(L-NAME, Calbiochem, La Jolla, CA) and
(NG-monomethyl-L-arginine
monoacetate (L-NMMA, Calbiochem), inhibitors of NO synthase
(18, 19), on morphine-induced M apoptosis. Equal numbers of
subconfluent M were washed twice with PBS and incubated in media
(DMEM + 1% FCS) containing either buffer (control), morphine
(10-8 M), L-NAME, (1 mM) or morphine
(10-8 M) + L-NAME (1 mM) for 24 h. Five
sets of experiments were conducted, each in triplicate. In parallel
experiments, equal numbers of subconfluent M were incubated in media
containing either buffer (control), morphine (10-6 M),
L-NMMA (10-6 M), or morphine
(10-6 M) + L-NMMA (10-6 M) for
24 h. Three sets of experiments were conducted, each in
triplicate.
To determine whether other NO-releasing agents can also modulate M
apoptosis, we evaluated the effect of sodium nitroprusside (Sigma) on
M apoptosis. Equal numbers of subconfluent M were incubated in
medium (DMEM + 1% FCS) containing either buffer (control) or
variable concentrations (0.125, 0.25, and 0.5 mM) of sodium
nitroprusside for 24 h. Three series of experiments were
performed, each in triplicate.
To determine whether morphine-induced M apoptosis is mediated
through the activation of IL-converting enzyme (ICE)-1, we evaluated
the effect of ICE-1 inhibitor (Calbiochem) on morphine-induced M
apoptosis. Equal numbers of subconfluent M were incubated in media
(DMEM + 1% FCS) containing either buffer (control), morphine
(10-6 M), ICE-1 inhibitor (200 pM), or morphine
(10-6 M) + ICE-1 inhibitor (200 pM) for 24 h. Twelve
sets of experiments were conducted.
At the end of the scheduled incubation periods, aliquots of methanol containing H-33342 (final concentration, 1 µg/ml) were added and incubated for 10 min at 37°C. Subsequently, cells (without a wash) were placed on ice, and propidium iodide (final concentration, 1 µg/ml) was added to each well. Cells were incubated with the dyes for 10 min on ice, protected from light, and then examined under ultraviolet light using a Hoechst filter (Nikon, Garden city, NY). The percentage of live, apoptotic, and necrosed cells were recorded in eight random fields by two observers unaware of the experimental conditions.
To determine percentage apoptosis of peritoneal M in control and
morphine-treated rats, M harvested from control and morphine treated
rats were stained with H-33342 and propidium iodide. The percentage of
apoptotic M was recorded as mentioned above.
Evaluation of the effect of morphine, sodium nitroprusside,
sulfo-NONOate disodium salt, and nitric oxide synthase (NOS) inhibitors
on M NO production
To determine the effect of morphine and NOS inhibitors on M
NO production, equal numbers of M (10,000 cells/well) were seeded in
24-well plates containing DMEM + 10% FCS for 48 h.
Subsequently, cells were washed twice with PBS and incubated in media
(DMEM + 1% FCS) containing either buffer (control), morphine
(10-8 M), morphine (10-10 M),
L-NAME (1 mM), morphine (10-8 M) +
L-NAME (1 mM), or morphine (10-10 M) +
L-NAME (1 mM) for 24 h. Four sets of experiments were
conducted. In parallel experiments, M were treated under identical
conditions. However, after 24 h of incubation under the conditions
mentioned above, cells were further treated with LPS (LPS, Sigma), 10
ng/ml. The incubation was continued for the next 6 h. Four sets of
experiments were performed.
To determine the effect of L-NMMA, another inhibitor of
NOS, on morphine-induced M NO production, equal numbers of
subconfluent M were incubated in media (DMEM + 1% FCS),
morphine (10-6 M), L-NMMA (10-6
M), or morphine (10-6 M) + L-NMMA
(10-6 M) for 24 h. At the end of 24 h, an
aliquot of LPS (10 ng/ml) was added to each well. Three sets of
experiments were conducted.
To determine the effect of naloxone (an opiate receptor antagonist) on
morphine-induced M NO production, equal numbers of subconfluent M
were incubated in media (DMEM + 1% FCS) containing either buffer
(control), morphine (10-8 M), naloxone (10-6
M), or morphine (10-8 M) + naloxone (10-6 M)
for 24 h. Subsequently, an aliquot of LPS (10 ng/ml) was added to
each well. Three sets of experiments were performed.
To determine the effect of NO-releasing agent (positive control) on
M NO production, equal numbers of subconfluent M were incubated
in medium containing either buffer (control) or variable concentrations
of sodium nitroprusside (0.125, 0.25, and 0.5 mM) for 24 h.
Subsequently, an aliquot of LPS (10 ng/ml) was added to each well.
Three sets of experiments were conducted.
To determine the effect of sulfo-NONOate-disodium salt (negative
control; produces nitrous oxide but no nitric oxide at physiologic pH;
Alexis Corp. San Diego, CA) on M NO production, equal numbers of
subconfluent M were incubated in media (DMEM + 1% FCS)
con-taining either buffer (control) or variable
concentrations of sulfoNONOate-disodium salt
(10-6 M to 10-3 M) for 24 h. At the end
of 24 h, an aliquot of LPS (10 ng/ml) was added to each
well. Three sets of experiments were conducted. At the end of
the incubation period, supernatants were collected, and NO
production was assayed by the Griess method as described
previously (20).
Detection of rat peritoneal M/human monocyte apoptosis by gel
electrophoresis
Gel electrophoresis is a simple method for detection of
apoptosis. Briefly, equal numbers of rat peritoneal M (harvested
from control and morphine-treated rats) were plated on 100-mm petri
dishes containing media for 24 h. In parallel experiments, human
monocytes were plated on 100-mm petri dishes containing either media
alone (control) or media + morphine (10-6 M or
10-4 M) for 24 h. At the end of the incubation period
M/monocytes were lysed in DNA lysis buffer, and DNA was extracted
(21). DNA was run on a 1.8% agarose gel electrophoresis at 5 V/cm in
0.5 x TE buffer (Tris 10 mM; EDTA 1mM, pH 8.0) containing 10
µg/ml ethidium bromide.
Detection of rat peritoneal M/human monocyte apoptosis by using
DNA end-labeling technique
DNA end-labeling is a more sensitive method for detection of
apoptosis (21). In brief, 5 µg of isolated peritoneal M DNA from
control and morphine-treated rats, as well as from control and
morphine-treated human monocytes (as described above), were end labeled
(21). The end-labeled DNA were electrophoresed on a 1.8% agarose gel,
and radiolabeled fragments were visualized by exposure to Kodak
(Rochester, NY) x-ray film at -70°C for 30 min to 3 h.
Protein extraction
Equal numbers (10,000) of J774.16 M were seeded in 100-mm
petri dishes and grown to subconfluence. Subsequently, M were washed
twice with PBS and incubated in medium containing either
buffer (control) or morphine (10-8 M to
10-4 M) for 4 h. At the end of the incubation
periods, cells were washed twice with PBS and incubated with 600 µl
of PBSTDS lysis buffer (Calbiochem; with 2 µg/ml leupeptin, 1 mM
EDTA, 1 µg/ml pepstatin, and 100 µg/ml PMSF) for 30 min at room
temperature. Cells were scraped and protein was assayed using a BCA kit
(Pierce, Rockford, IL).
Western blotting
The proteins (20 µg/lane) extracted from M lysates
were separated on a 4 to 20% gradient polyacrylamide gel and
transferred onto a nitrocellulose membrane using a Bio-Rad (Hercules,
CA) Western blotting apparatus. Nitrocellulose membranes were then
processed further for p53 and Bax using either mouse anti-p53
(Zymed, San Francisco, CA) or rabbit anti-Bax (PharMingen, San
Diego, CA) at a 1 µg/ml concentration; 1 h at room temperature
using HRP (horseradish peroxidase)-labeled secondary goat
anti-mouse Ab or goat anti-rabbit (Pierce); blots were
developed using enhanced chemiluminescence (ECL, Amersham).
Northern blotting
Equal numbers of subconfluent M grown in 100-mm petri dishes
were incubated in media (DMEM + 1% FCS) containing either buffer
or morphine (10-10 to 10-8 M) for
18 h. Subsequently, an aliquot of LPS, 10 ng/ml, was added to each
petri dish and the incubation was extended for 6 h. At the end of
the incubation period, cells were harvested. Total RNA was extracted,
and Northern blots were generated and probed with cDNA specific for
iNOS (a gift from Dr. Carl Nathan, Cornell University Medical College,
New York, NY) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Densitometric analysis was performed on these blots and ratios between
iNOS and GAPDH were calculated.
Statistical analysis
For comparison of mean values between two groups, the unpaired t test was used. To compare values between multiple groups, analysis of variance (ANOVA) was applied and a Newman-Keuls multiple range test was used to calculate a q value. All values are means ± SE except where otherwise indicated. Statistical significance was defined as p < 0.05.
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Results |
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apoptosis
As shown in Table I
, morphine in
concentrations of 10-8 M to 10-4 M
promoted murine M (J774.16) apoptosis. Representative photographs of
control and morphine (10-6 M)-treated M are shown in
Figure 1. This effect of morphine
(10-8 M) was time dependent (12 h, control 2.3 ±
0.2% and morphine 3.6 ± 0.2%* apoptotic cells/field; 24 h,
control 2.6 ± 0.1% and morphine 4.8 ± 0.1%** apoptotic
cells/field; 48 h, control 2.5 ± 0.1% and morphine-7.4
± 0.6%*** apoptotic cells/field; n = 3;
*p < 0.05 compared with control, 12 h;
**p < 0.001 compared with control, 24 h;
***p < 0.001 compared with morphine, 12 and 24
h). Morphine also enhanced apoptosis in mouse peritoneal M in a
dose-dependent manner (Table II).
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opiate receptors, we evaluated the effect of naloxone, an opiate
receptor antagonist, on morphine-induced M apoptosis. Naloxone
attenuated (p < 0.01) morphine
(10-8 M)-induced M apoptosis (control, 2.3 ±
0.2%; morphine, 4.8 ± 0.5%; morphine + naloxone, 2.6
± 0.4% apoptotic cells/field, n = 3).
To evaluate whether other opiate agonists and antagonists also modulate
M apoptosis, we determined the effect of agonists (ß-endorphin,
DAGO, DADLE and DPDPE) and antagonists (naloxone and naltrexone) on
M apoptosis. As shown in Table III,
µ receptor agonists (morphine, ß-endorphin, and DAGO) promoted M
apoptosis, whereas receptor agonists showed only a mild alteration
on M apoptosis. These results suggest that morphine-induced M
apoptosis may be mediated through µ opiate receptors. To determine
whether this effect of morphine is species specific, we evaluated the
effect of morphine on rat peritoneal M as well as monocytes isolated
from healthy volunteers. Peritoneal M isolated from morphine-treated
rats showed a greater percentage of apoptosis when compared with M
isolated from control rats (control rats, 2.6 ± 0.8% vs
morphine-treated rats, 13.5 ± 1.5% apoptotic M/field). As
shown in Figures 2 and
3, peritoneal M harvested from
morphine-treated rats showed DNA fragmentation (integer multiples of
180 base pairs) in the form of a ladder pattern (at 3 and 10 days) when
isolated DNA was run in agarose electrophoresis, as well as when
end-labeled, whereas peritoneal M harvested from untreated rats
(control) did not show DNA fragmentation. Similarly, morphine-treated
human monocytes showed a classic ladder pattern characteristic of
apoptosis (Figs. 4 and
5). These results suggest that the effect
of morphine on M apoptosis is not species specific.
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apoptosis is mediated through activation of ICE-1, we evaluated the
effect of ICE-1 inhibitor on morphine-induced M apoptosis. ICE-1
inhibitor attenuated (p < 0.001)
morphine-induced M apoptosis (control, 1.9 ± 0.1%; morphine,
10-6 M, 6.8 ± 0.2%; ICE-1 inhibitor, 200 pM,
3.0 ± 0.1%; morphine + ICE-1 inhibitor, 3.1 ± 0.1%
apoptotic cells/field; n = 12). These results indicate
that morphine may be promoting an apoptotic pathway through the
activation of ICE-1.
Role of NOS and NO in morphine-induced M apoptosis
To determine whether morphine-induced M apoptosis is mediated
through activation of NOS, we evaluated the effect of
L-NAME and L-NMMA, inhibitors of NOS on
morphine-induced M apoptosis. The effect of L-NAME is
shown in Figure 6.
L-NAME attenuated (p <
0.05) morphine (10-8 M)-induced M apoptosis (control,
2.35 ± 0.45%; morphine, 7.67 ± 2.1%; morphine +
L-NAME, 3.86 ± 0.88% apoptotic cells/field;
n = 5). As shown in Table IV, L-NMMA also attenuated
(p < 0.001) morphine-induced M apoptosis
(morphine, 7.9 ± 1.40 vs morphine + L-NMMA,
2.20 ± 0.39% apoptotic cells/field). These results suggest that
morphine-induced M apoptosis may be mediated through the activation
of M NOS. To determine whether other NO-generating agents can also
modulate M apoptosis, we evaluated the effect of sodium
nitroprusside on M apoptosis. As shown in Table V, sodium nitroprusside promoted M
apoptosis in a dose-dependent manner (control, 2.2 ± 0.6% vs
sodium nitroprusside, 0.5 mM, 11.9 ± 2.4% apoptotic cells/field;
p < 0.01). These results suggest that other
NO-generating agents may also induce M apoptosis.
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NO production
Since NO has been demonstrated to trigger M apoptosis
(15), we evaluated the effect of morphine on M NO production. To
study the role of NOS in morphine-induced NO production, we examined
the effect of L-NAME and L-NMMA (NOS
inhibitors) on morphine-induced M NO production. We also evaluated
the effect of sodium nitroprusside (positive control) and sulfo-NONOate
disodium salt (negative control) on M NO production. To evaluate
whether this effect of morphine is mediated through opiate receptors,
we examined the effect of naloxone (opiate receptor antagonist) on
morphine-induced M NO production.
The effects of morphine on the production of NO under basal as
well as LPS-activated states are shown in Tables
VI and VII.
Morphine (10-8 M) enhanced (p <
0.001) production of NO by M under a basal state (control, 0.37
± 0.02 vs morphine, 0.79 ± 0.02 nM; n = 4).
Morphine in concentrations of 10-10 M and
10-8 M increased (p < 0.001) the
synthesis of NO under an LPS-stimulated state. The production of
morphine-induced NO by M was manyfold higher under LPS-activated
states when compared with the basal state. As shown in Tables VI and
VII, L-NAME attenuated morphine-induced M NO production
under basal (Table VI) as well as LPS-activated (Table VII) states.
Similarly, L-NMMA, a specific inhibitor of iNOS, attenuated
(p < 0.001) morphine-induced M NO
production (control, 1.1 ± 0.03; morphine, 10-6 M,
3.0 ± 0.16; L-NMMA, 1.3 ± 0.18; morphine +
L-NMMA, 1.1 ± 0.09 nM nitrite). Sodium nitroprusside,
another NO-releasing agent, enhanced (p <
0.001) M NO production in a dose dependent manner (control,
0.86 ± 0.16; sodium nitroprusside, 0.125 mM, 1.43 ± 0.16;
sodium nitroprusside, 0.25 mM, 3.4 ± 0.13; sodium nitroprusside,
0.5 mM, 4.0 ± 0.15 nitrite nM; n = 3). On the
contrary, sulfo-NONOate disodium salt did not alter M NO production
(control, 1.10 ± 0.10; sulfo-NONOate disodium salt,
10-5 M, 0.93 ± 0.03; sulfo-NONOate disodium salt,
10-3 M, 1.03 ± 0.09 nitrite nM). However, naloxone,
an opiate receptor antagonist, attenuated (p <
0.01) morphine-induced M NO production (control, 1.1 ± 0.03;
morphine, 10-8 M, 2.3 ± 0.27; morphine +
naloxone, 1.1 ± 0.14 nitrite nM). These results indicate that
morphine promotes the generation of NO by M. This effect of morphine
seems to be mediated through the activation of NOS.
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apoptosis, we studied the effect of morphine on M mRNA expression of
iNOS. As shown in Figure 7, morphine
enhanced M mRNA expression of iNOS by fivefold (control 0.15;
morphine, 10-10 M, 0.75; morphine, 10-8 M,
0.84 iNOS/GAPDH ratios).
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The induction of certain genes, such as p53, has been shown to be
a requirement in apoptosis induced by DNA damage (23, 24); therefore,
we studied the effect of morphine on the synthesis of p53 protein by
M. The effects of morphine on M p53 accumulation is shown in
Figure 8. Morphine promoted the
accumulation of p53 protein when compared with untreated cells.
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, morphine promoted the accumulation of
Bax protein by M. These results suggest that morphine promotes the
effector phase of the apoptotic pathway through the generation of Bax
protein by M.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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apoptosis. This effect on M is not species specific.
Since naloxone, an opiate receptor antagonist, attenuated
morphine-induced M apoptosis, this effect of morphine appears to be
mediated through opiate receptors. Morphine promoted the production of
NO both under basal and LPS-activated states. L-NAME
and L-NMMA, inhibitors of NOS, attenuated the
morphine-induced M generation of NO. Since morphine-induced M
apoptosis was also inhibited by L-NAME and
L-NMMA, it appears that morphine-induced M apoptosis may
be mediated through the generation of NO. It was further substantiated
by morphine-induced enhanced M mRNA expression of iNOS.
Opiate receptor binding sites have been previously described on human
PBMC (29). Recently, these binding sites have been designated as
µ3 (30). Opiate alkaloid-selective µ3
binding sites have also been shown to be present on murine J 774.16
cells (31). These receptors have been postulated to mediate
morphine-induced inhibition of monocyte/M activation caused by
IL-1
, TNF-, IL-8, or N-FMLP. The µ3
receptor has been suggested to function in response to both
endogenously formed and exogenously administered opiate alkaloids (30).
In the present study, µ receptor agonists promoted M apoptosis,
whereas receptor agonists showed only a mild alteration of M
apoptosis.
Tubaro et al. demonstrated that morphine can attenuate mouse peritoneal
M count in a dose-dependent manner (12). These investigators suggest
that the morphine-induced decrease in M count may contribute to
morphine-associated immunomodulation. However, the mechanism of
morphine-induced decreased M count was not analyzed. The present
study provides an explanation for morphine-induced decreased M
count.
Peterson et al. and Tubaro et al. also demonstrated that morphine
decreases the respiratory burst in stimulated M and PBMC (9, 12). Peterson et al. further demonstrated that lymphocytes are the
prime target cells in opiate-mediated suppression of PBMC respiratory
burst activity (12). These investigators suggest that opiate-activated
lymphocytes release a product capable of inhibiting the respiratory
burst enzyme system of cultured monocytes. The bactericidal function of
mononuclear phagocytes is predominantly dependent upon their
respiratory burst, a response in which oxygen is rapidly metabolized to
provide a series of toxic intermediates. Morphine-induced decreased
respiratory burst activity has also been suggested to contribute to
morphine-induced immunomodulation.
Recently, NO has been reported to promote M apoptosis (15).
This effect of NO was mediated through the generation of p53 (15).
Expression of wild-type p53 appears to be linked to apoptosis promoted
by most DNA-damaging agents (32). p53 was originally characterized as a
tumor suppressor protein that acted as a checkpoint control in the cell
cycle, allowing the repair of damaged DNA. Interestingly, p53 also
signals apoptosis in the case of severe DNA damage. In the present
study, morphine promoted the production of NO by M. Inhibition of
M NO production by L-NAME attenuated morphine-induced
M apoptosis. Morphine also enhanced M synthesis of p53. We
speculate that morphine-induced M apoptosis may be mediated through
the generation of NO.
In the present study, morphine also promoted the accumulation of
Bax protein, a member of the Bcl-2 family. Bcl-2 is reported to be a
growing family of apoptosis regulatory gene products that may be either
death antagonists (Bcl-2, Bcl-XL, Bcl-w, Bfl-1, Brag-1, and
A1) or death agonists (Bax, Bak, Bcl-Xs, Bad, Bid, Bik, and
Hrk)(26, 27, 28). Bax, being a death agonist, may have contributed to the
effector phase of the morphine-induced apoptotic pathway. In addition,
morphine-induced activation of ICE may have pushed M to an
irreversible commitment of death.
While there is no controversy about morphine-induced immunomodulation,
the direct effect of morphine on the mononuclear phagocyte system is
far from clear. It has been suggested that morphine may be modulating
the function of monocytes through opiate receptors present on
lymphocytes (33, 34), indirectly via opiate receptors in the central
nervous system, or by activating the hypothalamic-pituitary-adrenal
axis (35) to secrete immunosuppressive glucocorticoids (36). The
present study shows the direct effect of morphine on M and thus
provides a basis for a hypothesis that morphine can directly modulate
immune function in patients with drug addiction.
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Footnotes |
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2 This work was presented at the 59th Annual Scientific Meeting of the College on Problems of Drug Dependence on June 18, 1997 in Nashville, TN.
3 Address correspondence and reprint requests to Dr. Pravin C. Singhal, Nephrology Division, Room 228, Long Island Jewish Medical Center, New Hyde Park, NY 11040.
4 Abbreviations used in this paper: M, macrophage; NO, nitric oxide; DPDPE, [D-Pen2, D-Pen5]enkephalin; DADLE, [DAla2, DLeu5]enkephalin; DAGO, [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin; H, Hoechst; L-NAME, NG-nitro-L-arginine methyl ester; L-NMMA, NG-monomethyl-L-arginine monoacetate; ICE, IL-converting enzyme; NOS, NO synthase; iNOS, inducible NOS; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication April 21, 1997. Accepted for publication October 30, 1997.
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