1677
Anesthesiology
2000; 921677-84
D 2000 American Society of Anesthesiologists, Inc.
Lippincott Williams & wilkins, Inc.
Morphine Inhibits NF-rcB Nuclear Binding in Human
Neutrophils and Monocytes by a Nitric
Oxide-dependent Mechanism
lngeborg D. Welters, M.D.,* Axel Menzebach, M.D.,t Yannick Goumon, Ph.D.,* Patrick Cadet, Ph.D.,§
Thilo Menges, M.D.,I( Thomas K. Hughes, Ph.D.,# Gunter Hempelmann, M.D., Ph.D.,**
George 6.Stefano, Ph.D. tt
Background: The transcription factor NF-KB plays a pivotal
role in gene expression of inflammatory mediators such as
cytokines or adhesion molecules. NF-&mediated transcriptional activationof these genes is inhibited by nitric oxide (NO)
in a variety of cells, including monocytes. Morphine mediates
* Staff Anesthesiologist and Postdoctoral Research Fellow in Anesthesia, Department of Anesthesiology and Operative Intensive Care
Medicine, Justus-Liebig-UniversityGiessen, Giessen, Germany; and
Neuroscience Research Institute, State University of New York at Old
Westbury, Old Westbury, New York.
t Resident in Anesthesia, Department of Anesthesiology and Operative Intensive Care Medicine,JustusLiebig-UniversityGiessen, Giessen,
Germany.
+
Postdoctoral Research Fellow in NKUroSCiKncK, Neuroscience RKsearch Institute, State University of New York at Old Westbury, Old
Westbury, New York.
5 Postdoctoral Fellow in Neuroscience/Molecular Biology, Neuroscience Research Institute, State University of New York at Old Westbury, Old Westbury, New York.
I/ Staff Anesthesiologist, Department of Anesthesiology and Operative Intensive Care Medicine,Justus-Liebig-UniversityGiessen, Giessen,
Germany.
# Full Professor of Microbiology, Department of Microbiology, University of Texas Medical Branch at Galveston, Galveston, Texas.
** Full Professor of Anesthesia, Department of Anesthesiology and
Operative Intensive Care Medicine, Justus-Liebig-UniversityGiessen,
Giessen, Germany.
tt Distinguished Professor of Biology, NeurOSCiKnCK Research Institute, State University of New York at Old Westbury, Old Westbury,
New York.
Received from the Department of Anesthesiology and Operative
Intensive Care Medicine, Justus-Liebig-University Giessen, Giessen,
Germany. Submitted for publication JUIY26, 1999. Accepted for publication January 14, 2000. Supported in part by grant no. DA 09010
from the National Institutes of Health, Bethesda, Maryland (to Dr.
Stefano), and a fellowship from the Deutsche Forschungsgemeinschaft
(We 2440-1/1), Bonn, Gemany (to Dr. Welters).
Address reprint requests to Dr. Welters: Department of Anaesthesiology and Intensive Care, Justus-Liebig-UniversitPt Giessen, RudolfBuchheim-Str. 7, 35385 Giessen, Germany. Address electronic mail to:
Ingeborg.D.Welters@chi~.med.uni-giessen.de
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NO release in a naloxone antagonizable manner in monocytes
and neutrophils.
Methods: The influence of morphine on NF-KB activation was
investigated in a whole-blood flow cytometric assay. A specific
antibody against the p65 subunit of NF-KB was used and detected by fluoresceine-isothiocyanatdabeled anti-iimunoglobulin G. Nuclei were stained with propidium iodide. Leukocyte subpopulations were evaluated by gating on neutrophils
and monocytes. The median fluorescence channel was determined. Different morphine concentrations (50 m, 50 PM, 1m ~ )
and incubation intervals (10-150 min) were used.
Results: Morphine inhibits lipopolysaccharide-induced
NF-KB nuclear binding in human blood neutrophils and monocytes in a time-, concentration-, and naloxone-sensitive-dependent manner. Similar effects were achieved with the NO donor
S-nitroso-N-acetyl-penciIlamineand the antioxidant N-acetylcysteine. The NO synthase inhibitors N,-nitro-r-arginine-methyl-esther and N,-nitro-L-arginine completely abolished the morphine-induced attenuation of NF-KB nuclear binding,
demonstrating that the inhibitory action is mediated by NO
release.
Conclusion: Morphine causes immunosuppression,at least in
part, via the NO-stimulated depression of NF-KBnuclear binding. (Key words: Leukocytes: opiates: transcription factors.)
MORPHINE has been shown to be an effective immuno-
modulator, acting, in part, by stimulating nitric oxide
(NO) production in neutrophils, monocytes, and endothelial cells.'-3 The N O synthase (NOS) inhibitor N,nitro-1.-arginine ( N U ) as well as the opiate antagonist
naloxone abrogates morphine-induced NO production
in these cells,' demonstrating the specificity of this process. The significance of these observations is increased
because NO also inhibits gene expression in inflammation in a variety of cells, including neutrophils and monocytes,*-"' suggesting that this linkage may be the basis of
the immune downregulating actions of morphine.
NF-KBis a transcription factor that plays a crucial role
in cytokine- and lipopolysaccharide (LPS)-induced gene
activation during inflammatory events. l 1 Incubation of
vascular endothelial cells with NO donors inhibited tu-
1678
WELTERS ET AL.
mor necrosis factor (TNF)-a-induced NF-KB activation
by induction and stabilization of the NF-KB inhibitor
IKBcx." In addition, NO donors can directly inhibit the
DNA binding activity of NF-KB.'~
Taken together, few studies have demonstrated the
activation of NF-KB in human blood monocytes and neutrophils in a whole-blood assay. Although morphine'*
and NO15 have been reported to inhibit leukocyte function, i.e., adherence, phagocytosis, and motility,'" the
exact intracellular molecular processes in the opiatemediated downregulation is not known. In the present
report, we determined the influence of morphine on the
activation of NF-KB in human blood neutrophils and
monocytes. Our data indicate that morphine attenuates
LPS-induced NF-KB activation by a NO-dependent and
naloxone-sensitive mechanism. NO donors mimicked
the inhibitory effects of morphine on nuclear binding of
NF-KB, whereas NOS inhibitors such as NLA and Nunitro-L-arginine-methyl-esther(L-NAME) abolished morphine effects on NF-KB activation. Thus, morphine
seems to invoke an intracellular molecular cascade, extending into nuclear events.
phine treatment. Control tubes were incubated with
0.9% saline. To induce NF-KB, blood cells were stimulated with 100 ng/ml LPS for 30 min in a 37°C water
bath. Controls were set up with phosphate-buffered saline. All aliquots were treated with 2 ml of a commercially available lysing solution (FACS Brand Lysing Solution; Becton Dickinson) for 30 min at room temperature
for lysis of erythrocytes. The cells were centrifuged at
200s for 5 min, and the supernatant was discarded.
Blood Samples
After an internal review board approved the project,
informed consent was obtained from 13 healthy male
volunteers. Ten milliliters of venous blood was collected
from each individual. The donors were nonsmokers and
had no history of infection or allergy, and they had never
been subjected to immunosuppressive therapy. The
mean age of the group was 28 -C 3 yr, and the mean
weight was 81 ? 6 kg.
Preparation of Cellsfor Flow Cytometry Detection
Preparation of nuclei for flow cytometric analysis was
performed as previously described. l 7 Briefly, cells were
washed once with phosphate-buffered saline before using staining reagents contained in a commercially available DNA staining kit (Cycletest Plus DNA Reagent Kit;
Becton Dickinson). After centrifugation, the aliquots
were resuspended in 3 ml citrate buffer and centrifuged
at 300g for 5 min. The supernatant was discarded, and
1.5 ml citrate buffer was added, followed by centrifugation at 300g for 5 min. After discarding the supernatant,
cells were resuspended in a mixture of 250 pl solution A
(trypsin in a spermine tetrahydrochloride detergent
buffer) and 200 pl of solution B (trypsin inhibitor and
ribonuclease A in citrate stabilizing buffer with spermine
tetrahydrochloride) for 10 min at room temperature. A
total of 40 pl NF-KB polyclonal rabbit antibody (Santa
Cruz Biotechnology, Heidelberg, Germany) was then
added to the aliquots for a 10-min incubation period at
room temperature. A total of 2.5 pl of a fluorescein isothio
cyanate- conjugated (FITC) antirabbit monoclonal antibody (Sigma Chemicals, Deisenhofen, Germany) was
added and incubated for further 10 min at room temperature. A total of 200 pl of a cold propidium iodide solution
was added to the aliquots and incubated for 10 min.
Incubation of whole Blood and Stimulation
with LPS
Whole blood was collected in EDTA and aliquoted in
tubes (Falcon tubes; Becton Dickinson, Heidelberg, Germany). A total of 100 pl of whole blood was treated with
either morphine sulfate (50 nM, 50 p ~1 ,mM) or with the
NO donor S-nitroso-N-acetyl-pencillamine
(SNAP) in a
water bath under steady shaking for different time intervals as indicated. To investigate the effects of NOS inhibitors, NLA (0.5 mM) or L-NAME(1 mM) was added to the
blood and incubated for 5 min at 37°C in a water bath
before treatment with 1 mM morphine sulfate. Opiate
receptor specificity was tested by preincubation of samples with naloxone (0.5 p~ and 0.5 mM) before mor-
Flow Cytometric Analysis
The flow cytometer (FACS-Calibur;Becton Dickinson)
was equipped with forward-scatter and side-scatter light
detectors that allow discrimination of cell size and complexity. It also has an optical filter to detect propidium
iodide-stained nuclei. These nuclei emit fluorescent
light between 580 and 650 nm that can be detected by
the fluorescence-2 filter. The fluorescence-1 detector
was used to detect NF-KB activation by measuring emission of green fluorescence at 530 nm corresponding
with FITC staining. Fluorescence caused by unspecific
binding of the secondary antibody was excluded by
incubation of whole blood with FITC-labeled immunoglobulin G alone. Unspecific binding of the p65-antibody
Materials and M e t h o d s
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1679
MORPHINE INHISITS NF-K6 NUCLEAR BlNDlNG VIA NO
MFI of PMN and monocytes after incubation
with different morphine concentrations
PMN
Timedependent inhibition of NF-163 by morphine
Monocytes
MFI
6oo
*
1
*#
*#
500
*#
400
1-
300
-m-
Morphine50 nM
200
100
0
0
0
1
2
3
4
5
1 = Control, 2 = LPS 100 nglml, 3 = MO 50 nM + LPS,
4 = MO 50 pM + LPS, 5 = MO 1 mM + LPS
Fig. 1. Whole blood was preincubated with morphine for 150
min. To induce NF-KBnuclear binding, samples were incubated
with 100 ng/ml lipopolysaccharidefor 30 min. NF-KB nuclear
binding was determined by flow cytometry. After gating either
on neutrophils or monocytes, the median of fluorescent intensity was determined. *P c: 0.05 compared with control; #P c:
0.05 compared with stimulationwith lipopolysaccharidealone,
was ruled out by western blot of cytosolic and nuclear
extracts of leukocytes. The set up was performed using
commercially available DNA particles (DNA Quality Control Particles Kit; Becton Dickinson). A total of 20,000
events were recorded for each sample. To evaluate leukocyte subpopulations to which nuclei belonged, we
analyzed a forward-scatter/side-scatterdot plot. Polymorphonuclear and mononuclear cells were analyzed for
FITC staining using histogram analysis of the fluorescence-1parameter. The median of intensity fluorescence
was used as an indicator for the intensity of nuclei
fluorescence.
Statistical Interpretation of Data
Statistical analysis was performed using the Friedman
test. To isolate the group or groups that differ from the
others, a pairwise multiple comparison procedure was
used. Dunnett’s method was chosen because it allows
multiple comparisons against a control group. P < 0.05
was considered significant.
Results
Morphine Alone Has No Effect on NF-KB Activation
Three different concentrations of morphine were used
to determine if there is an effect on NF-KB activation.
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20
8)
80
loo
Morphine incubation [min]
40
123
140
18)
Fig. 2. Effect of morphine incubation for different time periods.
Neutrophil NF-KBnuclear binding of six different blood donors
are shown. Morphine was used at a concentration of 50 n~
(black) and 50 PM (gray). NF-KB nuclear binding decreased
significantly with longer incubation times. Statistical analysis
revealed a significant effect of morphine on NF-KB nuclear
binding at any time interval when compared with lipopolysaccharide stimulation alone (0-min morphine).
None of the concentrations tested (50 nM, 50 p ~1 ,mM)
had any effect on NF-KB activation compared with control values (data not shown). Incubation with the NO
donor SNAP (1 mM and 10 p ~alone
)
also failed to induce
nuclear binding of NF-KB.Subsequent stimulation with
LPS (100 ng/ml) was essential to activate NF-KB translocation to the nucleus. As shown in figure 1, the percentage of cells showing nuclear binding of NF-KB increased
from 2% to 88% in neutrophils and from 2% to 86% in
monocytes, respectively, after stimulation with LPS (100
ng/ml; P < 0.05).
Morphine Attenuates LPS-induced Nuclear Binding
of NF-KB in a Time- and Concentration-dependent
Manner
Two series of experiments were performed to establish time- and concentration-dependent effects of morphine. After a 10-min preincubation with morphine (50
nM and 50 p ~ )only
,
a slight reduction of NF-KB activation was observed after stimulation with LPS (fig. 2).
However, after preincubation with 50 nM morphine for
2.5 h, the median of channel fluorescence was reduced
from 486 to 433 in neutrophils and from 287 to 247 in
monocytes, respectively (fig. 1;P < 0.05), indicating the
inhibition of LPS-induced NF-KBnuclear binding. This
effect became more pronounced at higher concentrations of morphine (50 p~ and 1 nm).
Preincubation with morphine at different time inter-
1680
WELTERS ET AL.
Influence of SNAP, morphine and NAC on
NF-kB nuclear binding in monocytes
Influence of SNAP, morphine and NAC on
NF-kB nuclear binding in PMN
MFI
600
MFI
-
600
500
*#
*#
*
_I
*#
400
400
300
300
200
200
100
100
*#
4#
*#
4
5
6
*#
0
0
1
2
3
4
5
6
7
1
2
3
1 =Control, 2 = LPS 100 ng/ml, 3 = SNAP (1 pM) + LPS. 4 = SNAP (10 nM)
1 = Control, 2 = LPS 100 ng/ml, 3 =SNAP (1 pM) + LPS, 4 =SNAP (10 nM)
+ LPS,5 = MO 50 pM + LPS, 6 = NAC (10 mM) + LPS, 7 = NAC (30 mM) + LPS
+ LPS,5 = MO 50 pM + LPS, 6
7
= NAC (10 mM) + LPS. 7 = NAC (30mM) + LPS
Fig. 3. Reduction of NF-KBnuclear binding by NO donor S-nitroso-N-acetyl-pencillamhe(SNAP), morphine, and N-acetyl-L-cysteine
(NAC) in polymorphonuclear cells (A) and monocytes(B). SNAP and NAC at different concentrationsas well as morphine were added
10 min before stimulation with lipopolysaccharide.*P < 0.05 compared with control; #P < 0.05 compared with stimulation with
Lipopolysaccharide alone.
vals showed that morphine-induced effects on attenuation of NF-KB activation increased with longer preincubation times (fig. 2). Because morphine is known to
modulate the activation of constitutive NOS in endothelial cells,'' we surmise that also in blood cells an ongoing
NO release at low levels may be responsible for a stronger attenuation of NF-KB nuclear binding after a 2.5-h
preincubation period, as was also suggested by the SNAP
experiments. The decrease in median of channel fluorescence correlated with a lower percentage of cells exerting nuclear binding of NF-KB.
NO Donors Attenuate LPS-induced Nuclear Binding
Of NF-KB
As was shown in previous studies, NO donors such as
sodium nitroprusside or SNAP can induce NO-associated
changes in various types of cells.' Our data show that 1
p~ SNAP seems to be more effective than 50 p~ morphine in inhibiting NF-KB activation (fig. 9,
because its
effect in the nanomolar range is comparable to the one
observed with 50 FM morphine. However, it has to be
taken into consideration that NO release induced by 1
~ L SNAP
M
is more than twice as high as the one induced
by 1 ~ L morphine
M
(unpublished data).
Morphine Attenuation of LPS-induced Nuclear
Binding of NF-KB Is Abolished by NO Antagonists
To determine whether morphine attenuates NF-KB activation via NO release. the NOS inhibitors L-NAMEand
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N L A were administered to the cells 10 min before incubation with morphine. L-NAME (1 mM) as well as NLA
(0.5 mM) completely inhibited morphine-induced attenuation of NF-KB nuclear binding (fig. 4). These data
demonstrate that morphine exerts its effects on NF-KB
activation by inducing NO release in both cell types.
Quantajication of Morphine-induced Effects by
Comparison with N-acetyl-cysteine
Conventional methods to determine nuclear binding of
NF-KB only allow a semiquantitative estimation of NF-KB
activation. In this study, the efficacy of morphine in
reducing NF-KB activation was compared with N-acetylcysteine (NAC). As previously described, NAC completely aborted NF-KBactivation at a concentration of 30
m ~ l 'and inhibits NF-KB- dependent gene transcription
in viuo." The effect of 10 mM NAC on nuclear binding
of NF-KB was comparable to the one observed after
preincubation with 50 p~ morphine (fig. 4). These data
suggest that morphine in the micromolar range can inhibit NF-KB activation as effective as NAC in the millimolar range.
The Effects of Morphine Are Mediated by a Classical
p Opiate Receptor
Because naltrindole was found to have little affinity for
the opiate receptors present on neutrophils" while naloxone was very effective in reversing morphine induced
1681
MORPHINE INHIBITS NF-KB NUCLEAR BINDING VIA NO
NOS inhibitors abolish morphine-induced
NF-KB attenuation
1
MFI
6oo
500
PMN
Monocytes
*
1
400
300
200
100
0
1
2
3
4
5
I= LPS, 2 = Control, 3 = morphine + LPS, 4 = morphine
+ L-NAME + LPS, 5 = morphine + NLA + LPS
Fig. 4. Dependence of morphine-inducedNF-KB inhibition on
nitric oxide (NO) release using two different NO inhibitors.
L-NAME(1 m ~ was
) as effective in completely antagonizing
morphine effects as was NLA (0.5 m ~ ) Morphine
.
was used at a
concentration of 50 p ~and
, NF-KB stimulation was performed
with 100 n g / d lipopolysaccharide. *P < 0.05 compared with
control; XP < 0.05 compared with stimulation with lipopolysaccharide alone.
changes, we chose naloxone to investigate whether morphine acts via a classical p-subtype opiate receptor.
Preincubation with naloxone at a 10-fold higher concentration than morphine completely reversed morphineinduced attenuation of NF-KB nuclear binding after LPS
stimulation (fig. 5). Naloxone alone did not influence
NF-KB activation after incubation of whole blood with
LPS. We thus conclude that morphine attenuates LPSinduced NF-KBactivation by the p 3 opiate receptor coupled to NO release on human granulocytes and monocytes because opioid peptides do not release NO or bind
to this opiate receptor subtype that these immunocytes
contain.20’2‘
Discussion
The present study demonstrates the following: (1)
morphine does not alter basal NF-KB nuclear binding
without subsequent stimulation; (2) LPS stimulates
NF-KB nuclear binding; (3) morphine inhibits LPS-induced NF-KB nuclear binding in a naloxone antagonizable manner; ( 4 ) NO donors also inhibit LPS-induced
NF-KB binding; and ( 5 ) NOS inhibitors block morphine
inhibition of LPS-induced NF-KB binding; demonstrating
that morphine initiates its actions via constitutive NOS
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(cNOS) stimulation, since its NO-stimulating activity occurs immediately. Taken together, we surmise that morphine, in part, causes immunosuppression via the NOstimulated depression of NF-KB nuclear binding.
The significance of NF-KB in immune modulation is
evident. It is a DNA-bindingfactor that plays an essential
role in the activation of several inflammatory mediators
such as adhesion molecules,” TNF-qZ3interleukin 0L)S,’* IL-lP, IL-2, and IL6.25The NF-KB complex consists
of two heterodimers, termed p50 (NF-KBI) and p65
(RelA). In most types of cells, the NF-KB-inhibitor I K Bis~
phosphorylated and proteolytically degraded within
minutes on activation by iilflammatory agents such as
TNF-cK’~
The p50 -p65 complex is released, migrates to
the nucleus, binds to the promoter region of target
genes, and subsequently induces gene transcripti~n.~’
In
addition, the NF-KB/Rel transcription factor family also
plays a crucial role in cytokine-induced gene activation
during inflammatory responses or after exposure to bacterial LPS,”328,29
as also noted in the present study.
In addition to NF-KB immune modulation, NO also has
been reported to modulate the downregulation of immunocytes,’” such as inhibiting the expression of proinflammatory mediators3’ and adhesion molecules,” and
the phenotypic reversion of activated cells to a shape
that is round and nonmotile.’”‘ Although many NO
effects are coupled to cyclic guanosine monophosphate- dependent pathways,31 recent data have demonstrated that inhibitory actions of NO on immunocytes are
mediated by inhibiting the binding of NF-KB to specific
DNA binding motifs in the promoter region of proidammatory cytokine genes,” linking NO and NF-KB in a
cascading process.
Interestingly, in both neutrophils and monocytes, morphine stimulates cNOS-derived NO release that initiates
the NO-associated cell rounding and the loss of immunocyte adherence.’.’* In the same reports, opioid peptides were demonstrated to be immunocyte-excitatory,
and they did not liberate NO. Binding studies on monocytes and neutrophils demonstrated that they expressed
the opiate alkaloid-selective and opioid peptide-insensitive opiate receptor subtype, designated p3.” Furthermore, we demonstrated that morphine activates cNOS, a
calcium/calmodulin-dependent enzyme, by increasing
intracellular calcium levels.’” In these studies, it is also
important to note that NO donors, i.e., SNAP, mimic the
effects of morphine and that the morphine-mediated
processes are naloxone-sensitive, indicating that this action is mediated via the p3 receptor subtype.
In other reports, by different laboratories, NO donors
1682
WELTERS ET AL.
Naloxone abolishes morphine-attenuated
NF-KB nuclear binding in PMN
Naloxone abolishes morphine-attenuated
NF-KBnuclear binding in monocytes
MFI
MFI
500
350
400
300
250
300
200
200
150
100
100
50
0
0
1
A
2
3
4
5
6
7
1 = Control, 2 = LPS, 3 = MO 50 nM + LPS. 4 = MO 50 pM + LPS,
5 = naloxone + MO 50 nM + LPS. 6 = naloxone + MO 50 W M+
LPS, 7 = naloxone + LPS
1
B
2
3
4
5
6
7
I = Control, 2 = LPS, 3 = MO 50 nM + LPS, 4 = MO 50 pM + LPS,
5 = naloxone + MO 50 nM + LPS, 6 = naloxone + MO 50 pM +
LPS, 7 = naloxone + LPS
Fig. 5. Reversal of morphine-inducedNF-KBinhibition by naloxone in polymorphonuclear cells ( A ) and monocytes (B). Morphine
was used at a concentration of 50 rn and 50 p ~Naloxone
.
was used at a 10-foldhigher concentration than morphine. Naloxone
effects on NF-KB activation were ruled out by incubation of whole blood with 0.5 rn naloxone followed by stimulation with 100
ng/ml lipopolysaccharide.
have also been shown to reduce LPS-elicited NF-KB acti~ation.~*-’*
Here, the end results include the inhibition
of inducible NOS (iNOS) mRNA, suggesting that NO
may limit its own production by a negative feedback
mechanism based on an inhibition of NF-KB activation
that results in diminished expression of iNOS as a NFKB- dependent gene.‘>j5 Thus, morphine-induced NO
release via activation of cNOS may at least partially
contribute in the regulation of NF-KB- dependent transcription and may thus limit inflammatory processes.
To our knowledge, this study constitutes the first demonstration that morphine inhibits NF-KB activation in
human neutrophils and monocytes by a NO-coupled
mechanism. In other studies, human peripheral-blood
mononuclear cells exposed to NO-generating compounds exhibited enhanced glucose uptake and TNF-a
secretion. 36 Furthermore, nuclear binding of NF-KB was
induced by SNAP and sodium nitroprusside. These results are contradictory to the inhibitory effects of NO
donors on NF-KB nuclear binding found in the present
study. However, because of different roles of leukocyte
subpopulations in inflammation, NO may activate other
signaling pathways in peripheral-blood mononuclear
cells and may thereby finally lead to an activation of
NF-KB in these cells.
In peritoneal mouse macrophages, nanomolar morphine concentrations were found to increase LPS-inAnesthesiology, V 92, N o 6 , Jun 2000
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duced NF-KB a~tivation,’~
which correlated with an increased in TNF-a and IL-6 synthesis. However,
micromolar concentrations led to a reduction of NF-KB
activation and suppressed TNF-a and IL-6 gene transcripti~n.~
These
’
results are contradictory to the concentration-dependent effects of morphine established in our
study. The different cell types used may contribute to
these contrasting results, because the expression of
NF-KB subunits p50 and p65 changes during differentiation of monocyte-derived macrophages.” In addition, a
100-fold higher LPS concentration was used, which may
override low-dose morphine effects. Attenuation of
NF-KB activation induced by nanomolar and even micromolar concentrations of morphine is of profound clinical
significance, because serum concentrations up to 5 p~
were determined after high-dose treatment with morphine. ”
Another outcome of the present study is that the flow
cytometric detection and quantification of NF-KBnuclear
binding methodology allows for a rapid and highly reproducible determination of NF-KB activation in different leukocyte populations. l7 Because a whole-blood assay is used, preactivation of leukocytes by cell separation
is avoided. Although, in isolated cells, interactions between different cell populations as well as the influence
of plasma proteins cannot be determined, the wholeblood assay provides a comparatively physiologic sur-
1683
MORPHINE INHIBITS NF-KB NUCLEAR BINDING VIA NO
rounding. Conventional techniques such as western blot
or electric mobility shift assay require at least 5 X lo6
cells to ensure collection of a certain amount of proteins
in nuclear extracts, whereas the flow cytometric assay
can be performed with as little as 100 pl of whole blood.
Furthermore, preparation of nuclear extracts from neutrophils is delicate, because the presence of certain antiproteinases is required to avoid proteolytic degradation
of ~ - K B . * In
' accordance with previous
we
could rule out unspecific binding of the polyclonal antibody against p65 used in our flow cytometric assay by
western blot of nuclear and cytosolic extracts of leukocytes, which revealed a single band corresponding to a
65-kd protein. Assay standardization with stained nuclei
ensures exclusive determination of nuclear NF-KB.
In summary, our data demonstrate that in human neutrophils and monocytes, morphine attenuates NF-KB activation by a NO-coupled mechanism. This signaling
pathway may at least partly mediate morphine-induced
immunosuppression.
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