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* Immunology and Inflammation Center, North Shore-Long Island Jewish Research Institute and Division of Kidney Diseases and Hypertension, Long Island Jewish Medical Center, New Hyde Park, NY 11040; and
University of Texas Health Science Center, San Antonio, TX 78229
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation,
and this phosphorylation was inhibited by SB 202190 as well as by SB
203580. Anti-TGF- Ab as well as naltrexone (an opiate receptor
antagonist) inhibited morphine-induced macrophage P38 MAPK
phosphorylation. Anti-TGF- Ab also attenuated morphine-induced p53
as well as inducible NO synthase expression; in contrast,
NG-nitro-L-arginine
methyl ester, an inhibitor of NO synthase, inhibited morphine-induced
P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the
expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab
prevented morphine-induced macrophage apoptosis. Moreover, naltrexone
inhibited morphine-induced FasL expression. In addition, macrophages
either deficient in FasL or lacking p53 showed resistance to the effect
of morphine. Inhibitors of both caspase-8 and caspase-9 partially
prevented the apoptotic effect of morphine on macrophages. In addition,
caspase-3 inhibitor prevented morphine-induced macrophage apoptosis.
These findings suggest that morphine-induced macrophage apoptosis
proceeds through opiate receptors via P38 MAPK phosphorylation. Both
TGF- and inducible NO synthase play an important role in
morphine-induced downstream signaling, which seems to activate proteins
involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax)
cell death pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Opiate addicts are prone to bacterial infections (1, 2, 3, 4). Because the mononuclear phagocyte system plays a critical role in the host’s defense against bacteria, modulation of monocyte function by morphine may be important. Opiates have been reported to modulate monocyte function in the form of decreased migration, phagocytosis, bacterial killing, inhibition of Ab formation, and altered production of cytokines (5, 6, 7, 8, 9, 10, 11). Previously, Nair et al. (12) and Yin et al. (13) demonstrated that morphine promotes the apoptosis of lymphocytes. Recently, we reported that morphine triggered macrophage/monocyte apoptosis (14, 15). In those studies we suggested that morphine-induced macrophage apoptosis is mediated through the activation of inducible NO synthase (iNOS),3 and expression of p53 and Bax (14). However, it appears that morphine uses multiple pathways to promote macrophage apoptosis.
We and other investigators previously reported that morphine stimulated
the production of TGF- by macrophages (15, 16, 17, 18). Because
TGF- has been reported to act as an immune modulator, many of the
effects of morphine may be mediated through the generation of TGF-
by monocytes (19, 20). TGF- has been reported to
trigger apoptosis in a variety of cells (21, 22, 23). TGF-
promotes the induction of apoptosis through downstream signaling by
phosphorylation of mitogen-activated protein kinases (MAPKs)
(24) and Smad proteins (25). Recently, we
demonstrated that TGF- also plays a role in morphine-induced
macrophage apoptosis (15). In this study, we examined the
role of P38 MAPK phosphorylation in morphine-induced macrophage
apoptosis. To establish a causal relationship, we also studied the
effect of SB 202190 and SB 203580, inhibitors of P38 MAPK
phosphorylation, on morphine-induced macrophage apoptosis. In addition,
we studied the effect of morphine on the expression of other
proapoptotic proteins such as Fas and Fas ligand (FasL). We have also
made an attempt to delineate other pathways by which morphine may be
tilting the balance toward apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used a murine macrophage cell line (J774 cells; American Type Culture Collection, Manassas, VA). Confluent macrophages were subcultured in DMEM (Life Technologies, Grand Island, NY) containing 10% FCS (Life Technologies), 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies).
To determine the role of FasL and p53, we evaluated the effect of morphine on peritoneal macrophages harvested from control (C57BL/6J, stock no. 000664; The Jackson Laboratory, Bar Harbor, ME), FasL-deficient (B6Smn.C3H-FasLgld, stock no. 001021; the background of this strain is statistically 96.9% C57BL/6J-like; The Jackson Laboratory), and p53-knockout (KO) (B6-129-TrP53tm1Tyj; The Jackson Laboratory) mice.
Experimental reagents
TGF-1 was obtained from Collaborative Biomedical Products
(Bedford, MA) and was used in concentrations of 1–10 ng/ml. Anti-P38
MAPK and Abs to phospho-P38 MAPK were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-TGF-1,
mouse monoclonal anti-p53, mouse monoclonal anti-Bax, rabbit
polyclonal anti-FasL, and goat polyclonal anti-Fas Abs, were
also obtained from Santa Cruz Biotechnology. Inhibitors of caspase-8,
-9, and -3, specific inhibitors of P38 MAPK phosphorylation (SB 202190
and SB 203580), and an inhibitor of mitogen-activated protein
(MAP)/extracellular signal-related kinase (ERK) kinase (PD
098059) were obtained from Calbiochem (La Jolla, CA). Mouse
monoclonal anti-iNOS Ab was obtained from BD Transduction
Laboratories (Lexington, KY). Morphine was dissolved in normal saline,
stocked at a concentration of 10-2 M, and used
in concentrations of
10-8–10-4 M (National
Institute on Drug Abuse, Bethesda, MD). Curcumin (inhibitor of
c-Jun-AP-1 complex) was used in a concentration of 5 µM
(Sigma-Aldrich, St. Louis, MO).
Apoptosis studies
Morphologic evaluation of macrophage apoptosis was performed by staining cells with H-33342 (Molecular Probes, Portland, OR) and propidium iodide (PI; Sigma-Aldrich). H-33342 stains the nuclei of live cells and identifies apoptotic cells by increased fluorescence. Double staining by these two agents provides the percentage of live, apoptotic, and necrotic cells (14, 26). Cells were prepared under control and experimental conditions. At the end of the incubation period, cells were treated with H-33342 (1 µg/ml) for 7 min at 37°C. Subsequently, PI (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 UV light. The percentage of live, apoptotic, and necrosed cells was recorded in eight random fields by two observers unaware of the experimental conditions.
To confirm the effect of morphine on macrophage apoptosis, control and morphine-treated macrophages (2 x 106 cells) were prepared for DNA isolation as described previously (27). DNA concentration was determined by reading the absorbance at 260 nm. DNA was electrophoresed on a 1.8% agarose gel at 5 V/cm in 0.5x Tris boric acid, EDTA buffer containing 10 µg/ml ethidium bromide.
Protein extraction and Western blotting for total and phosphorylated P38 MAPK
Cells were incubated under control and experimental conditions (for all conditions, cells were pretreated with 10 ng/ml LPS for 12 h). Subsequently, cells were washed twice with ice-cold PBS and lysed in a modified radioimmunoprecipitation assay lysis buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSf, aprotinin 4 µg/ml, 1 mMg orthovanadate, and a protease inhibitor mixture (Calbiochem). The cell lysates were centrifuged at 15,000 x g for 20 min and the supernatants of cell lysates containing 20 µg protein each were subjected to 10% SDS-PAGE and transferred onto Immobilon P membranes (Millipore, Marlboro, MA). The membranes were probed with anti-P38 MAPK (1:1,000; Santa Cruz Biotechnology), anti-phospho-P38 MAPK (1:1,000, New England Biolabs, Beverly, MA), anti-FasL (1:1,000; BD PharMingen, San Diego, CA), anti-Fas (1:1,000; Santa Cruz Biotechnology), anti-p53 (1:1,000; Santa Cruz Biotechnology), anti-iNOS (1:1,000; BD Transduction Laboratories), or anti-Bax (1:1,000; Santa Cruz Biotechnology) Abs. A HRP-conjugated appropriate secondary Ab (1:2,000) was used to detect immunoreactive bands using an ECL system from Pierce (Rockford, IL).
P38 MAPK assay
Equal amounts (150 µg) of cell lysates from control and
treated cells were immunoprecipitated by incubating with anti-P38
MAPK Ab (Cell Signaling Technology, Beverly, MA) at 4°C for 3 h.
Protein A-Sepharose beads were added and incubated at 4°C for 1
h. The immunobeads were washed three times in radioimmunoprecipitation
assay buffer (consisting of 20 mM Tris (pH 7.5), 150 mM sodium
chloride, 5 mM EDTA, 0.1 mM sodium orthovanadate, 1 mM PMSF, 0.1%
aprotinin, and 1% Nonidet P-40) and twice in PBS. Beads were incubated
with 2.5 mg/ml substrate, myelin basic protein (MBP), 25 µM cold ATP,
and 1 µCi of [
-32P]ATP in MAPK buffer (10
mM HEPES (pH 7.4), 10 mM magnesium chloride, 0.5 mM DTT, and 0.5 mM
sodium orthovanadate) for 30 min at 30°C. Reaction was stopped by the
addition of sample buffer. The reaction mixture was boiled for 5 min
and subjected to SDS-PAGE using a 15% gel. The gel was dried and
exposed to Kodak Biomax MR x-ray film (Kodak, Rochester, NY) with an
image-intensifying screen. Western blotting with anti-P38 MAPK Ab
was performed on an aliquot of assay lysates to assess loading.
Statistical analysis
For comparison of mean values between groups, the unpaired t test was used. To compare values among multiple groups, ANOVA was applied and a Newman-Keuls multiple range test was used to calculate a p value. All values are means ± SEM except where otherwise indicated. Statistical significance was defined as p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the role of P38 MAPK phosphorylation in
morphine-induced macrophage apoptosis, equal numbers of macrophages
were incubated in medium containing vehicle (control), morphine
(10-8 and 10-6 M), SB
202190 (5 µM), or morphine (10-8 and
10-6 M) plus SB 202190 for 24 h. At the end
of the incubation period, cells were stained with H-33342 and PI. As
shown in Fig. 1
A, morphine
promoted macrophage apoptosis. SB 202190, an inhibitor of P38 MAPK
phosphorylation, inhibited (p < 0.001) the
effect of morphine on macrophage apoptosis. These results suggest that
morphine-induced macrophage apoptosis is mediated through P38 MAPK
phosphorylation.
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B), whereas PD 098059 did not modulate the
effect of morphine (Fig. 1C).
To confirm the effect of morphine on macrophage apoptosis, equal
numbers of macrophages were incubated in medium containing buffer,
morphine (10-6 M), SB 202190 (1 µM), or SB
202190 plus morphine for 24 h. Subsequently cells were harvested
and DNA was extracted and electrophoresed. As shown in Fig. 1D, morphine-treated cells showed DNA fragmentation into
multiple integers of 180 bp (ladder pattern); however, control cells as
well as cells treated with SB 202190 did not show fragmentation of
DNA.
To determine the role of the c-Jun-AP-1 complex in morphine-induced
macrophage apoptosis, we studied the effect of curcumin, an inhibitor
of the c-Jun-AP-1 complex (28). Equal numbers of
macrophages were incubated in medium containing buffer (control),
morphine (10-8 and 10-6
M), curcumin (5 µM), or curcumin plus morphine for 24 h. At the
end of the incubation period, cells were assayed for apoptosis.
Curcumin partially inhibited the morphine-induced macrophage apoptosis
(Fig. 2A).
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To determine the effect of morphine on macrophage P38 MAPK
phosphorylation, equal numbers of macrophages in 100-mm petri dishes
were incubated in medium (DMEM plus 10% FCS) containing either vehicle
(control) or variable concentrations of morphine
(10-8–10-6 M) for 60
min. At the end of the incubation, cells were prepared for Western
blotting and probed with anti-P38 MAPK and phospho-P38 MAP Abs. As
shown in Fig. 3A, morphine
promoted macrophage P38 MAPK phosphorylation.
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by macrophages, equal numbers of macrophages in
100-mm petri dishes were incubated in medium (DMEM plus 10% FCS)
containing vehicle (control), variable concentrations of morphine
(10-8–10-6 M),
anti-TGF- Ab, or morphine plus anti-TGF- Ab for 60 min.
At the end of the incubation, cells were prepared for Western blotting
and probed with P38 MAPK and phospho-P38 MAPK Abs. As shown in Fig. 3B, morphine enhanced the phosphorylation of P38 MAPK;
however, anti-TGF- Ab inhibited the phosphorylation of P38 MAPK
under a morphine-stimulated state.
To determine the direct effect of TGF- on the macrophage P38 MAPK
pathway, equal numbers of J774 cells were incubated in medium (DMEM
plus 10% FCS) containing either vehicle (control) or variable
concentrations of TGF- (1, 5, and 10 ng/ml) for 60 min. At the end
of the incubation period, cells were prepared for Western blotting and
probed with anti-P38 MAPK or phospho-P38 MAPK Abs. As shown in Fig. 4A, TGF- enhanced the
phosphorylation of P38 MAPK.
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(5 ng/ml) with or without SB 202190 for 60 min. At
the end of the incubation period cells were prepared for Western
blotting and probed with anti-P38 MAPK and phospho-P38 MAPK Abs. As
shown in Fig. 4B, SB 202190 attenuated P38 MAPK
phosphorylation under a morphine-stimulated state.
To determine the role of TGF- in the constitutive activation of P38
MAPK, we determined the effect of anti-TGF- Ab on the activation
of P38 MAPK under a basal and a morphine-stimulated state. In addition,
we determined the direct effect of TGF- on P38 MAPK activation.
Equal numbers of macrophages were incubated in medium containing buffer
(control), morphine
(10-8–10-6 M),
anti-TGF-1 Ab (1 µg/ml), anti-TGF-1 Ab plus morphine
(10-8 M), anti-TGF-1 Ab plus morphine
(10-6 M), or TGF-1 (1 ng/ml) for 60 min.
Subsequently, cells were lysed and a P38 MAPK assay was performed. As
shown in Fig. 5, morphine enhanced P38
MAPK activity; however, anti-TGF-1 Ab attenuated this effect of
morphine. Interestingly, anti-TGF-1 Ab-treated cells showed a
lower P38 MAPK activity when compared with control cells, thus
suggesting that TGF- plays a role in the constitutive activation of
macrophage P38 MAPK. Moreover, TGF- directly enhanced macrophage P38
MAPK activation.
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We previously demonstrated that NOS inhibitors
NG-nitro-L-arginine
methyl ester (L-NAME) and
NG-monomethyl-L-arginine
monoacetate attenuated morphine-induced macrophage apoptosis
(14). In addition, sodium nitroprusside, an NO-generating
agent, promoted macrophage apoptosis (14). We asked
whether NOS has some relationship with morphine-induced macrophage P38
MAPK phosphorylation. Equal numbers of macrophages were incubated in
medium containing either buffer (control) or L-NAME (1 mM)
with or without morphine
(10-8–10-6 M) for 1
h. At the end of the incubation period cells were harvested, proteins
were extracted, and Western blots were generated and probed for P38
MAPK and phospho-P38 MAPKs. As shown in Fig. 6A,
L-NAME partially inhibited the morphine-induced
P38 MAPK phosphorylation.
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To determine the role of FasL in morphine-induced macrophage
apoptosis, equal numbers of macrophages were incubated in medium
containing buffer (control), morphine
(10-8–10-6 M),
anti-FasL Ab (1 µg/ml), or anti-FasL Ab plus morphine for
24 h. Subsequently, cells were stained for apoptosis. As shown in
Fig. 2B, anti-FasL Ab partially attenuated the
morphine-induced macrophage apoptosis.
To further confirm the role of FasL, we evaluated the effect of
morphine on peritoneal macrophages isolated from control and
FasL-deficient mice. Equal numbers of macrophages harvested
from control and FasL-deficient mice were incubated in
medium containing variable concentrations of morphine
(0–10-4 M) for 16 h. Subsequently, cells
were stained for apoptosis and necrosis. Macrophages isolated from
control mice showed greater apoptosis when compared with macrophages
harvested from FasL-deficient mice in response to morphine
(Fig. 2C).
To determine whether morphine promotes the expression of Fas and FasL,
equal numbers of macrophages were incubated in medium containing either
buffer (control) or variable concentrations of morphine
(10-8–10-6 M) for 60
min. Subsequently, cells were prepared for Western blots and probed for
Fas and FasL. As shown in Fig. 6, C and D,
morphine promoted macrophage expression of both FasL and Fas.
Role of p53 in morphine-induced macrophage apoptosis
We previously reported that morphine enhances the expression of
p53 in macrophages (14). To confirm the role of p53 in
morphine-induced macrophage apoptosis we evaluated the effect of
morphine on peritoneal macrophages isolated from control (C57BL/6J; The
Jackson Laboratory) and p53-KO
(B6-129-TrP53tm1Tyj; The Jackson Laboratory)
mice. Equal numbers of macrophages harvested from control macrophage
(C-M
) and p53KO-macrophage (p53KO-M)
mice were incubated in medium containing either buffer (control) or
variable concentrations of morphine
(10-8–10-6 M) for
16 h. At the end of the incubation period cells were stained for
apoptosis. As shown in Fig. 7A, morphine induced greater
apoptosis in C-M when compared with p53KO-M. These
results confirm that p53 also plays a role in morphine-induced
macrophage apoptosis.
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We previously reported that Bax protein plays a role in
morphine-induced macrophage apoptosis (14, 15). In
addition, morphine-induced macrophage Bax expression was inhibited by
anti-TGF- Ab (15). In this study, we evaluated the
relationship between morphine-iNOS-mediated downstream signaling and
Bax expression. Equal numbers of macrophages were incubated in medium
containing either buffer or L-NAME (1 mM) with or without
morphine (10-8 and 10-6
M) for 16 h. Subsequently, Western blots were prepared and probed
for Bax. As shown in Fig. 6B, morphine promoted Bax
expression; however, this effect of morphine was partially inhibited by
L-NAME. Thus it appears that iNOS-mediated signal
transduction also contributes to the mitochondrial pathway-induced
macrophage injury.
Role of caspases in morphine-induced macrophage apoptosis
To determine whether morphine-induced macrophage activation is
mediated through extrinsic (caspase-8) or intrinsic (caspase-9)
pathways, we studied the effect of inhibitors of caspase-8 and
caspase-9. Equal numbers of macrophages were incubated in medium
containing buffer (control), morphine (10-8 M),
caspase-8 inhibitor (1 µM), caspase-9 inhibitor (1 µM), morphine
plus caspase-8 inhibitor, or morphine plus caspase-9 inhibitor for
24 h. Subsequently, cells were stained for apoptosis. As shown in
Fig. 7B, inhibitors of caspase-8 and caspase-9 partially
inhibited the morphine-induced macrophage apoptosis.
To determine the role of caspase-3 in morphine-induced macrophage
apoptosis, equal numbers of macrophages were incubated in medium
containing either buffer (control) or morphine
(10-8–10-6 M) with or
without caspase-3 inhibitor (5 µM) for 16 h. At the end of the
incubation period, cells were stained for apoptosis. Caspase-3
inhibitor attenuated the morphine-induced macrophage apoptosis (Fig. 7C).
Role of TGF- in morphine-induced iNOS and p53 expression
To determine the relationship between morphine-induced p53/iNOS
expression and TGF-, we studied the effect of anti-TGF- Ab on
morphine-induced macrophage p53 and iNOS expression. Equal numbers of
macrophages were incubated in medium containing buffer (control),
morphine (10-8 and 10-6
M), or anti-TGF- (1 µg/ml) with or without morphine
(10-8 and 10-6 M) for
1 h. Subsequently, proteins were extracted and Western blots were
prepared and probed for p53 and iNOS. As shown in Fig. 8, morphine promoted the expression of
p53 and iNOS; however, these effects of morphine were inhibited by
anti-TGF- Ab.
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To determine the role of opiate receptors in morphine-induced
macrophage P38 MAPK phosphorylation, equal numbers of macrophages were
incubated in medium containing buffer (control), morphine
(10-8 M), naltrexone
(10-6 M), or morphine plus naltrexone for 60
min. Subsequently, proteins were extracted and Western blots were
prepared and probed for total and phospho-P38 MAPKs. As shown in Fig. 9A, morphine promoted
P38 MAPKphosphorylation. However, this effect of morphine was
partially inhibited by naltrexone. These results indicate that
morphine-induced P38 MAPK phosphorylation may be mediated through
opiate receptors.
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B). These results suggest that morphine-induced FasL
expression may be meditated through opiate receptors. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and iNOS-mediated
downstream signaling, which appears to have an ongoing
"cross-talk." Because morphine enhanced the expression of
Fas, FasL, p53, and Bax, it appears that morphine-induced
macrophage apoptosis is associated with the activation of proteins
involved in both extrinsic and intrinsic cell death pathways.
Welters et al. (29) recently reported that morphine
inhibits LPS-induced NF-
B activation in murine macrophages. NF-B
has been considered to be a transcription factor for genes associated
with survival. Roy et al. (30) also previously reported
that morphine at higher concentrations not only inhibits activation of
NF-B but also suppresses the production of TNF-
. TNF- has been
reported to inhibit macrophage apoptosis (31). It appears
that morphine acts as a double-edged sword, stimulating the production
of TGF-, a proapoptotic cytokine, while attenuating the production
of TNF-, an antiapoptotic cytokine.
Signals from various stimuli are conducted to the nucleus to regulate
gene expression through a distinct set of MAPK signal transduction
cascades. These include the ERKs, the c-Jun
NH2-terminal kinases, and P38 kinase (32, 33). With respect to cell growth, the important targets of MAPKs
are transcription factors such as c-fos and
c-jun. c-fos and c-jun families of
transcription factors form functional heterodimeric transcription
complexes known as AP-1 (34) and determine whether the
outcome of the signal will promote proliferation or apoptosis.
Inflammatory cytokines such as TGF- or other stress signals activate
serine/threonine kinase, which is related to the kinases of the ERK
pathway (33). The MAPKs are highly specific for their
transcription factor substrates; for example, ERK phosphorylates Elk-1
but not activated transcription factor 2, whereas c-Jun
NH2-terminal kinase phosphorylates c-Jun but not
c-fos. Stress signals can also stimulate another
protein kinase, P38 (35). Previously, morphine has been
shown to activate c-fos expression in the rat brain
(36); however, in these studies upstream and downstream
signaling was not investigated and thus it is difficult to comment on
the morphine-triggered upstream MAPK pathways (36).
It is the balance between phosphorylated and dephosphorylated forces
that determines whether the transcription of apoptosis-associated genes
will be sustained or switched off. In the present study, morphine
promoted P38 MAPK phosphorylation. Interestingly, morphine-induced P38
MAPK phosphorylation was inhibited by anti-TGF- Abs. Moreover,
TGF- directly enhanced the phosphorylation of P38 MAPK. Because
these kinases have been demonstrated to propagate proapoptotic signals
and promote the transcription of cell death-associated genes, we
suggest that TGF- mediates morphine-induced macrophage apoptosis
through the phosphorylation of P38 MAPK kinases. The specificity of
this effect of TGF- was confirmed by the inhibitory effect of SB
202190 on P38 MAPK phosphorylation under both the TGF- and
morphine-stimulated states. Alternatively, TGF- may have promoted
the phosphorylation of Smad proteins. Phosphorylated Smad proteins may
interact with c-Jun, thus activating the transcription of proapoptotic
proteins (24, 25).
Previously, various investigators reported that morphine enhances DNA
damage in lymphocytic cells (37, 38, 39). These investigators
suggest that opiates might be either decreasing the capacity to repair
the damaged DNA or enhancing sensitization for DNA damage
(37, 38, 39). Interestingly, TGF- inhibits the expression
of FasL in lymphocytes, and thus it appears that morphine-induced
lymphocyte apoptosis is unlikely to be mediated through TGF-
(40). However, in vivo, theoretically, morphine-induced
macrophage FasL expression may participate in the induction of
apoptosis of bystander lymphocytes (41).
It appears that morphine promotes macrophage apoptosis through multiple pathways. We previously reported not only that morphine promoted macrophage iNOS expression but that morphine-induced macrophage apoptosis was also inhibited by NOS inhibitors L-NAME and NG-monomethyl-L-arginine monoacetate (14). Other investigators have also reported the role of NO in the induction of macrophage apoptosis (42). We also demonstrated that morphine-induced macrophage apoptosis was associated with increased expression of p53 and Bax (14). Expression of wild-type p53 appears to be linked to apoptosis promoted by DNA-damaging agents (43). P53 has been demonstrated to be a direct transcriptional activator of the Bax gene, a cell death promoter gene. Thus it is plausible that morphine-induced expression of p53 and Bax are linked. Because Bax is a protein belonging to the Bcl-2 family, an important player of the mitochondrial (intrinsic) pathway, it appears that an intrinsic pathway also plays a role in morphine-induced macrophage apoptosis. This notion is further supported by morphine-induced enhanced expression of iNOS and p53 (proteins linked to the mitochondrial pathway).
In the present study, P38 MAPK activation appears to play an important
role in morphine-induced macrophage apoptosis. Both morphine and
TGF- promoted P38 MAPK activity, whereas anti-TGF- Ab
inhibited P38 MAPK activity in both the stimulated and basal states;
therefore, it appears that TGF- induces the constitutive activation
of P38 MAPK in macrophages. Because anti-TGF- Ab inhibited
morphine-induced P38 MAPK activation, it appears that morphine-induced
P38 MAPK activation is mediated through TGF- generation.
We previously reported that morphine stimulated the production of
TGF- by macrophages and that morphine-induced apoptosis was mediated
through TGF- (15). In those studies, anti-TGF-
Ab also inhibited morphine-induced Bax expression, thus suggesting the
role of TGF--mediated downstream signaling in the activation of the
mitochondrial pathway. In the present study we found that
morphine-induced expression of Bax was inhibited by L-NAME.
Thus, it appears that morphine-induced Bax production is also mediated
through iNOS. Because anti-TGF- Ab as well as L-NAME
inhibited the morphine-induced P38 MAPK phosphorylation it appears that
both TGF- and iNOS contribute to the phosphorylation of P38
MAPK.
The binding of FasL to Fas results in the recruitment of an adapter protein, Fas-associated death domain, which associates with a proenzyme form of caspase-8 and leads to the activation of caspase-8, leading to the activation of downstream caspases and subsequent degradation of nuclear proteins (44). In this study, morphine enhanced macrophage expression of both Fas and FasL, whereas anti-FasL Ab inhibited the morphine-induced macrophage apoptosis. In addition, peritoneal macrophages harvested from FasL-deficient mice showed resistance to the apoptotic effect of morphine. Thus, it appears that an extrinsic pathway (Fas and FasL interaction) may also be contributing to morphine-induced macrophage apoptosis.
Interestingly, morphine-induced FasL expression was inhibited by
L-NAME, thus suggesting a link to the iNOS-mediated
downstream signaling in the activation of the FasL-mediated (extrinsic)
pathway. In contrast, L-NAME also inhibited
morphine-induced Bax expression. These findings suggest that
iNOS-mediated downstream signaling contributes to the activation of
both the intrinsic (Bax, a protein belonging to intrinsic pathway) and
extrinsic (FasL, a protein participating in the extrinsic pathway)
pathways. Fig. 10 displays a
hypothetical scheme suggested for morphine-induced macrophage
apoptosis. These studies need to be further extended to delineate the
details of these pathways.
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We conclude that morphine promotes P38 MAPK phosphorylation via opiate
receptors through TGF-- and iNOS-mediated downstream signaling.
Because morphine activates proteins involved in extrinsic as well as
intrinsic cell death pathways it appears that both pathways contribute
to morphine-induced macrophage apoptosis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pravin C. Singhal, Division of Kidney Diseases and Hypertension, Long Island Jewish Medical Center, New Hyde Park, NY 11040. E-mail address: singhal{at}lij.edu
3 Abbreviations used in this paper: iNOS, inducible NO synthase; MAP, mitogen-activated protein; MAPK, MAP kinase; ERK, extracellular signal-related kinase; FasL, Fas ligand; L-NAME, NG-nitro-L-arginine methyl ester; PI, propidium iodide; KO, knockout; C-M, control macrophage; p53KO-M, p53KO macrophage.
Received for publication August 14, 2001. Accepted for publication February 19, 2002.
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  J. T. Mathew, H. Patni, A. N. Chaudhary, W. Liang, A. Gupta, P. N. Chander, G. Ding, and P. C. Singhal Aldosterone induces mesangial cell apoptosis both in vivo and in vitro Am J Physiol Renal Physiol, July 1, 2008; 295(1): F73 - F81. [Abstract] [Full Text] [PDF]
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 R. D. Sanders and M. Maze Translational Research: Addressing Problems Facing the Anesthesiologist Anesth. Analg., October 1, 2007; 105(4): 899 - 901. [Full Text] [PDF]
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 C. Spies, V. Eggers, G. Szabo, A. Lau, V. von Dossow, H. Schoenfeld, H. Althoff, K. Hegenscheid, B. Bohm, T. Schroeder, et al. Intervention at the Level of the Neuroendocrine-Immune Axis and Postoperative Pneumonia Rate in Long-term Alcoholics Am. J. Respir. Crit. Care Med., August 15, 2006; 174(4): 408 - 414. [Abstract] [Full Text] [PDF]
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 I. Tegeder and G. Geisslinger Opioids As Modulators of Cell Death and Survival--Unraveling Mechanisms and Revealing New Indications Pharmacol. Rev., September 1, 2004; 56(3): 351 - 369. [Abstract] [Full Text] [PDF]
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 A. Y. F. Kam, A. S. L. Chan, and Y. H. Wong {kappa}-Opioid Receptor Signals through Src and Focal Adhesion Kinase to Stimulate c-Jun N-Terminal Kinases in Transfected COS-7 Cells and Human Monocytic THP-1 Cells J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 301 - 310. [Abstract] [Full Text] [PDF]
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 R. S. Bhat, M. Bhaskaran, A. Mongia, N. Hitosugi, and P. C. Singhal Morphine-induced macrophage apoptosis: oxidative stress and strategies for modulation J. Leukoc. Biol., June 1, 2004; 75(6): 1131 - 1138. [Abstract] [Full Text] [PDF]
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 A. Porras, S. Zuluaga, E. Black, A. Valladares, A. M. Alvarez, C. Ambrosino, M. Benito, and A. R. Nebreda p38{alpha} Mitogen-activated Protein Kinase Sensitizes Cells to Apoptosis Induced by Different Stimuli Mol. Biol. Cell, February 1, 2004; 15(2): 922 - 933. [Abstract] [Full Text] [PDF]
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 G. Pelaia, G. Cuda, A. Vatrella, D. Fratto, R. D. Grembiale, P. Tagliaferri, R. Maselli, F. S. Costanzo, and S. A. Marsico Effects of Transforming Growth Factor-{beta} and Budesonide on Mitogen-Activated Protein Kinase Activation and Apoptosis in Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., July 1, 2003; 29(1): 12 - 18. [Abstract] [Full Text] [PDF]
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 I. Tegeder, S. Grosch, A. Schmidtko, A. Haussler, H. Schmidt, E. Niederberger, K. Scholich, and G. Geisslinger G Protein-independent G1 Cell Cycle Block and Apoptosis with Morphine in Adenocarcinoma Cells: Involvement of p53 Phosphorylation Cancer Res., April 15, 2003; 63(8): 1846 - 1852. [Abstract] [Full Text] [PDF]
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 G. Mace, M. Jaume, C. Blanpied, L. Stephan, J. D. Coudert, P. Druet, and G. Dietrich Anti-{micro}-opioid-receptor IgG antibodies are commonly present in serum from healthy blood donors: evidence for a role in apoptotic immune cell death Blood, October 16, 2002; 100(9): 3261 - 3268. [Abstract] [Full Text] [PDF]
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