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Human White Blood Cells Synthesize
Morphine: CYP2D6 Modulation
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J Immunol 2005; 175:7357-7362; ;
doi: 10.4049/jimmunol.175.11.7357
http://www.jimmunol.org/content/175/11/7357
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References
Wei Zhu, Patrick Cadet, Geert Baggerman, Kirk J. Mantione
and George B. Stefano
The Journal of Immunology
Human White Blood Cells Synthesize Morphine:
CYP2D6 Modulation1
Wei Zhu,* Patrick Cadet,* Geert Baggerman,† Kirk J. Mantione,* and George B. Stefano2*
T
he immune-regulatory effects of opioid chemical messengers are well established (1, 2). Exogenously administered opiates are immunosuppressive, inhibiting both cellular and humoral responses via their cytokine-like effects in both
the CNS and the periphery (2). In vitro exposure of human monocytes and granulocytes to morphine results in immunosuppression,
as evidenced by a marked reduction in chemotaxis, phagocytosis,
and responsiveness of cells to signal molecules (3). Furthermore,
receptors for many of the opioid peptides as well as morphine have
been shown to be expressed outside the CNS (2). In addition, endogenous morphine-like compounds have been identified in diverse organisms, including humans, and are released in response to
surgical, pathogenic, and psychological stress (4 – 6). In this regard, because endogenous morphine is constitutively expressed
and overexpressed following trauma, we have surmised that this
opiate alkaloid normally down-regulates immune, vascular, and
neural responsiveness on a basal level. In addition, it also limits
microenvironmental noise and postinflammatory immune responsiveness (7–9).
Our laboratory recently demonstrated that human monocytes
and granulocytes express a novel ␮ opiate receptor, ␮3, which is
morphine selective but opiate peptide insensitive (10). This G protein-coupled receptor, an alternatively spliced variant of the ␮ receptor gene, mediates downstream signaling via constitutive NO
synthase-derived NO release (10).
Human plasma contains low concentrations of morphine, which
can increase following trauma or exercise (4 – 6). Potential sources
*Neuroscience Research Institute, State University of New York College at Old Westbury, Old Westbury, NY 11568; and †Laboratory of Developmental Physiology and
Molecular Biology, Zoological Institute, Catholic University of Leuven, Leuven, Belgium
Received for publication August 9, 2005. Accepted for publication September
21, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
of plasma morphine include the adrenal gland and/or brain. Another important potential source is white blood cells (WBC;3 leukocytes) since morphine is found in plasma constitutively (4). Although the presence of morphine in human plasma has been
demonstrated, the source and biosynthetic pathways have not been
identified. We therefore undertook this study to 1) determine
whether human WBC have the capacity to synthesize morphine; 2)
identify the biosynthetic pathways for morphine synthesis in these
cells; and 3) determine whether such synthesis can result in modulation of immune activity in a physiologically relevant manner.
Materials and Methods
Human blood was obtained from the Long Island Blood Services (Melville,
NY). Human heparinized whole blood was immediately separated using
1-Step Polymorphs (Accurate Chemical and Scientific) gradient medium.
Five milliliters of heparinized blood was layered over 5 ml of Polymorphs
in a 14-ml round-bottom tube and then centrifuged for 35 min at 500 ⫻ g
in a swinging bucket rotor at 18°C. After centrifugation, the lower band
consisting of polymorphonuclear cells (PMN) were harvested in 14-ml
tubes and then washed with 10 ml of PBS (Invitrogen Life Technologies)
by centrifugation for 10 min at 400 ⫻ g. In addition, residual RBC were
lysed using ACK lysing buffer (0.15 M NH4Cl2, 1 mM KHCO3, 0.1 mM
Na2EDTA (ph 7.2)) (Current Protocols in Immunology).
A two-way ANOVA was used for statistical analysis after precursor
exposure to the cells. Each experiment was performed four times. The
mean value was combined with the mean value taken from four other
replicates. The SEM represents the variation of the mean of the means. All
drugs were purchased from Sigma-Aldrich, except bufuralol, which was
purchased from BD Biosciences Clontech. The medium containing the
PMN was then separated after and before precursor exposure at varying
concentrations for 1 h. Cells were washed and the endogenous morphine
content was determined.
Morphine determination
The morphine extraction, isolation, and identification and quantification
protocols were performed as described elsewhere in great detail for the past
15 years (11). Several blank HPLC purifications were performed between
each sample to prevent residual morphine contamination remaining on the
column. Furthermore, invertebrate molluscan mantle tissue was run as a
negative control, demonstrating a lack of contamination (12). All solutions,
1
This work was supported in part by grants from the National Institute of Mental
Health (Grant 47392) and National Institute on Drug Abuse (Grant 09010).
2
Address correspondence and reprint requests to Dr. George B. Stefano, Neuroscience Research Institute, State University of New York–College at Old Westbury,
P.O. Box 210, Old Westbury, NY 11568. E-mail address: [email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
3
Abbreviations used in this paper: WBC, white blood cell; PMN, polymorphonuclear
cell; L-DOPA, L-3,4-dihydroxyphenylalanine; FF, form factor; Q-TOF, quadrupole
time-of-flight; MS, mass spectrometry.
0022-1767/05/$02.00
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Human plasma contains low, but physiologically significant, concentrations of morphine that can increase following trauma or
exercise. We now demonstrate that normal, human white blood cells (WBC), specifically polymorphonuclear cells, contain and
have the ability to synthesize morphine. We also show that WBC express CYP2D6, an enzyme capable of synthesizing morphine
from tyramine, norlaudanosoline, and codeine. Significantly, we also show that morphine can be synthesized by another pathway
via L-3,4-dihydroxyphenylalanine (L-DOPA). Finally, we show that WBC release morphine into their environment. These studies
provide evidence that 1) the synthesis of morphine by various animal tissues is more widespread than previously thought and now
includes human immune cells. 2) Moreover, another pathway for morphine synthesis exists, via L-DOPA, demonstrating an
intersection between dopamine and morphine pathways. 3) WBC can release morphine into the environment to regulate themselves and other cells, suggesting involvement in autocrine signaling since these cells express the ␮3 opiate receptor subtype. The
Journal of Immunology, 2005, 175: 7357–7362.
7358
medium etc., were also examined for any presence of morphine. The results
of these tests revealed a lack of morphine contamination.
Nanoflow electrospray quadrupole time-of-flight mass spectrometry (QTOF-MS) offers an effective way of demonstrating endogenous alkaloids in
biological tissues (11, 13, 14). Q-TOF-MS was performed on a Q-TOF
hybrid system (Q-Tof2, Micromass). One microliter of acetonitrile/water/
formic acid (30:69:1, v/v/v) of the PMN HPLC extraction was loaded in a
gold-coated borosilicate capillary (Proxeon). Authentic morphine standard
was used as a control. This sample was sprayed at a flow rate of ⬃30
nl/min, giving extended analysis time in which MS spectra, as well as
MS/MS spectra, were obtained. During MS/MS or tandem MS, fragment
ions are generated from a selected precursor ion by collision-induced dissociation. The collision energy is typically varied between 20 and 35 eV so
that the parent ion is fragmented into a number of different daughter ions,
allowing a comparison to be made between the experimental and authentic
samples.
RIA determination
The morphine RIA determination is a solid-phase, quantitative RIA,
wherein 125I-labeled morphine competes for a fixed time with morphine in
the test sample for the Ab binding site. The detection limit was 0.5 ng/ml.
The commercial kit used was obtained from Diagnostic Products (11–13,
15, 16).
Human heparinized whole blood obtained from volunteer blood donors
(Long Island Blood Services) was prepared as described above.
chemical adhesion molecule alterations (19 –21) as well as cytokine
production (19 –21).
All pharmacological agents were purchased from Research Biochemicals or Sigma-Aldrich.
Results
In control (vehicle-exposed) WBC, morphine was identified at a
level of 12.33 ⫾ 5.64 pg/million cells ⫾ SEM (Fig. 1). Mass
spectrometric analysis demonstrated the molecular mass of the single charged ion of morphine, which is identical with authentic
morphine (Fig. 1, top and middle). It is important to note that these
cells were extensively washed in serum-free RPMI 1640, limiting
any plasma morphine that may be found on the cells. However, it
is possible that the cells nonspecifically accumulated morphine
from plasma. To determine whether WBC contain morphine due to
endogenous synthesis, cells were incubated with specific morphine
precursors, including tyramine, previously shown to be present in
whole animals (12, 16, 22) and previously shown to increase morphine synthesis in healthy neural tissue (16, 22). Tyramine, norlaudanosoline tetrahydropapaveroline (THP), reticuline, and L-3,4dihydroxyphenylalanine (L-DOPA) significantly increase WBC
morphine concentrations above those found in untreated cells in a
concentration-dependent manner (ANOVA test, p ⬍ 0.001; Fig. 1,
Isolation of total RNA
PMN (5 ⫻ 106) were pelleted by centrifugation and total RNA was isolated
with the RNeasy Mini kit (Qiagen). Pelleted cells were resuspended in 500
␮l of RLT buffer and homogenized by passing the lysate five times through
a 20-gauge needle fitted to a syringe. The samples were then processed
according to the manufacturer’s instructions. In the final step, the RNA was
eluted with 50 ␮l of RNase-free water by centrifugation for 1 min at
10,000 rpm.
RT-PCR
First-strand cDNA synthesis was performed using random primers (Invitrogen Life Technologies). One microgram of total RNA was denatured
at 95°C and reverse transcribed at 40°C for 1 h using Superscript III RNase
H-RT (Invitrogen Life Technologies). Ten microliters of the RT product
was added to the PCR mix containing specific primers for the CYP2D6
gene and Platinum TaqDNA polymerase (Invitrogen Life Technologies.
The forward primer sequence was 5⬘-AGGTGTGTCTCGAGGAGC
CCATTTGGTA-3⬘ and reverse primer was 5⬘-GCAGAAAGCCCGACTC
CTCCTTCA-3⬘. The PCR was denatured at 94°C for 5 min, followed by
40 cycles at 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min and then
an extension step cycle at 72°C for 10 min. PCR products were analyzed
on a 2% agarose gel (Sigma-Aldrich) stained with ethidium bromide. The
expected sizes of the PCR products were 700 bp, 300 bp, and others as
described by Zhuge and Yu (17).
Sequencing of the PCR product
The 300-bp bands obtained after running the agarose gel were excised and
purified with the Qiaquick gel extraction kit (Qiagen). The PCR product
(200 ng) and the forward primer were then sent to Lark Technologies for
direct sequencing.
Computer-assisted cell activity analysis
PMN, obtained as described earlier, were also processed for image analysis
of cell conformation (18). The morphological measurements of PMN are
based on cell area and perimeter determinations by the use of image analysis software (Compix). Form-factor (FF) calculations were performed as
previously described (19 –21). The observational area used for measurement determinations and frame grabbing was 0.4 ␮m in diameter. The
computer-assisted image analysis system (Zeiss Axiophot fitted with
Nomarski and phase-contrast optics) was the same as previously
described (19 –21).
The cells were analyzed for conformational changes indicative of either
activation (amoeboid and mobile) or inhibition (round and stationary) (19 –
21). The lower the FF number, the longer the perimeter and the more
amoeboid the cellular shape. The proportion of activated cells was determined as previously described (19 –21). The activated state (amoeboid conformation) of human and vertebrate cells is correlated to bio-
FIGURE 1. Morphine detection and enhancement following precursor
exposure. Top, Q-TOF analysis of authentic morphine extracted from
HPLC fractions. WBC morphine (top) showed the same MS as authentic
material (middle, 286.14). They have the same fragments. Routinely, various controls were run to ensure that contamination had not occurred (all
solutions, media, and blank runs between samples as well as negative tissue
controls ensuring residual material was not on the column were performed). All glassware, etc., was new. Bottom, Human WBC obtained
from a buffy coat were separately incubated with various morphine precursors at varying concentrations for 1 h. WBC morphine significantly
increased in a concentration-dependent manner when incubated with tyramine, norlaudanosoline (THP), reticuline, and L-DOPA (p ⬍ 0.001, oneway ANOVA at the 10⫺7–10⫺6 M concentrations compared with a control
value of 12.33 ⫾ 5.64 pg/106 cells ⫾ SEM). Each experiment was repeated
four times and the mean ⫾ SEM was graphed.
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CYP2D6 molecular demonstration
WBC SYNTHESIZE MORPHINE
The Journal of Immunology
7359
FIGURE 2. CYP2D6 involvement with PMN morphine synthesis. Top,
PMN (106 cells per treatment) obtained from a buffy coat were incubated
with various tyramines and a CYP2D6 substrate, bufuralol, demonstrating
that morphine biosynthesis occurs via the involvement of the cytochrome
P450 isoform. The tyramine PMN morphine levels were diminished significantly in increasing concentrations of bufuralol (p ⬍ 0.001, one-way
ANOVA). Each experiment was repeated three times and the mean ⫾ SEM
was graphed. Middle, CYP2D6-specific inhibitors, quinidine (Q) and paroxetine, inhibit tyramine-, norlaudanosoline (THP)-, and codeine-stimulated morphine synthesis in human PMN. As shown, the inhibitors were
used at their demonstrated efficacious concentrations (10⫺6 M), inhibiting
tyramine (10⫺6 M)- and THP (10⫺7 M)-stimulated morphine production
(p ⬍ 0.001, one-way ANOVA compared with tyramine- and THP-stimulated PMN morphine levels, respectively). Codeine was a positive control
because this step was shown to be CYP2D6 dependent. Each experiment
was repeated five times and the mean ⫾ SEM was graphed. Bottom left,
Image of 2% agarose gel analysis of PCR product from human blood cells.
The CYP2D6 primers used in the PCR amplified an ⬃306-bp fragment in
both cell types tested. Lane 1, 100-bp DNA ladder; lane 2, negative control; and lane 3, blood cell PCR product. Bottom right, CYP2D6 sequence
(306 bp) obtained from human blood cells using RT-PCR exhibits 100%
sequence identity with known material.
bottom). Morphine concentrations in cells incubated with precursors were 90.25 ⫾ 10.42, 136.04 ⫾ 8.71, and 146.5 ⫾ 12.43 pg/
million cells ⫾ SEM after 1 h for THP, reticuline, and L-DOPA,
respectively. Furthermore, we found that morphine concentrations
increased with exposure to precursors in a concentration-dependent manner (Fig. 1, bottom). However, WBC incubated with the
serotonin precursors (23), either tryptophan or 5-hydroxytryptophan, in the range of 1 nM to 10 ␮M did not exhibit endogenous
FIGURE 3. Morphine release from PMN and its behavioral modification of naive cells. Top, Testing medium for morphine after the cells have
been removed for morphine. Values obtained after 1 h of incubating three
million WBC in medium either alone or with the respective morphine
precursor as indicated. Values are means ⫾ SEM via a one-way ANOVA,
which determined that all treatments are statistically significant at the p ⬍
0.05 level of confidence when compared with control. Bottom, PMN induced to make morphine limit cytokine (IL-1␤, 2 ng/ml)-stimulated activation when the two groups of cells are mixed. The following are the
treatments: 1, PMN activity level after 60 min, no treatment; 2, PMN
incubated with IL-1␤, which statistically increases the number of activated
cells; 3, PMN incubated with 10⫺6 M L-DOPA; 4, incubated with 50%
L-DOPA-exposed cells and 50% IL-1␤-exposed cells for 1 h, exhibiting
a lower level of activation compared to treatment 2; 5, PMN incubated
with 50% L-DOPA-exposed cells and 50% IL-1␤-exposed cells for 1 h;
5 min before being exposed to morphine they were exposed to 10⫺6 M
naloxone, resulting in an increase in cellular activation (ANOVA of
treatments 3 and 5 vary by p ⬍ 0.001 from treatment 1; treatment 4 by
p ⬍ 0.001 from treatments 3 and 5). Cells mixed without treatment
from the two groups exhibited only a 6% increase over that of their
respective controls. Each experiment was replicated four times and the
mean ⫾ SEM was graphed.
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morphine level increases or decreases, demonstrating a level of
specificity in morphine synthesis (data not shown). Regardless of
the concentration, these results demonstrate that WBC contain low
but physiologically significant quantities of morphine and that exposure of these cells to morphine precursors increases morphine
synthesis.
To identify a specific population of WBC capable of synthesizing morphine, we more closely examined PMN. Morphine was
found in these cells at the level of 11.2 ⫾ 4.21 pg/million cells ⫾
SEM (Fig. 2). Exposing PMN to morphine precursors including
tyramine, at levels previously shown to increase WBC morphine
concentrations, resulted in a statistically significant increase in
morphine concentrations in PMN (Fig. 2).
To determine whether CYP2D6 is involved in morphine synthesis in PMN, we incubated PMN with tyramine and the CYP2D6
substrate bufuralol and found significantly diminished synthesis of
morphine ( p ⬍ 0.001 compared with precursor augmentation levels; Fig. 2, top). Additionally, the CYP2D6 inhibitor, quinidine,
blocked morphine synthesis when tyramine, THP, or codeine was
exposed to PMN, further demonstrating CYP2D6 presence and
modulation (Fig. 2, middle). In addition, we found that CYP2D6 is
expressed in these cells, as exemplified by RT-PCR expression
analysis that amplified a 300-bp fragment corresponding to the
enzyme in PMN (Fig. 2, bottom right and left). Sequence analysis
of this fragment demonstrates 100% homology with human
CYP2D6 (Fig. 2, bottom right). These results demonstrate that
CYP2D6 is expressed in human PMN and that it is involved in
morphine synthesis.
7360
Possible roles for PMN-derived morphine include self-regulation as well as regulation of cells in the local environment or
distantly. It was of interest therefore to determine whether morphine found in PMN would also be found in the PMN incubation medium following exposure to morphine precursors (Fig.
3, top). We found that following exposure to morphine precursors, concentrations of morphine detected in the medium increased significantly compared with untreated cells (Fig. 3).
Before incubation with PMN, the incubation medium was found
to contain morphine at the lower detection limit of the assay
(Fig. 3).
To examine a possible physiological role of PMN-derived morphine, we then incubated precursor-treated PMN with other PMN
that had been exposed to different experimental protocols and evaluated their activity level via computer-assisted image analysis.
Specifically, we aimed to examine the effect of precursor-treated
PMN on PMN that had been activated by IL-1␤ (Fig. 3, bottom).
Untreated PMN exhibit an activity level of 7.3 ⫾ 2.1% activated
(FF ⬎0.6) compared with 43.4 ⫾ 5.7% for cells treated with IL-1␤
(2 ng/ml) after 1 h. PMN incubated with L-DOPA (10⫺6 M; 106
cells) exhibited a 3.7 ⫾ 0.4% level of activation. After washing
PMN separately and mixing the populations (L-DOPA treated and
IL-1␤ treated) in a 1:1 ratio, the percentage of activated cells decreased to 12.5 ⫾ 3.7% in the mixed PMN population (Fig. 3,
bottom). In performing the same experiment but cotreating the
IL-1␤ cells with naloxone and then mixing them with the L-DOPA-treated PMN, the activity level was 35.2 ⫾ 6.3% activated,
indicating that morphine mediated this reduced level of activation
since naloxone significantly blocked its action.
Discussion
In the current report, we demonstrate that morphine is present and
can be synthesized in human WBC and PMN from its precursors,
L-DOPA, reticuline, THP, and tyramine, in a concentration-dependent manner. In part, this finding provides further evidence for this
signaling molecule acting in a hormonal capacity as well as acting
within the confines of an autocrine loop, since PMN express the ␮3
opiate receptor subtype that is coupled to constitutive NO release
(10, 20, 24, 25). We envision the autocrine process to be inhibitory
in nature since morphine exposure to monocytes, granulocytes,
and platelets results in constitutive NO release, resulting in inactive rounded cells, which do not adhere (3, 7, 8, 26 –32). Taken
together, we surmise endogenous morphine normally performs this
immune down-regulating function.
The CYP2D6 isoenzyme, found on chromosome 22, is part of
the CYP2 family and is expressed in neural, immune, and hepatic
tissues (33). CYP is involved with the metabolism of many endogenous compounds such as biogenic amines, steroids, leukotrienes, fatty acids, and PG, where it may generate inactive or active
compounds (33). The CYP system is involved in the oxidative
metabolism of pharmaceutical compounds as well. For example,
anti-arrhythmics, ␤ blockers, antipsychotics, vasodilators, and, as
demonstrated in the present report (Fig. 4), analgesics are also
substrates for this enzyme (33).
In this report we show that CYP2D6 appears to act at critical
steps of the morphine biosynthetic pathway in PMN (12, 34, 35)
as others have done with different animal tissues (36 –38). We
show that bufuralol, a CYP2D6 substrate, as well as a CYP2D6
inhibitor, diminished morphine synthesis in PMN exposed to
tyramine. This provides additional support for the ability of this
tissue to synthesize morphine via dopamine since we and others
have shown that this enzyme is expressed in PMN (7, 38, 39).
Therefore, our data show that a key enzyme family, which is
also present in mammalian brain, is expressed in WBC and is
capable of synthesizing morphine via tyramine (Fig. 4). These
results, taken together with previous studies show that both
brain and WBC express both CYP2D6 and the ␮3 receptor subtype (10, 34, 39).
The morphine-enhancing effect of L-DOPA and its key position in animals in both the dopamine and morphine biosynthesis
pathways suggest that morphine may be involved in a regulatory step (7, 12) (Fig. 2). The observation that L-DOPA can also
lead to increased PMN morphine concentrations suggests that
an additional pathway for morphine synthesis exists via tyrosine (7). It is important to note that WBC also contain dopaminergic signaling components (40 – 42), supporting this
hypothesis.
Reticuline and THP have both been shown to be endogenously
synthesized in animal tissues (12, 16, 22), supporting their role in
the present pathway as precursors of morphine synthesis. Interestingly, each is believed to exert specific pharmacological effects.
However, given their presence in the biochemical morphine synthesis pathway their independent actions must be reexamined (12,
15, 16).
We further demonstrate that PMN exposed to morphine precursors eventually release morphine into their environment,
which can influence the activity state of the same cells as well
as other cells not exposed to the precursors. The mechanism of
morphine release is unknown, however. Excited cells may elaborate a factor that specifically stimulates the release of morphine from WBC. However, in cells not exposed to the precursors morphine can still be found in the environment, suggesting
that once synthesis starts it is designed to be released since this
may only occur at appropriate times, i.e., the presence of high
levels of these precursors. Further demonstrating that this is the
case in the ability of naloxone to block morphine’s action on
activated PMN in the vicinity of morphine precursor-loaded
WBC. What, however, is the physiological function of low
plasma concentrations of morphine? Perhaps, it provides a basal
level that may help down-regulate immune and vascular tissues
tonally via NO, preventing their activation by nonspecific microenvironmental noise (43).
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FIGURE 4. In general CYP2D6 mediates hydroxylation, demethylation, O-dealkylation, and arylamine N-oxidation (37). Top. Tyramine is
hydroxylated to form dopamine (37). Middle. (R)-reticuline is metabolized
through a p-ortho-oxidative coupling of (R)-reticuline, which is catalyzed
by a microsomal cytochrome P450 enzyme (48). Bottom. The human cytochrome P450 2D6 is involved in the demethylation of codeine to morphine (35).
WBC SYNTHESIZE MORPHINE
The Journal of Immunology
Disclosures
The authors have no financial conflict of interest.
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20. Stefano, G. B., A. Digenis, S. Spector, M. K. Leung, T. V. Bilfinger,
M. H. Makman, B. Scharrer, and N. N. Abumrad. 1993. Opiate-like substances
in an invertebrate, a novel opiate receptor on invertebrate and human immunocytes, and a role in immunosuppression. Proc. Natl. Acad. Sci. USA 90:
11099 –11103.
21. Stefano, G. B., and T. V. Bilfinger. 1993. Human neutrophil and macrophage
chemokinesis induced by cardiopulmonary bypass: loss of DAME and IL-1 chemotaxis. J. Neuroimmunol. 47: 189 –198.
22. Zhu, W., K. J. Mantione, L. Shen, and G. B. Stefano. 2005. In vivo and in vitro
L-DOPA exposure increases ganglionic morphine levels. Med. Sci. Monitor 11:
MS1–MS5.
23. Iversen, L. L., S. D. Iversen, and S. H. Snyder. Biochemistry of Biogenic Amines.
Plenum, New York.
24. Stefano, G. B., W. Zhu, P. Cadet, K. Mantione, T. V. Bilfinger, E. Bianchi, and
M. Guarna. 2002. A hormonal role for endogenous opiate alkaloids: vascular
tissues. Neuroendocrinol. Lett. 23: 21–26.
25. Stefano, G. B., W. Zhu, P. Cadet, and K. Mantione. 2004. Morphine enhances
nitric oxide release in the mammalian gastrointestinal tract via the ␮3 opiate
receptor subtype: a hormonal role for endogenous morphine. J. Physiol. Pharmacol. 55: 279 –288.
26. Stefano, G. B. 1994. Pharmacological and binding evidence for opioid receptors
on vertebrate and invertebrate blood cells. In Neuropeptides and Immunoregulation. B. Scharrer, E. M. Smith, and G. B. Stefano, eds. Springer-Verlag, New
York, pp. 139 –151.
27. Stefano, G. B., V. Kushnerik, M. Rodriquez, and T. V. Bilfinger. 1994. Inhibitory
effect of morphine on granulocyte stimulation of tumor necrosis factor and substance P. Int. J. Immunopharmacol. 16: 329.
28. Makman, M. H., T. V. Bilfinger, and G. B. Stefano. 1995. Human granulocytes
contain an opiate receptor mediating inhibition of cytokine-induced activation
and chemotaxis. J. Immunol. 154: 1323–1330.
29. Stefano, G. B., A. Hartman, T. V. Bilfinger, H. I. Magazine, Y. Liu, F. Casares,
and M. S. Goligorsky. 1995. Presence of the ␮3 opiate receptor in endothelial
cells: coupling to nitric oxide production and vasodilation. J. Biol. Chem. 270:
30290 –30293.
30. Bilfinger, T. V., A. Hartman, Y. Liu, H. I. Magazine, and G. B. Stefano. 1997.
Cryopreserved veins used for myocardial revascularization: a 5 year experience
and a possible mechanism for their increased failure. Ann. Thorac. Surg. 63:
1063–1069.
31. Bilfinger, T. V., A. Hartman, Y. Liu, H. I. Magazine, and G. B. Stefano. 1997.
Nitric oxide in homograft vein function. Ann. Thorac. Surg. 64: 1524 –1525.
32. Magazine, H. I., J. Chang, Y. Goumon, and G. B. Stefano. 2000. Rebound from
nitric oxide inhibition triggers enhanced monocyte activation and chemotaxis.
J. Immunol. 165: 102–107.
33. Touw, D. J. 1997. Clinical implications of genetic polymorphisms and drug interactions mediated by cytochrome P-450 enzymes. Drug Metab. Drug Interact.
14: 55– 82.
34. Pai, H. V., R. P. Kommaddi, S. J. Chinta, T. Mori, M. R. Boyd, and
V. Ravindranath. 2004. A frameshift mutation and alternate splicing in human
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In terms of an autocrine role for morphine, endogenously synthesized morphine could act via ␮3 receptors expressed on PMN
(10). In addition to demonstrating the immunomodulating effect of
morphine on PMN at the cellular level, our laboratory recently
identified, using expression microarray analysis, that morphine altered expression of RNA in key pathways such as TNF signaling,
IL-2 signaling, apoptosis, and oxidative stress response (44). Furthermore, in malignant histiocytosis, which is characterized by
hyperactive immune cells, a condition that results in death, the
␮3 opiate receptor subtype was not expressed on human immunocytes, resulting in cells which could not be down-regulated
by morphine (45). In general, morphine tended to down-regulate proinflammatory gene expression and up-regulate genes associated with signaling and immune down-regulation. These
observations support the evidence that suggests morphine’s role
in down-regulating tissue excitation via a constitutive NO synthase-derived NO pathway (10). The presence of opiate alkaloids in the circulation and of special opiate receptors on immunocytes, shown in both vertebrates and invertebrates,
provides a direct role for these compounds in both autocrine and
immunomodulatory pathways.
Dopamine can significantly effect the immune system (reviewed in Ref. 46). Depending on the environment (in vitro vs
in vivo) and cell type, dopamine has activating and suppressive
effects on cytokines such as IL-1␤, IL-2, IL-6, TNF, and IFN.
How these effects are mediated is unknown; however, our data
suggest a potential role for morphine signaling in some of these
processes.
In addition to a direct metabolic link, there are alternative hypotheses regarding the interaction between dopamine and morphine. For example, dopamine may be acting via cell surface dopamine receptors, supported by the observation that dopamine
receptor antagonists can block morphine-induced immunomodulation (47). The time course for these observed changes is likely
much slower than the metabolic link between dopamine and morphine that we have described in this study.
In summary, our data support the coupling of the morphine
and dopamine biosynthetic pathways, a finding that has tremendous biomedical significance that crosses many physiological
systems. Further studies that more clearly elucidate the potential interplay between these two regulatory chemical messengers and their effects on the immune system are currently
under way.
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2003. Gene expression of cytochrome P450 1B1 and 2D6 in leukocytes in human
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affinity dopamine binding to mouse thymocytes and Mytilus edulis (Bivalvia)
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WBC SYNTHESIZE MORPHINE
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immune system by electrospray ionization mass spectrometry. Rapid Commun.
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43. Stefano, G. B., Y. Goumon, T. V. Bilfinger, I. Welters, and P. Cadet. 2000. Basal
nitric oxide limits immune, nervous and cardiovascular excitation: human endothelia express a ␮ opiate receptor. Prog. Neurobiol. 60: 531–544.
44. Stefano, G. B., J. D. Burrill, S. Labur, J. Blake, and P. Cadet. 2005. Regulation
of various genes in human leukocytes acutely exposed to morphine: expression
microarray analysis. Med. Sci. Monitor 11: MS35–MS42.
45. Fricchione, G. L., L. Cytryn, T. V. Bilfinger, and G. B. Stefano. 1997. Cell
behavior and signal molecule involvement in a case study of malignant histiocytosis: a negative model of morphine as an immunoregulator. Am. J. Hematol.
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46. Basu, S., and P. S. Dasgupta. 2000. Dopamine, a neurotransmitter, influences the
immune system. J. Neuroimmunol. 102: 113–124.
47. Saurer, T. B., K. A. Carrigan, S. G. Ijames, and D. T. Lysle. 2004. Morphineinduced alterations of immune status are blocked by the dopamine D2-like receptor agonist 7-OH-DPAT. J. Neuroimmunol. 148: 54 – 62.
48. Gerady, R., and M. H. Zenk. 1992. Formation of salutaridine from (R)-reticuline
by a membrane-bound cytochrome P-450 enzyme from Papaver somniferum.
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