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CUTTING EDGE |
Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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1.5 nM for this PMN opioid receptor.
Nociceptin evoked PMN chemotaxis with maximal activity at 100 pM,
without intracellular Ca2+ mobilization. When injected in
murine air pouches, nociceptin elicited leukocyte infiltration in a
concentration-dependent fashion. Nociceptin-stimulated PMN infiltration
was inhibited by treating mice with a synthetic analog of the
aspirin-triggered lipid mediator 15-epi-lipoxin A4. The
present results identify nociceptin as a potent chemoattractant and
provide a novel link between the neural and immune systems that are
blocked by aspirin-triggered lipid mediators and may be relevant in
neurogenic inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The neuropeptide orphanin FQ/nociceptin is an endogenous ligand of the human opioid receptor-like, ORL1, (hereafter termed nociceptin receptor (Noci-R)5) (4), a member of the opioid receptors family (5). Nociceptin and its receptor are present in brain and are involved in the processing of pain signals (6, 7). Nociceptin is also present in the periphery at primary afferent nerve fibers (8) and may thus act on tissues such as the skin, visceral organs, and blood vessels, suggesting that additional physiological functions of this neuropeptide have yet to be appreciated.
In the present report, we demonstrate that Noci-R is also present and
functional in human leukocytes, and questioned whether nociceptin acts
on these cells. Because polymorphonuclear leukocyte (PMN) migration is
a critical component of inflammation and is held to play a role in
neuropathic pain, it was of interest to characterize the potential
relationship between these key cells of host defense and the neural
network. Aspirin (ASA) is well appreciated for its analgesic and
antipyretic actions (9). ASA therapeutic mechanism of
action includes inhibition of prostaglandin formation,
thought to be critical in its both analgesic and anti-inflammatory
properties. Cyclooxygenase (COX)-2, an isoform of COX that generates
prostanoids, is constitutively expressed in neuronal systems
(9). Recently, we have found that, when acetylated by ASA,
the ability of COX-2 to generate prostanoids is blocked yet stimulates
the biosynthesis of ASA-triggered-15-epi-lipoxin
A4 (ATL). These novel lipid mediators are the
carbon 15 epimers of the native lipoxins (10). ATL can
contribute to the beneficial action of ASA in that they down-regulate
key events in acute inflammation (10). The present
findings indicate that peripheral blood PMNs express Noci-R
(Kd 1.5 nM), and that its high affinity
ligand nociceptin stimulates chemotaxis and recruitment in vivo.
Moreover, a fluorinated ATL stable analog, namely
15-epi-16-(para-fluoro)-phenoxy-lipoxin
A4 (ATL-1) is a potent inhibitor of
nociceptin-stimulated leukocyte recruitment in vivo. Therefore,
nociceptin may constitute a novel mediator of neurogenic inflammation
that can be regulated by ATL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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PMNs from healthy volunteers were obtained as originally described (11), with modifications (12). Monocytes were isolated according to (13). PMN chemotaxis was performed using a microchamber technique as in Falk et al. (14), with modifications (15). The cells migrating across the filter (Neuroprobe, Gaithersburg, MD) onto the lower surface were counted in four high power fields under light microscopy in triplicate. Results are presented as the chemotactic index calculated by the following ratio: cells migrating to chemoattractants/cells migrating to vehicle. Nociceptin was purchased from Phoenix Pharmaceuticals (Belmont, CA) and fMLP was obtained from Sigma (St. Louis, MO); both were used throughout. Intracellular Ca2+ ([Ca2+]i) levels were monitored as in (16).
RT-PCR and sequencing
Total RNA from PMNs or monocytes was extracted with TRIzol (Life Technologies, Grand Island, NY), according to the manufacturer’s instructions. Total RNA (1 µg) was used in each reaction using Titan One tube RT-PCR (Roche Molecular Biochemicals, Indianapolis, IN). RT-PCR was performed according to the following profile: 50°C for 30 min for reverse transcription, then 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, repeated 35 times for PCR, followed by a final extension at 72°C for 10 min. Internal primers for human Noci-R were 5'-GAT GAA GAG ATC GAG TGC CTG GTG-3' (P1) and 5'-GGC CTT GAA GTT CTC ATC CAG GAA-3' (P2); with an expected PCR product of 402 bp in length. These primers were designed in view of the originally reported human nucleotide sequence Noci-R (5) to involve two exons (separated by an intron of 118 bp), which allowed discrimination between genomic DNA and expressed mRNA. Primers used to amplify the full coding sequence of Noci-R were 5'-ACC TGT CGT CGA CTG CCA GCC GGC-3' (P3) and 5'-CAC CAG GAG GGC AGC TTT AGT CTG-3' (P4), with an expected PCR product of 1471 bp in length according to the cDNA sequence. The latter primers were derived from the consensus sequence resulting from reported human sequences (GenBank and EMBL accession nos. X77130, U30185, NM_000913, and L40949).6 Primers were used in paired combinations to obtain overlapping fragments of 782 bp (P1-P4) and 1090 bp (P3-P2) in length, which together with the 402 bp fragment, span the entire coding region of the receptor. RT-PCR products were chromatographed in 1% agarose gel containing ethidium bromide and photographs of the gels were taken under ultraviolet illumination. Bands extracted from the gel (Qiaex II gel extraction kit; Qiagen, Valencia, CA) were analyzed by fluorescent dideoxy terminator-automated sequencing (BWH Automatic Sequencing and Genotyping Core Facility, Boston, MA).
Nociceptin binding with human PMN
[125I]-Labeled nociceptin binding
was performed with freshly isolated human PMN. Cells were suspended in
Dulbecco’s PBS with CaCl2 and
MgCl2 (DPBS2+). Aliquots
(10 x 106/0.1 ml PMN) were incubated with
0.25 nM of [125I]-labeled nociceptin (specific
activity 2000 Ci/mmol; Amersham Pharmacia Biotech, Buckinghamshire,
U.K.) in the absence or presence of increasing concentrations of
unlabeled nociceptin (Phoenix Pharmaceuticals) for 1 h at 14°C.
Incubations were terminated by adding 1 ml ice-cold
DPBS2+. The bound and unbound radioligands were
separated by brief centrifugation. Cell pellets were washed with 1 ml
of ice-cold DPBS2+. The radioactivity associated
with the cell pellets was determined using a scintillation counter
(Beckman Coulter, Fullerton, CA). Nonspecific binding was determined in
the presence of 100 nM of unlabeled nociceptin.
Murine air pouches
Six-day murine air pouches were raised on the dorsal skin of 6-wk-old BALB/c mice and experiments were performed essentially as in Ref. 17 . Briefly, 1.5 h after local injection of nociceptin into the air pouch cavity, mice were sacrificed and individual air pouches were lavaged twice with 2 ml sterile PBS. The lavage exudates were centrifuged and the cell pellets were suspended in PBS and enumerated. The synthetic analog of 15-epi-lipoxin A4, ATL-1 (18), was a generous gift from D. Perez, W. Guilford, and J. Parkinson (Berlex Biosciences, Richmond, CA) and its integrity was quantitated by liquid chromatography-tandem mass spectrometry as in Ref. 18 . The ATL-1 analog (10 µg), diluted in sterile PBS, was delivered as a bolus injection either into the tail vein or locally into the 6-day air pouch immediately before nociceptin injection.
Statistical analyses
Statistical analyses were performed using the ANOVA test, and significance was considered to be attained at p < 0.05.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Total RNA was extracted from freshly isolated human PMNs and
monocytes for RT-PCR was performed using specific primers for the human
Noci-R (see Materials and Methods). Both PMNs and monocytes
gave a band with the expected length, 400 bp (Fig. 1
), supporting the notion that both cell
types express this opioid-like receptor. To verify its identity, we
sequenced the Noci-R-like cDNA from isolated PMN. Using a combination
of primers (see Materials and Methods) with PMN cDNA
permitted sequencing of a continuous segment of this receptor that
spans the entire coding region (5) of the putative PMN
Noci-R (not shown). Sequence alignment of the PMN-derived cDNA revealed
identity with the reported sequence obtained from brainstem tissues
(5). Because both monocytes and PMN possess mRNA for
Noci-R that was cell ratio- (PMN/monocyte) dependent (n
= 3; not shown), we conducted direct specific binding experiments with
125I-labeled nociceptin and PMN. Human PMN
displayed specific [125I]-labeled nociceptin
binding with apparent Kd 1.5 nM.
Fig. 2 shows representative results from
three separate donors. Nonspecific binding accounted for 48% of the
total binding for PMN with the commercially isolated
[125I]-labeled nociceptin. These results
indicated that circulating PMNs express Noci-R mRNA and displayed high
affinity recognition sites, raising the question of whether these
receptors are functional on PMN.
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Next, we determined whether synthetic nociceptin evoked PMN
chemotaxis, using a microchamber migration assay with isolated human
PMNs. Nociceptin-induced chemotaxis was evident within a wide
concentration range
(10-14–10-6 M) giving a
steep rise and maximum effect at 10-10 M (Fig. 3). At the optimal concentration of
10-10 M, the chemotactic index was similar to
that obtained with 10-8 M of fMLP, a surrogate
of microbial-derived N-formylated proteins and a
well-appreciated chemoattractant for PMNs (19) that was
used here as a reference agonist for purpose of direct comparison.
These results are in line with the involvement of other neuropeptides
such as neurotransmitters in leukocyte chemotaxis, as recently reported
for norepinephrine (20), which stimulates chemotaxis of
monocytes, at an optimal concentration of 10-10
M. At these potential physiologically relevant concentrations
(10-14–10-6 M),
nociceptin is apparently not recognized by other opioid receptors nor
does the Noci-R bind other opioid ligands (4, 21).
Specific antagonists for the Noci-R that are devoid of agonist activity
are not currently available (22). Although it is possible
that other receptors present in PMNs might mediate nociceptin’s direct
chemotactic activity, the present results suggest that this
neuropeptide acts via engagement of its own recognized receptor Noci-R
(4, 6) present on PMN (Figs. 1
and 2). Also, the
proposed antagonist
[Phel-Psi(CH2)-NH)Gly(2)]nociceptin-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)NH2
(22) did compete at 14°C for specific binding in the
10-8–10-5 M range with human PMN (data not
shown).
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10,000-fold
the potency of substance P (23), and 1,000-fold that of
bradykinin (24) in a flexor-reflex pain model. In the
present experiments, nociceptin’s action on PMN chemotaxis showed a
peak in activity at a concentration of 100 pM, which is 100- to
1,000-fold more potent than that observed for the synthetic
chemoattractant peptide fMLP (19). Thus, our results
affirm that nociceptin is a highly potent biopeptide and indicate that
its actions extend to the immune system. Nociceptin acts on PMNs independently of [Ca2+]i mobilization
PMN chemoattractants typically stimulate production of
superoxide anions and induce
[Ca2+]i mobilization
(25). No detectable superoxide anion production in PMNs
was observed following exposure of PMN to nociceptin (n
= 3; data not shown). Also, when PMNs were exposed to a broad range of
concentrations of nociceptin
(10-14–10-6 M) it did
not stimulate a rise in
[Ca2+]i. Although
nociceptin did not alter the
[Ca2+]i responses to
either the chemoattractants fMLP (Fig. 4)
or IL-8 (not shown), this neuropeptide did potentiate in a very modest
but statistically significant manner
[Ca2+]i mobilization in
response to leukotriene B4
(LTB4) (Table I).
These results, together with 125I-labeled
nociceptin specific binding, suggest that nociceptin may represent a
new class of neural-derived chemoattractant molecules, possessing a
distinct profile of leukocyte activation than classical
chemoattractants (e.g., soluble PMN agonists) such as fMLP,
LTB4, or IL-8 that each evoke the rapid
mobilization of Ca2+ (25).
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To establish whether this neuropeptide can act as a
chemoattractant in vivo, we examined nociceptin in the murine dorsal
air pouch, an established animal model for evaluating leukocyte
infiltration (26, 27). When injected locally into the air
pouch, nociceptin elicited leukocyte infiltration in a
concentration-dependent manner (Fig. 5A). Statistically significant
leukocyte migration into the air pouch was observed with doses as low
as 10 ng nociceptin and the number of cells within the exudates
increased with higher amounts of this peptide, up to 1 µg, above
which there was no additional increase in PMN numbers. Leukocyte
recruitment values obtained with 1 µg nociceptin were within the same
range (Fig. 5B) of those induced by local injection of
recombinant murine TNF-
, a well appreciated stimulus for PMN
recruitment in this model (27). These findings, together
with results of direct specific binding with human PMN, suggest that
nociceptin, when present in peripheral tissues, can stimulate
diapedesis and leukocyte accumulation. Because ATL are bioactive
anti-inflammatory lipids that may mediate some of ASA beneficial
actions (10), we tested whether an ATL stable analog could
regulate nociceptin-stimulated leukocyte recruitment. Local delivery of
10 µg ATL-1 into the air pouch or by i.v. injection potently
inhibited nociceptin-driven leukocyte infiltration (Fig. 5B). To determine whether 1–100 nM ATL could directly
inhibit chemotaxis of human PMN, isolated cells were exposed to ATL
followed by nociceptin, and chemotaxis was assessed as in Fig. 3. In
these experiments, previous exposure to ATL did not block
nociceptin-stimulated PMN chemotaxis in vitro (n = 3;
not shown). LXA4, ATL, and their analogs inhibit
isolated PMN degranulation in response to immune complexes
(28) and inhibit PMN responses to certain soluble agonists
directly as well as indirectly by, for example, inhibiting
TNF--stimulated chemokine production in vivo (17).
Nociceptin directly stimulated PMN chemotaxis with isolated cells (Fig. 3) and stimulated recruitment of leukocytes in vivo that could involve
direct nociceptin-stimulated PMN chemotaxis as well as
nociceptin-stimulated generation of other endogenous chemoattractants
in vivo (Fig. 5), the formation of which appears to be blocked by ATL
in vivo. Together, these results identify a novel link between the
nervous and the immune system, namely the stimulation of leukocyte
trafficking by nociceptin.
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, which
can evoke leukocyte infiltration (27). In view of the
present results it is possible that nociceptin, which can be released
from afferent nerve fibers, contributes to leukocyte trafficking,
particularly PMN, toward a site of nociception, where it may serve as a
signal to amplify, both directly and/or indirectly via potential
release of additional endogenous chemoattractants, the recruitment of
PMNs to the site of inflammation that in turn evoke pain. In summary, the present results further elucidate the complex dialogue that can occur between immune and neural systems (30). Along these lines, it is of interest that recent data indicate that hematopoietic and neural lineages may be more closely associated than previously recognized (31). Hence, these results provide evidence that nociceptin evokes PMN recruitment that is likely to be relevant in the genesis of neurogenic inflammation, and may provide a basis for new therapeutic approaches to manage pain and inflammation, based on ATL structures.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Charles N. Serhan, Center for Experimental Therapeutics and Reperfusion Injury, Thorn Building for Medical Research, 7th Floor, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115.
3 Current address: Departamento de Farmacologia e Psicobiologia, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Av. 28 de Setembro 87 fnds 5o andar, Vila Izabel, Rio de Janeiro, Rio de Janeiro, Brazil, CEP: 20551-030.
4 Current address: Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, CHUL, Québec, Canada, G1V 4G2.
5 Abbreviations used in this paper: Noci-R, nociceptin receptor; ASA, aspirin; ATL, ASA-triggered-15-epi-lipoxin A4; ATL-1, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4; [Ca2+]i, intracellular Ca2+; LTB4, leukotriene B4; PMN, polymorphonuclear leukocyte; COX, cyclooxygenase; DPBS2+, Dulbecco’s PBS with CaCl2 and MgCl2.
6 The sequence presented in this paper has been deposited in the EMBL Nucleotide Sequence Database (accession no. AF348323).
Received for publication October 24, 2000. Accepted for publication January 19, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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RIIIB (CD16) and CD11b in response to inflammatory microcrystals. FASEB J. 12:209.-initiated neutrophil responses and trafficking: regulators of a cytokine-chemokine axis. J. Exp. Med. 189:1923.. J. Immunol. 1:3595.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
).
Bound and unbound radioligands were separated by centrifugation and
specific binding was determined. Results are representative of one
experiment of three separate donors.
denotes fMLP at
10-8 M. *, Significantly different
(p < 0.05) compared with PMN exposed to vehicle
alone (control). **, Significant differences of
p < 0.01.
