Pain and Regional Anesthesia
Neurokinin-1 Receptor Antagonists Inhibit the Recruitment of Opioid-containing Leukocytes and Impair Peripheral Antinociception
Rittner, Heike L. M.D., D.E.S.A.*; Lux, Christian†; Labuz, Dominika Ph.D.‡; Mousa, Shaaban A. Ph.D.*; Schäfer, Michael M.D.§; Stein, Christoph M.D.∥; Brack, Alexander M.D., D.E.S.A.*
Background: Neurokinins (e.g., substance P) contribute to pain transmission in the central nervous system, peripheral neurogenic inflammation, and leukocyte recruitment in inflammation. Leukocyte recruitment involves (1) up-regulation of adhesion molecule expression through neurokinin-1 (NK1) receptors on endothelial cells, (2) augmented chemokine production, or (3) chemotaxis through NK1 receptors on leukocytes. In inflammation, leukocytes can trigger endogenous antinociception through release of opioid peptides and activation of opioid receptors on peripheral sensory neurons. The authors hypothesized that NK1 receptor antagonists impair recruitment of opioid-containing leukocytes and stress-induced antinociception.
Methods: Rats were treated intraperitoneally and intrathecally with peripherally restricted (SR140333) or blood-brain barrier–penetrating (L-733,060) NK1 receptor antagonists and were evaluated for paw pressure thresholds, numbers of infiltrating opioid-containing leukocytes and leukocyte subpopulations, expression of adhesion molecules, NK1 receptors, and chemokines 24–48 h after complete Freund adjuvant–induced hind paw inflammation.
Results: Systemic and peripherally selective, but not intrathecal, NK1 receptor blockade reduced stress-induced antinociception (control: 177 ± 9 g, L-733,060: 117 ± 8 g, and control: 166 ± 30 g, SR140333: 89 ± 3 g; both P < 0.05, t test) without affecting baseline hyperalgesia. In parallel, local recruitment of opioid-containing leukocytes was decreased (L-733,060 and SR140333: 56.0 ± 4.3 and 59.1 ± 7.9% of control; both P < 0.05, t test). NK1 receptors were expressed on peripheral neurons, infiltrating leukocytes and endothelial cells. Peripheral NK1 receptor blockade did not alter endothelial expression of intercellular adhesion molecule-1 or local chemokine and cytokine production, but decreased polymorphonuclear cell and macrophage recruitment.
Conclusions: Endogenous inhibition of inflammatory pain is dependent on NK1 receptor–mediated recruitment of opioid-containing leukocytes.
MANY cellular inflammatory mediators contribute to the generation of pain.1,2
However, leukocyte recruitment also contributes to peripheral antinociception.3
In complete Freund adjuvant (CFA)–induced inflammation, leukocytes containing opioid peptides (e.g.
, β-endorphin and metenkephalin) migrate to inflamed tissue. After exposure to a cold water swim stress (CWS) or local injection of various releasing agents, opioid peptides can be liberated from leukocytes and bind to nearby opioid receptors on peripheral sensory neurons resulting in unilateral antinociception.3–5
Both polymorphonuclear leukocytes (PMNs) and monocytes/macrophages cause opioid-mediated antinociception in different stages of inflammation.6–8
Blockade of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) or selectins inhibits the recruitment of opioid-containing leukocytes into the paw and CWS-induced antinociception.9–11
In early stages (i.e.
, 0–12 h after CFA injection), chemokines (i.e.
, CXCR2 ligands) are produced at the site of inflammation, mediate the recruitment of opioid-containing PMNs, directly trigger the release of opioid peptides from PMNs, and induce peripheral opioid-mediated antinociception.5,6
In contrast, at later stages (i.e.
, 24–96 h after CFA), molecular mechanisms for the recruitment of opioid-containing leukocytes have not yet been identified. PMNs are predominantly recruited in early inflammation (0–12 h after CFA), whereas macrophages are the principal leukocyte subpopulation at later stages (beyond 24 h after CFA).8
Leukocyte recruitment is classically mediated by chemokines, but other mediators, such as complement or neuropeptides, can also act as chemoattractants. Substance P is one of the neuropeptides and was originally described as a mediator in pain transmission in the central nervous system and in neurogenic inflammation in peripheral tissue.12,13
Three neurokinin (NK1–3
) receptors have been identified. Substance P preferentially binds to NK1
receptors are expressed on neurons in the peripheral and central nervous system as well as on leukocytes, endothelial cells, and keratinocytes.16–18
Neurokinin receptor agonists enhance leukocyte migration by three distinct mechanisms: (1) direct chemotactic effects on monocytes and PMNs,19–22
(2) binding to NK receptors on endothelial cells and increasing the expression of ICAM-1 and several selectins,17,23,24
and (3) augmentation of local chemokine production (e.g.
, CC chemokine ligand 2 [CCL2]; synonym: monocyte chemoattractant protein 1).25
In this study, we examined whether the blockade of NK1
receptors (1) impairs opioid-mediated peripheral antinociception; (2) reduces recruitment of opioid-containing leukocytes; and (3) influences endothelial adhesion molecule expression, local chemokine production, and/or migration of PMNs or monocytes/macrophages at 24 or 48 h of CFA inflammation. To further characterize the site of action of NK1
receptor agonists, we used systemic administration of blood-brain barrier–penetrating (L-733,060) and nonpenetrating NK1
receptor antagonists (SR140333) as well as intrathecal injection.26–28
Materials and Methods
Animals and Model of Inflammation
Animal protocols were approved by local authorities and are in accordance with the guidelines of the International Association for the Study of Pain.29
Male Wistar rats weighing 180–240 g were injected with 150 μl CFA (Calbiochem, La Jolla, CA) into the right hind paw (intraplantar) during brief isoflurane anesthesia and developed an inflammation confined to the inoculated paw. Experiments were conducted at 0–48 h after inoculation of CFA. All further injections were also performed during brief isoflurane anesthesia.
Measurement of Paw Pressure Threshold
Mechanical nociceptive thresholds were assessed using the paw pressure algesiometer (modified Randall-Selitto test; Ugo Basile, Comerio, Italy).30
Rats were handled for 4 days before testing. On the day of testing, rats were held under paper wadding, and incremental pressure was applied via
a wedge-shaped, blunt piston onto the dorsal surface of the hind paw by an automated gauge. The pressure required to elicit paw withdrawal, the paw pressure threshold, was recorded (cutoff at 250 g). The average of three trials, separated by 10-s intervals, was calculated. The same measurement was performed on the contralateral paw in alternated sequence to preclude order effects. To examine endogenous mechanisms of antinociception, paw pressure thresholds were determined at baseline conditions and 1 min after swimming in 2°–4°C cold water for 1 min.8
An increase in paw pressure threshold was interpreted as mechanical antinociception. All behavioral experiments were performed by an examiner blinded to the treatment protocol.
Implantation of Spinal Catheters
Implantation was performed under continuous isoflurane anesthesia as described.31
In brief, a 150-mm polyethylene tube (PE 10; Portex, Hythe, United Kingdom) was inserted intrathecally for 25 mm in cervical direction through an incision at the L3–L4 level. During the recovery period of 2 days, animals showing neurologic damage (e.g.
, paralysis of the hind limbs) were excluded from the study. To ensure intrathecal localization of the catheter, 10 μl lidocaine, 2% (Braun, Melsungen, Germany), followed by a 10-μl solvent flush was applied, and the animals were monitored for development and reversal of paralysis of both hind limbs.
Fluorescence-activated Cell Sorting
All hematopoietic cells were stained by mouse anti-rat CD45 Cy5 phycoerythrin monoclonal antibody (clone OX-1, identifies the leukocyte common antigen, 4 μg/ml; BD Biosciences, Heidelberg, Germany).32
PMNs were identified by mouse anti-rat RP-1 phycoerythrin monoclonal antibody (12 μg/ml). Macrophages were stained by mouse anti-rat CD68 fluorescein isothiocyanate monoclonal antibody (formerly called ED1; 2 μg/ml; Serotec, Oxford, United Kingdom). Opioid-containing cells were labeled by mouse 3E7 monoclonal antibody (recognizing the pan opioid sequence, 20 μg/ml; Gramsch Laboratories, Schwabhausen, Germany) followed by rabbit anti-mouse immunoglobulin (Ig)G2a+b
phycoerythrin antibody (15 μg/ml; BD Biosciences).5,7
receptor was stained by rabbit anti-rat NK1
receptor serum (1:100; Chemicon, Hampshire, United Kingdom) at 4°C for 30 min and subsequently by goat anti-rabbit IgG- fluorescein isothiocyanate antitbody (15 μg/ml; Vector Laboratories, Burlingame, CA). Specificity of staining was controlled by isotype-matched control antibodies (mouse IgG2a
, 20 μg/ml, BD Biosciences; and rabbit IgG 4 μg/ml, Santa Cruz, Santa Cruz, CA).
Cell suspensions from paw tissue were prepared and stained as described.5,7,32
Briefly, subcutaneous paw tissue was enzymatically digested and was pressed through a 70-μm nylon filter (BD Biosciences). Staining with anti-rat CD45 Cy5 phycoerythrin was done without permeabilization. For intracellular stains using 3E7, anti-rat–CD68 fluorescein isothiocyanate, and anti-rat RP-1 phycoerythrin, cells were fixed with 1% paraformaldehyde and permeabilized with saponin buffer. Permeabilized cells were incubated with the aforementioned primary and secondary antibodies. Replacement of the primary antibodies with isotype-matched irrelevant antibodies was used for negative controls.11
To calculate absolute numbers of cells per paw, fluorescence-activated cell sorting events from fluorescent TruCOUNT beads and CD45+ stained cells were collected simultaneously, and the number of CD45+ cells per tube was calculated accordingly. For quantification, 70,000 fluorescence-activated cell sorting events were acquired. Data were analyzed using CellQuest software (all BD Biosciences).
Enzyme-linked Immunosorbent Assay
All experiments were performed 0–48 h after intraplantar injection of CFA. Paw tissue was retrieved, the skin was removed, and subcutaneous tissue was cut into small pieces and processed as described.6
Substance P, CXC chemokine ligand 1 (CXCL1; synonym: keratinocyte-derived chemokine), CCL2, and interleukin-1β (IL-1β) concentrations were measured by commercially available substance P, mouse CXCL1, rat CCL2, and rat IL-1β enzyme-linked immunosorbent assay kits according to manufacturers' instructions (Bachem/Peninsula, London, United Kingdom; Biosource International, Nivelles, Belgium; and R&D, Minneapolis, MN, respectively). To measure CXCL1, a rat CXCL1 peptide standard was used as described.6
Optical density was measured by Spectra Max (Molecular Devices, Ismaning, Germany). Data were analyzed by the Softmax program (Molecular Devices).
The expression of ICAM-1 and NK1
receptor in inflamed paw tissue was analyzed in rats (n = 5/group) treated with NK1
receptor antagonists immediately before induction of inflammation and in control animals. Twenty-four hours later, rats were deeply anesthetized with halothane and perfused transcardially (0.1 m phosphate-buffered saline [PBS], followed by fixative solution: PBS containing 4% paraformaldehyde, pH 7.4). The skin with adjacent subcutaneous tissue was removed from both hind paws, postfixed for 30 min at 4°C in the fixative solution, and cryoprotected overnight at 4°C in PBS containing 10% sucrose. The tissue was embedded in Tissue Tek compound (OCT; Miles, Elkhart, IN), frozen, cut into 7-μm sections, mounted onto gelatin-coated slides, and processed for immunofluorescence.33
The sections were incubated with mouse anti–ICAM-1 (1:200) alone or in combination with rabbit polyclonal anti–NK1
receptor antibody (1:500) and then with secondary antibodies. After incubation with primary antibodies, the tissue sections were washed with PBS and then incubated with the appropriate secondary antibodies: a Texas red conjugated goat anti-rabbit antibody in combination with fluorescein isothiocyanate conjugated donkey anti-mouse. The sections were then washed with PBS, mounted in Vectashield or Vectashield mounting media containing 4′,6 diamidino-2-phenylindole (DAPI) (both Vector Laboratories, Burlingame, CA), and viewed under Zeiss LSM510META confocal laser scanning system (Carl Zeiss Imaging, Thornwood, NY). To demonstrate specificity of staining, the following controls were included as mentioned in details elsewhere10
by omission of either the primary antisera/antibodies or the secondary antibodies. Cell types were identified based on morphologic criteria. The number of ICAM and NK1
receptor expression in endothelial cells was quantified by an observer blinded to the experimental protocol using a Zeiss microscope (objective 20×).10
The mean number of ICAM– or NK1
receptor–positive vessels per section was calculated from counting 3 sections per animal and 5 squares (384 mm2
Systemic Application of Nk1 Receptor Antagonists.
Rats were injected intraperitoneally every 12 h with 20 mg/kg L-733,060 (dissolved in sterile water and further diluted in up to 500 μl normal saline; Tocris, Bristol, United Kingdom) or 10 mg/kg SR140333 (dissolved in dimethyl sulfoxide and further diluted in up to 500 μl normal saline; Sanofi, Paris, France). Doses were chosen based on previous publications34,35
and our pilot data. Control animals received solvent only (sterile water or dimethyl sulfoxide in normal saline, respectively). CFA was injected intraplantarly after the first injection, and experiments were performed 24–48 h later.
Intrathecal Application of Nk1 Receptor Antagonists.
Separate groups of rats were injected intrathecally with 0.5 mg/kg L-733,060 (20 μl every 12 h) followed by 10 μl NaCl (0.9%) flushes.36
Doses were chosen in pilot experiments and did not produce any toxic effects. Control animals received solvent only. Immediately after the first injection, paw inflammation was induced by CFA, and tissue was harvested 24 h later.
Data are presented as raw values or percentage control of baseline (mean ± SEM). Missing values are due to injection-related hematoma formation distorting fluorescence-activated cell sorting quantification of leukocyte subpopulations or due to accidental death by isoflurane overdose. Normally distributed data were analyzed by t test. Otherwise, the Mann–Whitney rank sum test was used. Multiple measurements were analyzed by one-way analysis of variance for normally distributed data and by two-way repeated-measures analysis of variance when dependent and independent factors were present. Post hoc comparisons were performed by the Student-Newman-Keuls and Holm-Sidak method, respectively. Differences were considered significant if P < 0.05.
Stress-induced Antinociception Is Reduced by NK1 Receptor Antagonists
Neurokinin-1 receptor blockade did not significantly change baseline paw pressure threshold in inflamed (fig. 1
) or noninflamed paws (data not shown). At 24 h of inflammation, CWS-induced antinociception was significantly reduced by both intraperitoneal L-733,060 and intraperitoneal SR140333 treatments (figs. 1A and B
), but not by intrathecal L-733,060 (fig. 1C
). Furthermore, systemic treatment with L-733,060 did not alter baseline hyperalgesia at 48 h of inflammation, whereas it significantly reduced CWS-induced antinociception (table 1
Recruitment of Opioid-containing Leukocytes Is Reduced by Blockade of NK1 Receptors
Cold water swim stress–induced antinociception is mediated by opioid-containing leukocytes at the site of inflammation.8,30
In the inflamed paw, 213 ± 11 × 103
opioid-containing leukocytes per paw were present at 24 h after CFA, as quantified by flow cytometry. At 24 h of inflammation, intraperitoneal L-733,060 or intraperitoneal SR140333 administration significantly reduced the number of opioid-containing leukocytes in the paw by 41% and 44%, respectively (figs. 2A and B
), whereas no effect was observed after intrathecal L-733,060 treatment (fig. 2C
). Systemic treatment (L-733,060 intraperitoneally) also significantly decreased the number of opioid-containing leukocytes in the paw at 48 h after CFA (control: 100 ± 7.4% vs.
intraperitoneal L-733,060: 55 ± 7.2%; P
< 0.05, t
Expression of NK1 Receptors and Substance P at the Site of Inflammation
Levels of substance P were low in noninflamed paws (fig. 3A
). At 24 and 48 h of inflammation, substance P content in the paw was significantly increased, with a 17.5-fold increase at 24 h. NK1
receptor expression was observed on endothelial cells as shown by colocalization with ICAM-1, peripheral neurons, and on CD45+
leukocytes in the inflamed paw (figs. 3B–D
Endothelial Adhesion Molecule Expression and Local Chemokine/Cytokine Content after NK1 Receptor Blockade
Intercellular adhesion molecule 1 and NK1
receptor immunoreactivities on endothelial cells were quantified. Both were significantly up-regulated at 24 h of inflammation (fig. 4
, representative examples; and table 2
). Blockade of NK1
receptors using intraperitoneal SR140333 treatment did not alter ICAM-1 or NK1
receptor expression at 24 h (table 2
). At 24 h inflammation, intraperitoneal SR140333 treatment did not alter the content of CXCL1 (PMN specific), CCL2 (monocyte specific), or IL-1β (table 3
NK1 Receptor Blockade Reduces Migration of PMNs and Macrophages
At 24 h of CFA, similar numbers of monocytes/macrophages and PMNs per paw were detected by flow cytometry (245 ± 18 × 103
macrophages per paw and 219 ± 16 × 103
PMNs per paw; fig. 5
). Treatment with intraperitoneal L-733,060 or intraperitoneal SR140333 significantly reduced infiltrating PMNs and macrophages (CFA 24 h: figs. 5A, B, D, and E
; CFA 48 h: table 4
lymphocytes did not change (data not shown). Intrathecal administration of L-733,060 did not influence overall leukocyte migration or recruitment of leukocyte subpopulation at 24 h after CFA (figs. 5C and F
In this study, we demonstrated that NK1 receptor antagonists (1) impaired stress-induced peripheral opioid-mediated antinociception but did not alter baseline inflammatory hyperalgesia and (2) reduced migration of opioid-containing leukocytes without changing expression of ICAM-1 on endothelial cells or local chemokine/cytokine production. Systemic administration of blood-brain barrier–penetrating (SR140333) and nonpenetrating (L-733,060) NK1 receptor antagonists was equally effective, but intrathecal injection (L-733,060) was ineffective. Taken together, NK1 receptor antagonists seem to act peripherally by directly inhibiting the recruitment of opioid-containing leukocytes to the site of inflammation.
Baseline inflammatory hyperalgesia was not reduced by administration of NK1
receptor antagonists (figs. 1A and B
and table 1
). These findings are in line with previous animal studies15,37
as well as studies in human pain patients.12
Intrathecal application of NK1
receptor antagonists was also ineffective in our study (fig. 1C
), but it has been reported to reduce hyperalgesia in other models.37–39
In those models, antihyperalgesic effects were short lasting (maximum of 6 h after injection), whereas we measured nociceptive thresholds at 24–48 h of inflammation and 12 h after the last injection of the NK1
receptor antagonist. Long-lasting antihyperalgesic effects were only achieved by intrathecal injection of saporin-conjugated substance P that destroys NK1
receptor–expressing spinal neurons.40
Inflammatory hyperalgesia is induced by pronociceptive mediators,2,41
including cytokines (e.g.
) and chemokines (e.g.
). In our studies, NK1
receptor blockade did not change the local expression of representative hyperalgesic cytokines or chemokines (table 3
), in line with the unaltered baseline nociceptive thresholds (fig. 1
and table 1
In contrast to this lack of change in baseline hyperalgesia, stress (CWS)–induced antinociception was reduced by NK1
receptor antagonists (figs. 1A and B
and table 1
). Our previous studies demonstrated that this form of antinociception is mediated by opioid peptide release from leukocytes at the site of inflammation and is fully blocked by opioid receptor antagonists in late stages of inflammation.8,30,46
In the current study, we observed a comparable decrease in opioid-containing leukocytes in the inflamed paw using both blood-brain barrier–penetrating (L-733,060) and nonpenetrating (SR140333) NK1
receptor antagonists, whereas intrathecal injection (L-733,060) was ineffective. The intrathecally injected dose of L-733,060 was previously used in rats and was shown to effectively inhibit NR2B tyrosine phosphorylation in the spinal cord in the CFA model.36
In addition to intrathecal injection of L-733,060, we also examined intrathecal injection of SR140333 (up to 6 μg/kg). This treatment did not reduce migration of opioid-containing leukocytes or stress-induced antinociception either, but we did not present the data because we observed neurotoxicity (e.g.
, paralysis) at the highest doses. CWS-induced antinociception is mediated exclusively by locally secreted opioid peptides in subcutaneous inflammation.30,47
In another paradigm, swim stress has been shown to involve IL-1 receptor activation,48
but major differences exist between the two models: (1) presence versus
absence of inflammation, (2) species (rats vs.
mice), (3) type of swim stress (cold vs.
warm water), and (4) tests of nociception (Randall-Selitto vs.
hot plate). In particular, in our model CWS-induced antinociception cannot be blocked by an antibody against IL-1 or by an IL-1 receptor antagonist, indicating that this form of antinociception is not IL-1 dependent.49
Taken together, our data show that NK1
receptor antagonist treatment correlates with impaired recruitment of opioid-containing leukocytes as well as a decrease in opioid-mediated antinociception. This seems to involve a peripheral site of action (i.e.
, outside the central nervous system). Conceivably, peripheral NK1
receptors could be blocked on leukocytes, endothelial cells. or peripheral sensory neurons.
To further elucidate the mechanisms of reduced migration of opioid-containing leukocytes, we measured substance P content and NK1
receptor expression in the inflamed paw. Local substance P content increased during CFA-induced inflammation (fig. 3A
). Potential sources of substance P are the peripheral sensory neurons and infiltrating leukocytes.13,50,51
In addition to increased substance P amounts, we detected NK1
receptor expression on all infiltrating leukocytes, on neurons, and on endothelial cells (figs. 3B–D
), confirming previous studies.16,17,20,52
While neurogenic inflammation contributes to leukocyte infiltration, it is unclear whether blockade of NK1
receptors on peripheral sensory neurons influences leukocyte recruitment.53–55
receptors have previously been shown to enhance leukocyte recruitment by increasing adhesion molecule expression on endothelial cells,17,23,24
by augmenting chemokine production25
or by directly inducing chemotaxis of leukocytes.19–22
Adhesion molecules (i.e.
, ICAM-1, selectins, and integrins) have previously been shown to mediate the recruitment of opioid-containing leukocytes, and their blockade can impair peripheral opioid-mediated antinociception during CFA-induced inflammation.9,11
In the current study, expression of ICAM-1 on endothelial cells was increased at later stages of inflammation (24 h after CFA; table 2
). Neither the CFA-induced up-regulation of ICAM-1 nor that of NK1
receptors on endothelial cells was altered by peripheral NK1
receptor blockade (fig. 4
and table 2
). Previous in vitro
studies have demonstrated that substance P increased the expression of ICAM-116,17
as well as NK1
In in vivo
models of inflammation, the effects of NK1
receptor antagonists vary between experimental models: They decreased NK1
receptor but not ICAM-1 expression in a mouse model of pancreatitis.58
In contrast, they reduced ICAM-1 expression in experimental autoimmune encephalomyelitis.59
In our model, NK1
receptor antagonists reduced the recruitment of opioid-containing leukocytes, but they did not alter the expression of ICAM-1 or NK1
receptors on the inflamed endothelium. We cannot fully exclude the possibility that NK1 receptor antagonists alter the expression of other adhesion molecules such as selectins or integrins and, thereby, impair peripheral antinociception.
As a second hypothesis, NK1
receptor antagonists might decrease local chemokine production and thereby impair selective leukocyte recruitment. Substance P has been shown to up-regulate chemokine production in leukocytes and epithelial cells in vitro
In experimental pancreatitis, local chemokine expression was reduced by an NK1
We examined a PMN- and a monocyte/macrophage-specific chemokine in our model (i.e.
respectively), and their expression was not altered by NK1
receptor antagonists (table 3
). Multiple chemokines are responsible for PMN and monocyte/macrophage recruitment,65
and alterations in other chemokines might occur after treatment with NK1
receptor antagonists. Although this possibility cannot be excluded, our previous study demonstrated that PMN-specific chemokines (i.e.
, CXCR2 ligands) were regulated as a group and did not show relevant differences between individual chemokines.6
Taken together, in our model NK1
receptor antagonists reduce the migration of opioid-containing leukocytes, but this effect does not seem to be mediated through alterations in adhesion molecule expression on endothelial cells or in local chemokine production.
Third, substance P binding to NK1
receptors on leukocytes might directly induce migration of opioid-containing leukocytes. This migration might be reduced by NK1
receptor blockade. Several studies demonstrated that NK1
receptor agonists induce chemotaxis of PMNs and monocytes/macrophages in vitro
Previous studies have shown that PMNs and monocyte/macrophage migration to the site of inflammation is impaired after treatment with NK1
receptor antagonists or in NK1
In line with these studies, the number of infiltrating macrophages and PMNs as well as of opioid-containing leukocytes was reduced in our model (figs. 2 and 5
and table 4
In conclusion, we have shown that NK1
receptor antagonists reduce the recruitment of opioid-containing leukocytes into the inflamed paw and thereby impair opioid-mediated antinociception in CFA inflammation. NK1
receptor agonists such as substance P seem to directly induce preferential recruitment of opioid-containing monocytes/macrophages while no indirect effects (i.e.
, alterations in adhesion molecule expression or chemokine production) are observed. Most likely, NK1
receptor antagonists act directly on NK1
receptor–expressing, opioid-containing leukocytes, but the effects might also be mediated indirectly through NK1
receptors on peripheral neurons. Taken together, NK1
receptor agonists are important in the recruitment of opioid-containing leukocytes and the generation of peripheral opioid-mediated antinociception. The impairment of endogenous opioid-mediated peripheral analgesia might be an additional explanation for the lack of efficacy of NK1
receptor antagonist in human studies.12
The authors thank Susanne Kotré and Katharina Hopp (both Technicians, Department of Anesthesiology, Charité, Berlin, Germany) for expert technical assistance.
1. Julius D, Basbaum AI: Molecular mechanisms of nociception. Nature 2001; 413:203–10
2. Rittner HL, Machelska H, Stein C: Leukocytes in the regulation of pain and analgesia. J Leukoc Biol 2005; 79:1215–22
3. Stein C, Schäfer M, Machelska H: Attacking pain at its source: New perspectives on opioids. Nat Med 2003; 9:1003–8
4. Schäfer M, Mousa SA, Stein C: Corticotropin-releasing factor in antinociception and inflammation. Eur J Pharmacol 1997; 323:1–10
5. Rittner HL, Labuz D, Schaefer M, Mousa SA, Schulz S, Schäfer M, Stein C, Brack A: Pain control by CXCR2 ligands through Ca2+-regulated release of opioid peptides from polymorphonuclear cells. FASEB J 2006; 20:2627–9
6. Brack A, Rittner HL, Machelska H, Leder K, Mousa SA, Schäfer M, Stein C: Control of inflammatory pain by chemokine-mediated recruitment of opioid-containing polymorphonuclear cells. Pain 2004; 112:229–38
7. Rittner HL, Brack A, Machelska H, Mousa SA, Bauer M, Schäfer M, Stein C: Opioid peptide expressing leukocytes: Identification, recruitment and simultaneously increasing inhibition of inflammatory pain. Anesthesiology 2001; 95:500–8
8. Brack A, Labuz D, Schlitz A, Rittner HL, Machelska H, Schäfer M, Rezka R, Stein C: Tissue monocytes/macrophages in inflammation: Hyperalgesia versus
opioid-mediated peripheral antinociception. Anesthesiology 2004; 101:204–11
9. Machelska H, Cabot PJ, Mousa SA, Zhang Q, Stein C: Pain control in inflammation governed by selectins. Nat Med 1998; 4:1425–8
10. Machelska H, Mousa SA, Brack A, Schopohl JK, Rittner HL, Schäfer M, Stein C: Opioid control of inflammatory pain regulated by intercellular adhesion molecule-1. J Neurosci 2002; 22:5588–96
11. Machelska H, Brack A, Mousa SA, Schopohl JK, Rittner HL, Schäfer M, Stein C: Selectins and integrins but not platelet-endothelial cell adhesion molecule-1 regulate opioid inhibition of inflammatory pain. Br J Pharmacol 2004; 142:772–80
12. Hill R: NK1 (substance P) receptor antagonists: Why are they not analgesic in humans? Trends Pharmacol Sci 2000; 21:244–6
13. O'Connor, TM O'Connell J, O'Brien DI, Goode T, Bredin CP, Shanahan F: The role of substance P in inflammatory disease. J Cell Physiol 2004; 201:167–80
14. Cao YQ, Mantyh PW, Carlson EJ, Gillespie AM, Epstein CJ, Basbaum AI: Primary afferent tachykinins are required to experience moderate to intense pain. Nature 1998; 392:390–4
15. De Felipe C, Herrero JF, JA OB, Palmer JA, Doyle CA, Smith AJ, Laird JM, Belmonte C, Cervero F, Hunt SP: Altered nociception, analgesia and aggression in mice lacking the receptor for substance P. Nature 1998; 392:394–7
16. Ho WZ, Lai JP, Zhu XH, Uvaydova M, Douglas SD: Human monocytes and macrophages express substance P and neurokinin-1 receptor. J Immunol 1997; 159:5654–60
17. Quinlan KL, Song IS, Bunnett NW, Letran E, Steinhoff M, Harten B, Olerud JE, Armstrong CA, Wright Caughman S, Ansel JC: Neuropeptide regulation of human dermal microvascular endothelial cell ICAM-1 expression and function. Am J Physiol 1998; 275:C1580–90
18. Harrison S, Geppetti P: Substance P. Int J Biochem Cell Biol 2001; 33:555–76
19. Schratzberger P, Reinisch N, Prodinger WM, Kahler CM, Sitte BA, Bellmann R, Fischer-Colbrie R, Winkler H, Wiedermann CJ: Differential chemotactic activities of sensory neuropeptides for human peripheral blood mononuclear cells. J Immunol 1997; 158:3895–901
20. Cao T, Pinter E, Al-Rashed S, Gerard N, Hoult JR, Brain SD: Neurokinin-1 receptor agonists are involved in mediating neutrophil accumulation in the inflamed, but not normal, cutaneous microvasculature: An in vivo
study using neurokinin-1 receptor knockout mice. J Immunol 2000; 164:5424–9
21. Souza DG, Mendonca VA, de A Castro MS, Poole S, Teixeira MM: Role of tachykinin NK receptors on the local and remote injuries following ischaemia and reperfusion of the superior mesenteric artery in the rat. Br J Pharmacol 2002; 135:303–12
22. Santos JM, Tatsuo MA, Turchetti-Maia RM, Lisboa MC, de Francischi JN: Leukocyte recruitment to peritoneal cavity of rats following formalin injection: Role of tachykinin receptors. J Pharmacol Sci 2004; 94:384–92
23. Miyazaki Y, Satoh T, Nishioka K, Yokozeki H: STAT-6-mediated control of P-selectin by substance P and interleukin-4 in human dermal endothelial cells. Am J Pathol 2006; 169:697–707
24. Scholzen TE, Steinhoff M, Sindrilaru A, Schwarz A, Bunnett NW, Luger TA, Armstrong CA, Ansel JC: Cutaneous allergic contact dermatitis responses are diminished in mice deficient in neurokinin 1 receptors and augmented by neurokinin 2 receptor blockage. FASEB J 2004; 18:1007–9
25. Ramnath RD, Bhatia M: Substance P treatment stimulates chemokine synthesis in pancreatic acinar cells via
the activation of NF-κB. Am J Physiol Gastrointest Liver Physiol 2006; 291:G1113–9
26. Rupniak NM, Carlson EC, Harrison T, Oates B, Seward E, Owen S, de Felipe C, Hunt S, Wheeldon A: Pharmacological blockade or genetic deletion of substance P (NK(1)) receptors attenuates neonatal vocalisation in guinea-pigs and mice. Neuropharmacology 2000; 39:1413–21
27. Rupniak NM, Carlson EJ, Shepheard S, Bentley G, Williams AR, Hill A, Swain C, Mills SG, Di Salvo, J Kilburn R, Cascieri MA, Kurtz MM, Tsao KL, Gould SL, Chicchi GG: Comparison of the functional blockade of rat substance P (NK1) receptors by GR205171, RP67580, SR140333 and NKP-608. Neuropharmacology 2003; 45:231–41
28. Rupniak NM, Carlson E, Boyce S, Webb JK, Hill RG: Enantioselective inhibition of the formalin paw late phase by the NK1 receptor antagonist L-733,060 in gerbils. Pain 1996; 67:189–95
29. Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983; 16:109–10
30. Stein C, Hassan AH, Przewlocki R, Gramsch C, Peter K, Herz A: Opioids from immunocytes interact with receptors on sensory nerves to inhibit nociception in inflammation. Proc Natl Acad Sci U S A 1990; 87:5935–9
31. Schmitt TK, Mousa SA, Brack A, Schmidt DK, Rittner HL, Welte M, Schäfer M, Stein C: Modulation of peripheral endogenous opioid analgesia by central afferent blockade. Anesthesiology 2003; 98:195–202
32. Rittner HL, Mousa SA, Labuz D, Beschmann K, Schäfer M, Stein C, Brack A: Selective local PMN recruitment by CXCL1 or CXCL2/3 injection does not cause inflammatory pain. J Leukoc Biol 2006; 79:1022–32
33. Mousa SA, Machelska H, Schäfer M, Stein C: Co-expression of beta-endorphin with adhesion molecules in a model of inflammatory pain. J Neuroimmunol 2000; 108:160–70
34. Bang R, Sass G, Kiemer AK, Vollmar AM, Neuhuber WL, Tiegs G: Neurokinin-1 receptor antagonists CP-96,345 and L-733,060 protect mice from cytokine-mediated liver injury. J Pharmacol Exp Ther 2003; 305:31–9
35. Luccarini P, Henry M, Alvarez P, Gaydier AM, Dallel R: Contribution of neurokinin 1 receptors in the cutaneous orofacial inflammatory pain. Naunyn Schmiedebergs Arch Pharmacol 2003; 368:320–3
36. Guo W, Zou S, Guan Y, Ikeda T, Tal M, Dubner R, Ren K: Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia. J Neurosci 2002; 22:6208–17
37. Sluka KA, Milton MA, Willis WD, Westlund KN: Differential roles of neurokinin 1 and neurokinin 2 receptors in the development and maintenance of heat hyperalgesia induced by acute inflammation. Br J Pharmacol 1997; 120:1263–73
38. Ren K, Iadarola MJ, Dubner R: An isobolographic analysis of the effects of N-methyl-D-aspartate and NK1 tachykinin receptor antagonists on inflammatory hyperalgesia in the rat. Br J Pharmacol 1996; 117:196–202
39. Traub RJ: The spinal contribution of substance P to the generation and maintenance of inflammatory hyperalgesia in the rat. Pain 1996; 67:151–61
40. Nichols ML, Allen BJ, Rogers SD, Ghilardi JR, Honore P, Luger NM, Finke MP, Li J, Lappi DA, Simone DA, Mantyh PW: Transmission of chronic nociception by spinal neurons expressing the substance P receptor. Science 1999; 286:1558–61
41. Rittner HL, Brack A: Chemokines and pain. Curr Opin Investig Drugs 2006; 7:643–6
42. Honore P, Wade CL, Zhong C, Harris RR, Wu C, Ghayur T, Iwakura Y, Decker MW, Faltynek C, Sullivan J, Jarvis MF: Interleukin-1αβ gene-deficient mice show reduced nociceptive sensitivity in models of inflammatory and neuropathic pain but not post-operative pain. Behav Brain Res 2006; 167:355–64
43. Cunha TM, Verri WA Jr, Silva JS, Poole S, Cunha FQ, Ferreira SH: A cascade of cytokines mediates mechanical inflammatory hypernociception in mice. Proc Natl Acad Sci U S A 2005; 102:1755–60
44. White FA, Sun J, Waters SM, Ma C, Ren D, Ripsch M, Steflik J, Cortright DN, Lamotte RH, Miller RJ: Excitatory monocyte chemoattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root ganglion. Proc Natl Acad Sci U S A 2005; 102:14092–7
45. Abbadie C, Lindia JA, Cumiskey AM, Peterson LB, Mudgett JS, Bayne EK, DeMartino JA, MacIntyre DE, Forrest MJ: Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. Proc Natl Acad Sci U S A 2003; 100:7947–52
46. Machelska H, Schopohl JK, Mousa SA, Labuz D, Schäfer M, Stein C: Different mechanisms of intrinsic pain inhibition in early and late inflammation. J Neuroimmunol 2003; 141:30–9
47. Stein C, Gramsch C, Herz A: Intrinsic mechanisms of antinociception in inflammation: Local opioid receptors and beta-endorphin. J Neurosci 1990; 10:1292–8
48. Wolf G, Yirmiya R, Kreisel T, Goshen I, Weidenfeld J, Poole S, Shavit Y: Interleukin-1 signaling modulates stress-induced analgesia. Brain Behav Immun 2007; 21:652–9
49. Schäfer M, Carter L, Stein C: Interleukin 1 beta and corticotropin-releasing factor inhibit pain by releasing opioids from immune cells in inflamed tissue. Proc Natl Acad Sci U S A 1994; 91:4219–23
50. Donnerer J, Schuligoi R, Stein C: Increased content and transport of substance P and calcitonin gene-related peptide in sensory nerves innervating inflamed tissue: Evidence for a regulatory function of nerve growth factor in vivo
. Neuroscience 1992; 49:693–8
51. Safieh-Garabedian B, Poole S, Allchorne A, Winter J, Woolf CJ: Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia. Br J Pharmacol 1995; 115:1265–75
52. Simeonidis S, Castagliuolo I, Pan A, Liu J, Wang CC, Mykoniatis A, Pasha A, Valenick L, Sougioultzis S, Zhao D, Pothoulakis C: Regulation of the NK-1 receptor gene expression in human macrophage cells via
an NF-kappa B site on its promoter. Proc Natl Acad Sci U S A 2003; 100:2957–62
53. Fristad I, Kvinnsland IH, Jonsson R, Heyeraas KJ: Effect of intermittent long-lasting electrical tooth stimulation on pulpal blood flow and immunocompetent cells: A hemodynamic and immunohistochemical study in young rat molars. Exp Neurol 1997; 146:230–9
54. Chavolla-Calderon M, Bayer MK, Fontan JJ: Bone marrow transplantation reveals an essential synergy between neuronal and hemopoietic cell neurokinin production in pulmonary inflammation. J Clin Invest 2003; 111:973–80
55. Razavi R, Chan Y, Afifiyan FN, Liu XJ, Wan X, Yantha J, Tsui H, Tang L, Tsai S, Santamaria P, Driver JP, Serreze D, Salter MW, Dosch HM: TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes. Cell 2006; 127:1123–35
56. Lau HY, Bhatia M: The effect of CP96,345 on the expression of tachykinins and neurokinin receptors in acute pancreatitis. J Pathol 2006; 208:364–71
57. Liu JY, Hu JH, Zhu QG, Li FQ, Sun HJ: Substance P receptor expression in human skin keratinocytes and fibroblasts. Br J Dermatol 2006; 155:657–62
58. Lau HY, Bhatia M: Effect of CP 96,345 on the expression of adhesion molecules in acute pancreatitis in mice. Am J Physiol Gastrointest Liver Physiol 2007; 292:G1283–92
59. Nessler S, Stadelmann C, Bittner A, Schlegel K, Gronen F, Brueck W, Hemmer B, Sommer N: Suppression of autoimmune encephalomyelitis by a neurokinin-1 receptor antagonist: A putative role for substance P in CNS inflammation. J Neuroimmunol 2006; 179:1–8
60. Guo CJ, Lai JP, Luo HM, Douglas SD, Ho WZ: Substance P up-regulates macrophage inflammatory protein-1beta expression in human T lymphocytes. J Neuroimmunol 2002; 131:160–7
61. Koon HW, Zhao D, Zhan Y, Simeonidis S, Moyer MP, Pothoulakis C: Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves protein kinase CΔ activation. J Pharmacol Exp Ther 2005; 314:1393–400
62. Sun J, Bhatia M: Blockade of neurokinin 1 receptor attenuates CC and CXC chemokine production in experimental acute pancreatitis and associated lung injury. Am J Physiol Gastrointest Liver Physiol 2006; 2006:27
63. Frevert CW, Huang S, Danaee H, Paulauskis JD, Kobzik L: Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation. J Immunol 1995; 154:335–44
64. Matsushima K, Larsen CG, DuBois GC, Oppenheim JJ: Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line. J Exp Med 1989; 169:1485–90
65. Murphy PM, Baggiolini M, Charo IF, Hebert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, Power CA: International Union of Pharmacology: XXII. Nomenclature for chemokine receptors. Pharmacol Rev 2000; 52:145–76
66. Kolasinski SL, Haines KA, Siegel EL, Cronstein BN, Abramson SB: Neuropeptides and inflammation: A somatostatin analog as a selective antagonist of neutrophil activation by substance P. Arthritis Rheum 1992; 35:369–75
67. Hood VC, Cruwys SC, Urban L, Kidd BL: Differential role of neurokinin receptors in human lymphocyte and monocyte chemotaxis. Regul Pept 2000; 96:17–21
68. Auais A, Adkins B, Napchan G, Piedimonte G: Immunomodulatory effects of sensory nerves during respiratory syncytial virus infection in rats. Am J Physiol Lung Cell Mol Physiol 2003; 285:L105–13
69. Costa SK, Yshii LM, Poston RN, Muscara MN, Brain SD: Pivotal role of endogenous tachykinins and the NK(1) receptor in mediating leukocyte accumulation, in the absence of oedema formation, in response to TNFα in the cutaneous microvasculature. J Neuroimmunol 2006; 171:99–109
This article has been cited 9 time(s).
Brain Research BulletinSubstance P induces gastric mucosal protection at supraspinal level via increasing the level of endomorphin-2 in ratsBrain Research Bulletin
Life SciencesEndogenous opioids regulate alveolar bone loss in a periodontal disease modelLife Sciences
Zeitschrift Fur RheumatologieOpioids in musculoskelettal painZeitschrift Fur Rheumatologie
Brain Behavior and ImmunityOpioid receptors and opioid peptide-producing leukocytes in inflammatory pain - Basic and therapeutic aspectsBrain Behavior and Immunity
Current Opinion in PharmacologyPeripheral mechanisms of opioid analgesiaCurrent Opinion in Pharmacology
Journal of Biological ChemistryNeurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent MechanismJournal of Biological Chemistry
Brain Research ReviewsPeripheral mechanisms of pain and analgesiaBrain Research Reviews
PeptidesEndogenous opiates and behavior: 2007Peptides
British Journal of AnaesthesiaPain and the immune systemBritish Journal of Anaesthesia
© 2007 American Society of Anesthesiologists, Inc.
Publication of an advertisement in Anesthesiology Online does not constitute endorsement by the American Society of Anesthesiologists, Inc. or Lippincott Williams & Wilkins, Inc. of the product or service being advertised.