Peripheral administration of morphine attenuates postincisional pain by regulating macrophage polarization through COX-2-dependent pathway
© Godai et al.; licensee BioMed Central Ltd. 2014
Received: 14 January 2014
Accepted: 9 June 2014
Published: 14 June 2014
Macrophage infiltration to inflammatory sites promotes wound repair and may be involved in pain hypersensitivity after surgical incision. We recently reported that the development of hyperalgesia during chronic inflammation is regulated by macrophage polarity, often referred to as proinflammatory (M1) or anti-inflammatory (M2) macrophages. Although opioids such as morphine are known to alter the inflammatory milieu of incisional wounds through interactions with immunocytes, the macrophage-mediated effects of morphine on the development of postincisional pain have not been well investigated. In this study, we examined how morphine alters pain hypersensitivity through phenotypic shifts in local macrophages during the course of incision-induced inflammation.
Local administration of morphine in the early phase, but not in the late phase alleviated mechanical hyperalgesia, and this effect was reversed by clodronate-induced peripheral depletion of local macrophages. At the morphine-injected incisional sites, the number of pro-inflammatory F4/80+iNOS+M1 macrophages was decreased during the course of pain development whereas increased infiltration of wound healing F4/80+CD206+M2 macrophages was observed during the early phase. Morphine increased the gene expression of endogenous opioid, proenkephalin, and decreased the pronociceptive cytokine, interleukin-1β. Heme oxygenase (HO)-1 promotes the differentiation of macrophages to the M2 phenotype. An inhibitor of HO-1, tin protoporphyrin reversed morphine-induced analgesic effects and the changes in macrophage phenotype. However, local expression levels of HO-1 were not altered by morphine. Conversely, cyclooxygenase (COX)-2, primarily produced from peripheral macrophages in acute inflammation states, was up-regulated in the early phase at morphine-injected sites. In addition, the analgesic effects and a phenotype switching of infiltrated macrophages by morphine was reversed by local administration of a COX inhibitor, indomethacin.
Local administration of morphine alleviated the development of postincisional pain, possibly by altering macrophage polarity at the incisional sites. A morphine-induced shift in macrophage phenotype may be mediated by a COX-2-dependent mechanism. Therefore, μ-opioid receptor signaling in macrophages may be a potential therapeutic target during the early phase of postincisional pain development.
KeywordsMorphine Postoperative pain M1/M2 macrophages Cyclooxygenase-2 Heme oxygenase-1
Peripheral neuroimmune interactions play an important role in the development of pain hypersensitivity and in the process of wound healing after surgery. Macrophages, predominately activated during the early stage of the postoperative periods, eliminate necrotic tissue and protect the wound from post-surgical infection by increasing their phagocytic activity .
Macrophages can acquire distinct functional phenotypes depending on their microenvironment, such as is present at inflamed sites. Two well-established polarized macrophage phenotypes are proinflammatory (M1) and wound healing (M2) macrophages. M1 macrophages produce high levels of toxic intermediates associated with increased phagocytic activity and pro-nociceptive mediators, such as inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 , whereas M2 macrophages display homeostatic functions linked to wound healing . Mice lacking CCR2, a marker for the M1 phenotype and a receptor for macrophage-specific chemo-attractant macrophage chemoattractant protein-1, showed impaired inflammatory pain development and decreased macrophage infiltration to complete Freund’s adjuvant (CFA)-induced sites of inflammation , suggesting that M1 macrophages exacerbate the development of hyperalgesia after inflammation. The influx of M2 macrophages in the late phase is preceded by an influx of M1 macrophages . The phenotypic shift in macrophages toward an M2 phenotype is predominantly promoted by heme oxygenase (HO)-1, a stress-responsive enzyme with potent antioxidant and anti-inflammatory activities that is induced immediately after incision and that has antihyperalgesic effects against inflammatory pain after formalin injection [6–8]. We recently demonstrated that the activation of peroxisome proliferator-activated receptor (PPAR)γ signaling promotes macrophage polarization towards the M2 phenotype through an HO-1-dependent pathway, attenuating the development of CFA-induced inflammatory pain . In addition, PPARγ agonist alleviated postincisonal pain by regulating macrophage phenotype . Therefore, the balance between these two subsets of macrophages plays a crucial role in regulating the inflammation and pain development processes.
Peripherally-applied morphine can attenuate inflammatory pain induced by carrageenan  and CFA . Morphine regulates the production of neurotransmitters involved in nociception, such as substance P and iNOS, by phagocytes at inflamed sites [12, 13]. Recently, the peripheral actions of opioids on immune cells during the course of postoperative wound repair have been suggested. It was reported that morphine inhibited the monocyte-macrophage conversion phase, resulting in delayed migration of monocytes at the sites of injury . Peripheral administration of morphine suppressed the phagocytic activity of macrophages and promoted apoptosis by an HO-1-dependent mechanism [15, 16]. These data suggest that morphine might regulate peripheral sensitization through local neuro-inflammatory processes, particularly macrophages.
In closing wounds that were locally treated with morphine sulfate, macrophage infiltration was decreased in the early phase but increased in the late phase of the wound healing process . In addition, acute morphine administration reduced peri-incisional expression of proinflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α , which are major nociceptive mediators produced by M1 macrophages . Therefore, these reports support our hypothesis that morphine alters the M1/M2 balance in incised sites during the postoperative period.
Although acute peripheral actions of morphine have been suggested, the macrophage-mediated effects of morphine on the development of postincisional pain have not been well investigated. Therefore, we first examined whether repeated administrations of morphine to incised sites had analgesic effects, and then evaluated whether the effects of morphine were mediated by a shift in macrophage polarity at the incision sites.
Results and discussion
Local administration of morphine in the early phase of postincisional pain development ameliorates mechanical hyperalgesia
Long-lasting analgesic effects of morphine on mechanical hyperalgesia are mediated by local macrophages
Morphine alters macrophage polarity in incision-induced local inflammation
Morphine attenuates mechanical hyperalgesia by acting downstream of HO-1
Analgesic effects of morphine on mechanical stimuli was reversed by COX inhibitor
Morphine alters the phenotype of local macrophages through COX-2-dependent mechanism
We demonstrated that local administration of morphine in the early phase attenuated post-incisional mechanical hyperalgesia with a concomitant decrease in M1 macrophages and an increase in M2 macrophages. Depletion of macrophages, inhibition of HO-1 or COX all resulted in the reversal of morphine-induced analgesia, with an alteration in macrophage phenotype.
Previous studies have shown that infiltration of M1 macrophages preceded that of M2 macrophages during the course of pain development, resulting in the late onset of M2 macrophage-derived analgesic effects [5, 6]. Although the phenotype shift of macrophages was observed on POD2 (Figure 3), morphine had no analgesic effects during PODs 1–5 (Figure 1A). This might be explained by the changes in the expression levels of IL-1β and enkephalin on POD7, but not POD2 (Figure 4). These data suggest that functional changes in macrophages require additional time after phenotype shift by the administration of morphine, which may also explain why levels of enkephalin were increased on POD7 (Figure 5) although the number of M2 macrophages was unchanged by morphine on POD7 (Figure 3). Another possibility is that the transient increase in pronociceptive PGE2 by morphine on POD2 (Figure 7B) may have resulted in a small analgesic effect in the early phase. Wolf et.al. reported that IL-1β-deficient mice exhibited complete abolishment of post-incisional pain behavior . Intraplantar injection of small doses (0.1-100 pg) of IL-1β decreased the mechanical threshold . Taken together with our data, IL-1β might be the critical mediator for the development of mechanical hyperalgesia, partly regulated by μ opioid receptor signaling (Figure 4).
Although it has been reported that a single administration of morphine into the hindpaw attenuated paw edema and thermal hyperalgesia in the acute phase of a carrageenan-induced inflammatory pain model , intraplantar morphine had no effect on paw edema when carrageenan was repeatedly administered . Supporting these previous reports, in the present study peripherally administered morphine had no effect on paw edema or hyperalgesia to heat stimuli from 1 day after incision (Figure 1). We previously reported that a phenotype shift to M2 macrophages by PPARγ signaling altered the threshold to mechanical, but not thermal stimuli in a CFA-induced inflammatory pain model . Thus, the development of thermal hyperalgesia may be modulated by macrophage-independent mechanisms.
In the present study, morphine was repeatedly administered for 3 days (days 0–2 or 5–7) after incision. Therefore, peripheral morphine tolerance is a concern for this study. Although the analgesic response was eliminated in mice receiving topical morphine alone for 3 days , Zollner et al. reported that mice receiving chronic morphine treatment (10 mg/kg subcutaneously twice daily for 4 days) did not develop tolerance at the peripheral μ-opioid receptors in the presence of CFA-induced paw inflammation . Furthermore, they demonstrated that tolerance ensued when endogenous opioid peptides in inflamed tissue were removed by antibodies or by depleting opioid-producing leukocytes with cyclophosphamide. Because the majority of opioid-containing leukocytes were ED1+ monocytes/macrophages in the late stage of CFA–induced inflammation , the possibility that the phenotype of local macrophages might have played a role in peripheral opioid tolerance during the development of inflammatory pain could not be excluded. Further investigation is needed to clarify the involvement of macrophage polarization in opioid tolerance.
The expression of COX-2 was increased in the early phase by morphine administration, and the analgesic effects of morphine in the late phase were reversed by coadministration of indomethacin (Figure 7). The phenotypic shift of local macrophages by morphine was mediated by COX-dependent mechanism (Figure 8). COX-2/PGE2 is known to be a pronociceptive mediator mainly released by local macrophages during acute inflammation. In response to peripheral inflammatory challenges by the administration of carrageenan and CFA, mice lacking the ATP-gated P2X4 channel did not elicit pain hypersensitivity and lacked the COX-dependent release of PGE2 , suggesting that COX-dependent release of PGE2 from macrophages is essential for the development of inflammatory pain. Conversely, previous reports have demonstrated that PGE2 promoted the differentiation of macrophages to the anti-inflammatory M2 phenotype [33, 34]. PGE2 release was enhanced from peritoneal macrophages isolated from morphine-dependent rats . Although the expression levels of Ptgs2 were not different between vehicle- and morphine-injected sites on POD7, morphine-injected mice exhibited less hypersensitivity to mechanical stimuli in the late phase. Therefore, we speculate that peripheral COX-2 in the microenvironment might have pronociceptive effects in the early phase, but inhibit the development of chronic pain by altering macrophage phenotype in the late phase.
We demonstrated that local administration of morphine attenuates the development of postincisional hyperalgesia through macrophage-dependent mechanisms. Phenotype shifts of local macrophages were induced by morphine administered early in the course of pain development, possibly through a COX2/PGE2-dependent pathway. Therefore, peripheral μ opioid receptors in macrophages could be a potential new therapeutic target for the development of postoperative pain therapies.
Male C57BL6 mice aged 8–10 weeks were obtained from CLEA Japan, Inc. (Tokyo, Japan). The Animal Research Committee of Kagoshima University approved all experimental procedures, which were implemented according to the guidelines of the National Institutes of Health and the International Association for the Study of Pain . Mice were housed in groups of four or five per cage in a 12 hour light–dark cycle.
Paw incision model
The mouse hindpaw plantar incision model was created as described previously . Mice were deeply anesthetized by inhalation of 1.5–2.0% isoflurane (Abbott, Tokyo, Japan) via a nose cone. A 5-mm longitudinal incision was made with a No. 11 blade through the skin and fascia of the plantar foot. The incision was started 2 mm from the proximal edge of the heel and extended toward the toes. The underlying muscle was elevated with a curved forceps leaving the muscle origin and insertion intact. The skin was apposed with a single mattress suture of 8–0 nylon. Morphine (Shionogi & Co. LTD., Japan) was dissolved in phosphate-buffered saline (PBS, pH 7.2), SnPP (Tocris Bioscience, Bristol, UK) was diluted in dimethyl sulfoxide (DMSO), and indomethacin (Nacalai Tesque, Kyoto, Japan) was diluted in Tris buffer (TB, pH8.0). Morphine (3 μg/20 μL or 10 μg/20 μL), naloxone (5 μg/20 μL, Wako, Osaka, Japan), SnPP (400 nmol/20 μL) or indomethacin (50 μg/20 μL) were injected locally to the incisional sites 1 hour after the skin was sutured, and on PODs 1 and 2, or on PODs 5–7. The total amount of solution injected to the hind paws was 20 μL/paw for all experiments. The suture was removed at the end of POD2.
All behavioral experiments were performed by the same tester in a blinded manner. Withdrawal latencies to heat stimuli were assessed by applying a focused radiant heat source to an unrestrained mouse placed on a heat-tempered glass floor using the Paw Thermal Stimulator (UCSD, San Diego, CA, USA). A thermal stimulus was then applied to the plantar surface of each hind paw. Each mouse was tested at an interval of 2–3 minutes. The latencies to thermal stimuli were calculated as the mean of three trials. A cut-off time was set at 20.5 s to avoid tissue damage. To evaluate tactile allodynia, calibrated von Frey filaments (0.08–2.00 g) were applied to the plantar surface of the hindpaw from underneath the mesh floor. The 50% paw withdrawal threshold was determined using the updown method . Behavioral experiments were performed before the administration of reagents to hind paws on PODs 1, 2, 5, 7, 10, 12, and 14.
Measurement of paw edema
Post-incisional edema, reflected by an increase in dorsal-to-ventral paw thickness, was measured by a micro-caliper (Shinwa Measuring Tools; Niigata, Japan). The mean of three measurements at each time-point was used for analysis.
Depletion of local macrophages
For macrophage depletion, 10 μL of clodronate encapsulated in liposomes (Clophosome-A) or empty control liposomes (Formu Max, Palo Alto, CA, USA) were locally injected into the incisional sites 1 hour after the skin was sutured, and on PODs 1 and 2.
Mice were deeply anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) and transcardially perfused with saline. Tissues were fixed in 4% paraformaldehyde overnight at 4°C and placed in 30% sucrose solution for 24 h at 4°C. Sections (30 μm thick) were incubated overnight with primary antibodies to pan-macrophage marker, F4/80 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), iNOS (1:500; Abcam, Cambridge, UK), or CD206 (1:100; Santa Cruz Biotechnology) at 4°C overnight and then incubated for 1 hour at room temperature with Alexa Fluor 488- or Alexa Fluor 546-conjugated secondary antibody (1:500; Invitrogen, Carlsbad, CA, USA) followed by nuclear staining with DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained using an LSM700 imaging system (Carl Zeiss, Aalen, Germany). The intensity of F4/80 immunofluorescence at clodronate-treated sites, the number of total F4/80+, F4/80+iNOS+, or F4/80+CD206+ cells with clearly visible nuclei stained by DAPI were evaluated using Image J 1.43u 2010 software (National Institutes of Health, Bethesda, MD, USA).
Total RNA of hind paws was extracted from the hindpaw using Sepazol reagent (Nacalai Tesque, Kyoto, Japan). The synthesis of first-strand cDNA was performed using High Capacity RNA-to-cDNA (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions. Quantitative PCR was performed on an ABI Prism StepOnePlus real-time PCR System (Applied Biosystems) TaqMan assays were performed for quantification of Il-1β (assay ID:Mm00434228_m1), Tnf (assay ID:Mm00443260_g1), Penk (assay ID:Mm012128758_m1), and Hmox-1 (assay ID:Mm00516005_m1) using TaqMan Fast Advanced Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Target gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase.
Data are presented as mean ± SEM. Differences among groups were analyzed using one-way or two-way ANOVA followed by Bonferroni post hoc testing (Graphpad Prism 5.0, La Jolla, CA, USA). A value of P < 0.05 was considered significant.
Analysis of variance
C-C chemokine receptor 2
Complete Freund’s adjuvant
Inducible nitric oxide synthase
Peroxisome proliferators-activated receptor γ
Prostaglandin-endoperoxide synthase 2
Tumor necrosis factor.
This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to M.H.-M.).
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