Interleukin-4 Induces the Release of Opioid Peptides from M1 Macrophages in Pathological Pain.

Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can be protective in inflammatory and neurologic disorders, and can alleviate pain. Classically, IL-4 diminishes pain by blocking the production of proinflammatory cytokines. Here, we uncovered that IL-4 induces acute antinociception by IL-4 receptor α (IL-4Rα)-dependent release of opioid peptides from M1 macrophages at injured nerves. As a model of pathologic pain, we used a chronic constriction injury (CCI) of the sciatic nerve in male mice. A single application of IL-4 at the injured nerves (14 d following CCI) attenuated mechanical hypersensitivity evaluated by von Frey filaments, which was reversed by co-injected antibody to IL-4Rα, antibodies to opioid peptides such as Met-enkephalin (ENK), ß-endorphin and dynorphin A 1-17, and selective antagonists of δ-opioid, µ-opioid, and κ-opioid receptors. Injured nerves were predominately infiltrated by proinflammatory M1 macrophages and IL-4 did not change their numbers or the phenotype, assessed by flow cytometry and qRT-PCR, respectively. Macrophages isolated from damaged nerves by immunomagnetic separation (IMS) and stimulated with IL-4 dose dependently secreted all three opioid peptides measured by immunoassays. The IL-4-induced release of ENK was diminished by IL-4Rα antibody, intracellular Ca2+ chelator, and inhibitors of protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), and ryanodine receptors. Together, we identified a new opioid mechanism underlying the IL-4-induced antinociception that involves PKA-mediated, PI3K-mediated, ryanodine receptor-mediated, and intracellular Ca2+-mediated release from M1 macrophages of opioid peptides, which activate peripheral opioid receptors in injured tissue.SIGNIFICANCE STATEMENT Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can ameliorate pain. The IL-4-mediated effects are considered to mostly result from the inhibition of the production of proinflammatory mediators (e.g., IL-1ß, tumor necrosis factor, prostaglandin E2). Here, we found that IL-4 injected at the injured nerves attenuates pain by releasing opioid peptides from the infiltrating macrophages in mice. The opioids were secreted by IL-4 in the intracellular Ca2+-dependent manner and activated local peripheral opioid receptors. These actions represent a novel mode of IL-4 action, since its releasing properties have not been so far reported. Importantly, our findings suggest that the IL-4-opioid system should be targeted in the peripheral damaged tissue, since this can be devoid of central and systemic side effects.

IL-4 induces M2 macrophages to produce sustained analgesia via opioids.

IL-4 is a pleiotropic antiinflammatory cytokine, which can be neuroprotective after nervous system injury. The beneficial actions of IL-4 are thought to result from the blunting of action of inflammatory mediators, such as proinflammatory cytokines. Here, we demonstrate that IL-4 induces M2 macrophages to continuously produce opioid peptides and ameliorate pain. IL-4 application at injured nerves in mice shifted F4/80+ macrophages from the proinflammatory M1 to the antiinflammatory M2 phenotype, which synthesized opioid peptides (Met-enkephalin, ß-endorphin, and dynorphin A 1-17). These effects were accompanied by a long-lasting attenuation of neuropathy-induced mechanical hypersensitivity, beyond the IL-4 treatment. This IL-4-induced analgesia was decreased by opioid peptide antibodies and opioid receptor (δ, µ, κ) antagonists applied at injured nerves, which confirms the involvement of the local opioid system. The participation of M2 macrophages was supported by analgesia in recipient mice injected at injured nerves with F4/80+ macrophages from IL-4-treated donors. Together, IL-4-induced M2 macrophages at injured nerves produced opioid peptides, which activated peripheral opioid receptors to diminish pain. Fostering the opioid-mediated actions of intrinsic M2 macrophages may be a strategy to tackle pathological pain.

Adoptive transfer of M2 macrophages reduces neuropathic pain via opioid peptides.

BACKGROUND: During the inflammation which occurs following nerve damage, macrophages are recruited to the site of injury. Phenotypic diversity is a hallmark of the macrophage lineage and includes pro-inflammatory M1 and anti-inflammatory M2 populations. Our aim in this study was to investigate the ability of polarized M0, M1, and M2 macrophages to secrete opioid peptides and to examine their relative contribution to the modulation of neuropathic pain. METHODS: Mouse bone marrow-derived cells were cultured as unstimulated M0 macrophages or were stimulated into an M1 phenotype using lipopolysaccharide and interferon-γ or into an M2 phenotype using interleukin-4. The macrophage phenotypes were verified using flow cytometry for surface marker analysis and cytokine bead array for cytokine profile assessment. Opioid peptide levels were measured by radioimmunoassay and enzyme immunoassay. As a model of neuropathic pain, a chronic constriction injury (CCI) of the sciatic nerve was employed. Polarized M0, M1, and M2 macrophages (5 × 105 cells) were injected perineurally twice, on days 14 and 15 following CCI or sham surgery. Mechanical and heat sensitivity were measured using the von Frey and Hargreaves tests, respectively. To track the injected macrophages, we also transferred fluorescently stained polarized cells and analyzed the surface marker profile of endogenous and injected cells in the nerves ex vivo. RESULTS: Compared to M0 and M1 cells, M2 macrophages contained and released higher amounts of opioid peptides, including Met-enkephalin, dynorphin A (1-17), and ß-endorphin. M2 cells transferred perineurally at the nerve injury site reduced mechanical, but not heat hypersensitivity following the second injection. The analgesic effect was reversed by the perineurally applied opioid receptor antagonist naloxone methiodide. M2 cells did not affect sensitivity following sham surgery. Neither M0 nor M1 cells altered mechanical and heat sensitivity in CCI or sham-operated animals. Tracing the fluorescently labeled M0, M1, and M2 cells ex vivo showed that they remained in the nerve and preserved their phenotype. CONCLUSIONS: Perineural transplantation of M2 macrophages resulted in opioid-mediated amelioration of neuropathy-induced mechanical hypersensitivity, while M1 macrophages did not exacerbate pain. Therefore, rather than focusing on macrophage-induced pain generation, promoting opioid-mediated M2 actions may be more relevant for pain control.

Pain inhibition by blocking leukocytic and neuronal opioid peptidases in peripheral inflamed tissue.

Inflammatory pain can be controlled by endogenous opioid peptides. Here we blocked the degradation of opioids in peripheral injured tissue to locally augment this physiological system. In rats with hindpaw inflammation, inhibitors of aminopeptidase N (APN; bestatin) or neutral endopeptidase (NEP; thiorphan), and a dual inhibitor, NH(2)-CH-Ph-P(O)(OH)CH(2)-CH-CH(2)Ph(p-Ph)-CONH-CH-CH(3)-COOH (P8B), were applied to injured paws. Combined bestatin (1.25-5 mg)/thiorphan (0.2-0.8 mg) or P8B (0.0625-1 mg) alone elevated mechanical nociceptive thresholds to 307 and 227% of vehicle-treated controls, respectively. This analgesia was abolished by antibodies to methionine-enkephalin, leucine-enkephalin, and dynorphin A 1-17, by peripherally restricted and by selective µ-, δ-, and κ-opioid receptor antagonists. Flow cytometry and photospectrometry revealed expression and metabolic activity of APN and NEP on macrophages, granulocytes, and sciatic nerves from inflamed tissue. Radioimmunoassays showed that inhibition of leukocytic APN and NEP by bestatin (5-500 µM)/thiorphan (1-100 µM) combinations or by P8B (1-100 µM) prevented the degradation of enkephalins. Blockade of neuronal peptidases by bestatin (0.5-10 mM)/thiorphan (0.1-5 mM) or by P8B (0.1-10 mM) additionally hindered dynorphin A 1-17 catabolism. Thus, leukocytes and peripheral nerves are important sources of APN and NEP in inflamed tissue, and their blockade promotes peripheral opioid analgesia.

Peripheral non-viral MIDGE vector-driven delivery of beta-endorphin in inflammatory pain.

BACKGROUND: Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as beta-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE) vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS)-coupled MIDGE encoding the beta-endorphin precursor proopiomelanocortin (POMC) on complete Freund's adjuvant-induced inflammatory pain in rats. RESULTS: POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of beta-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing beta-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of beta-endorphin. CONCLUSION: Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or beta-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.

Immune cell-derived opioids protect against neuropathic pain in mice.

The analgesic effects of leukocyte-derived opioids have been exclusively demonstrated for somatic inflammatory pain, for example, the pain associated with surgery and arthritis. Neuropathic pain results from injury to nerves, is often resistant to current treatments, and can seriously impair a patient's quality of life. Although it has been recognized that neuronal damage can involve inflammation, it is generally assumed that immune cells act predominately as generators of neuropathic pain. However, in this study we have demonstrated that leukocytes containing opioids are essential regulators of pain in a mouse model of neuropathy. About 30%-40% of immune cells that accumulated at injured nerves expressed opioid peptides such as beta-endorphin, Met-enkephalin, and dynorphin A. Selective stimulation of these cells by local application of corticotropin-releasing factor led to opioid peptide-mediated activation of opioid receptors in damaged nerves. This ultimately abolished tactile allodynia, a highly debilitating heightened response to normally innocuous mechanical stimuli, which is symptomatic of neuropathy. Our findings suggest that selective targeting of opioid-containing immune cells promotes endogenous pain control and offers novel opportunities for management of painful neuropathies.

CXCR1/2 ligands induce p38 MAPK-dependent translocation and release of opioid peptides from primary granules in vitro and in vivo.

Polymorphonuclear leukocytes (PMN) can release opioid peptides which bind to opioid receptors on sensory neurons and inhibit inflammatory pain. This release can be triggered by chemokine receptor 1/2 (CXCR1/2) ligands. Our aim was to identify the granule subpopulation containing opioid peptides and to assess whether MAPK mediate the CXCR1/2 ligand-induced release of these peptides. Using double immunofluorescence confocal microscopy, we showed that beta-endorphin (END) and Met-enkephalin (ENK) were colocalized with the primary (azurophil) granule markers CD63 and myeloperoxidase (MPO) within PMN. END and ENK release triggered by a CXCR1/2 ligand in vitro was dependent on the presence of cytochalasin B (CyB) and on p38 MAPK, but not on p42/44 MAPK. In addition, translocation of END and ENK containing primary granules to submembranous regions of the cell was abolished by the p38 MAPK inhibitor SB203580. In vivo CXCL2/3 reduced pain in rats with complete Freund's adjuvant (CFA)-induced hindpaw inflammation. This effect was attenuated by intraplantar (i.pl.) antibodies against END and ENK and by i.pl. p38 MAPK inhibitor treatment. Taken together, these findings indicate that END and ENK are contained in primary granules of PMN, and that CXCR1/2 ligands induce p38-dependent translocation and release of these opioid peptides to inhibit inflammatory pain.

Lymphocytes upregulate signal sequence-encoding proopiomelanocortin mRNA and beta-endorphin during painful inflammation in vivo.

Proopiomelanocortin (POMC)-derived beta-endorphin1-31 (END) released from immune cells inhibits inflammatory pain. We examined the expression of END and POMC mRNA encoding the signal sequence required for entry of the nascent polypeptide into the regulated secretory pathway in lymphocytes of rats with inflamed hindpaws. Within 12 h of inflammation, END increased in popliteal lymph nodes and at 96 h the intraplantar neutralization of END exacerbated pain. Lymphocytes expressed POMC, END, and full-length POMC mRNA. Semi-nested PCR revealed 8-fold increased exon 2-3 spanning POMC mRNA. Thus, painful inflammation enhances signal sequence-encoding lymphocytic POMC mRNA needed for regulated secretion of functionally active END.

Mu-opioid receptor activation modulates transient receptor potential vanilloid 1 (TRPV1) currents in sensory neurons in a model of inflammatory pain.

Current therapy for inflammatory pain includes the peripheral application of opioid receptor agonists. Activation of opioid receptors modulates voltage-gated ion channels, but it is unclear whether opioids can also influence ligand-gated ion channels [e.g., the transient receptor potential vanilloid type 1 (TRPV1)]. TRPV1 channels are involved in the development of thermal hypersensitivity associated with tissue inflammation. In this study, we investigated mu-opioid receptor and TRPV1 expression in primary afferent neurons in the dorsal root ganglion (DRG) in complete Freund's adjuvant (CFA)-induced paw inflammation. In addition, the present study examined whether the activity of TRPV1 in DRG neurons can be inhibited by mu-opioid receptor (mu-receptor) ligands and whether this inhibition is increased after CFA inflammation. Immunohistochemistry demonstrated colocalization of TRPV1 and mu-receptors in DRG neurons. CFA-induced inflammation increased significantly the number of TRPV1- and mu-receptor-positive DRG neurons, as well as TRPV1 binding sites. In whole-cell patch clamp studies, opioids significantly decreased capsaicin-induced TRPV1 currents in a naloxone- and pertussis toxinsensitive manner. The inhibitory effect of morphine on TRPV1 was abolished by forskolin and 8-bromo-cAMP. During inflammation, an increase in TRPV1 is apparently rivaled by an increase of mu-receptors. However, in single dissociated DRG neurons, the inhibitory effects of morphine are not different between animals with and without CFA inflammation. In in vivo experiments, we found that locally applied morphine reduced capsaicin-induced thermal allodynia. In summary, our results indicate that mu-receptor activation can inhibit the activity of TRPV1 via G(i/o) proteins and the cAMP pathway. These observations demonstrate an important new mechanism underlying the analgesic efficacy of peripherally acting mu-receptor ligands in inflammatory pain.

Selective local PMN recruitment by CXCL1 or CXCL2/3 injection does not cause inflammatory pain.

Polymorphonuclear cells (PMN) are recruited in early inflammation and are believed to contribute to inflammatory pain. However, studies demonstrating a hyperalgesic role of PMN did not examine selective PMN recruitment or did not document effective PMN recruitment. We hypothesized that hyperalgesia does not develop after chemokine-induced PMN selective recruitment and is independent of PMN infiltration in complete Freund's adjuvant (CFA)-induced, local inflammation. PMN were recruited by intraplantar injection of CXC chemokine ligand 1 (CXCL1; keratinocyte-derived chemokine), CXCL2/3 (macrophage inflammatory protein-2), or CFA, with or without preceding systemic PMN depletion. Chemokine inoculation resulted in dose (0-30 microg)- and time (0-12 h)-dependent, selective recruitment of PMN as quantified by flow cytometry. CXCL2/3, but not CXCL1, was less effective at high doses, probably as a result of significant down-regulation of CXC chemokine receptor 2 expression on blood PMN. Neither chemokine caused mechanical or thermal hyperalgesia as determined by the Randall-Selitto and Hargreaves test, respectively, despite comparable expression of activation markers (i.e., CD11b, CD18, and L-selectin) on infiltrating PMN. In contrast, CFA injection induced hyperalgesia, independent of PMN recruitment. c-Fos mRNA and immunoreactivity in the spinal cord were increased significantly after inoculation of CFA-independent of PMN-migration but not of CXCL2/3. Measurement of potential hyperalgesic mediators showed that hyperalgesia correlated with local prostaglandin E2 (PGE2) but not with interleukin-1beta production. In summary, hyperalgesia, local PGE2 production, and spinal c-Fos expression occur after CFA-induced inflammation but not after CXCL1- or CXCL2/3-induced, selective PMN recruitment. Thus, PMN seem to be less important in inflammatory hyperalgesia than previously thought.

Tissue monocytes/macrophages in inflammation: hyperalgesia versus opioid-mediated peripheral antinociception.

BACKGROUND: Opioid-containing leukocytes migrate to peripheral sites of inflammation. On exposure to stress, opioid peptides are released, bind to opioid receptors on peripheral sensory neurons, and induce endogenous antinociception. In later stages of Freund's complete adjuvant-induced local inflammation, monocytes/macrophages are a major opioid-containing leukocyte subpopulation, but these cells also produce proalgesic cytokines. In this study, the role of tissue monocytes/macrophages in hyperalgesia and in peripheral opioid-mediated antinociception was investigated. METHODS: After intraplantar injection of Freund's adjuvant, leukocyte subpopulations and opioid-containing leukocytes were analyzed by flow cytometry in the inflamed paw in the presence or absence of monocyte/macrophage depletion by intraplantar injection of clodronate-containing liposomes (phosphate-buffered saline and empty liposomes served as controls). Paw volume was measured with a plethysmometer. Hyperalgesia was determined by measuring heat-induced paw withdrawal latency and paw pressure threshold. Paw pressure threshold was also measured after swim stress and injection of fentanyl. RESULTS: At 48 and 96 h of inflammation, it was found that (1). monocytes/macrophages were the largest leukocyte subpopulation (> 55% of all leukocytes) and the predominant producers of opioid peptides (71-77% of all opioid-containing leukocytes in the paw), (2). clodronate-containing liposomes depleted monocytes/macrophages by 30-35% (P < 0.05), (3). hyperalgesia was unaltered by liposome injection (P > 0.05), and (4) opioid-containing leukocytes and swim stress but not fentanyl-induced antinociception were significantly decreased by clodronate-containing liposomes (P < 0.05, P > 0.05, all by t test; opioid-containing cells and swim stress-induced increase of paw pressure threshold were reduced by 35-42% and 20%, respectively). CONCLUSION: Partial depletion of tissue monocytes/macrophages impairs peripheral endogenous opioid-mediated antinociception without affecting hyperalgesia.

Endomorphin-2, deltorphin II and their analogs suppress formalin-induced nociception and c-Fos expression in the rat spinal cord.

In this study, we evaluated the effects of intrathecally administered agonists of mu- and delta-opioid receptor and their analogs on the pain-induced behavior and expression of c-Fos immunoreactivity in the spinal cord, elicited by intraplantar injection of 12% formalin to the hindpaw of the rat. Previous report from our laboratory and other author's study indicated that intrathecal administration of mu agonists morphine and endomorphin-2 and delta-opioid agonist deltorphin II produced a dose-dependent antinociceptive effects in acute and inflammatory pain. In this study, intrathecal injection of morphine (10 microg), endomorphin-2 (5 microg) and its analog Dmt-endomorphin-2 (10 microg) significantly decreased the formalin-induced pain behavior, and lowered a number of c-Fos positive neurons in the laminae I, II and III of the spinal cord by about 40%, 30% and 40%, respectively. Significant reduction of formalin-induced behavioral responses was also observed after i.th. administration of deltorphin II (15 microg) and its analog ile-deltorphin II (15 microg). Agonists of delta-opioid receptor significantly reduced a number of c-Fos positive neurons by about 28% and 40%, respectively. Analog of endomorphin-2 and analog of deltorphin II suppressed more potently expression of c-Fos in the dorsal horn of the spinal cord than the parent peptides. Our study indicates that new analogs of mu- and delta-opioid receptor exhibit strong antinociceptive potency similar or even higher than the parent peptides, and that their effect is positively correlated with the inhibition of c-Fos expression.

Formalin-induced pain and mu-opioid receptor density in brain and spinal cord are modulated by A1 and A2a adenosine agonists in mice.

The effects of adenosine analogues on pain have been shown to depend on the subtype receptor involved as well as on the nociceptive stimuli and on the route of administration. In the first experiment of the present study intraperitoneal administration of the A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) (0.015, 0.03, 0.09, 0.15, 0.21, 0.3 mg/kg) induced dose-dependent analgesia to formalin pain in both phases characterizing the test. The A(2a) receptor agonist 2-[p-2-(carbonyl-ethyl)-phenyethylamino]-5'-N-ethylcarboxaminoadenosine (CGS21680) (0.025, 0.05, 0.1, 0.15 mg/kg) significantly affected behavioral responses to formalin only during the early phase. In the second experiment the interaction between adenosine and the opioid system was investigated through both behavioral and neurochemical studies. The opioid antagonist naltrexone (0.1 mg/kg) did not affect the antinociception induced by CPA (0.21 mg/kg) and CGS21680 (0.05 mg/kg). Autoradiographic studies showed that formalin administration significantly modified mu-opioid receptor density in the superficial laminae of the spinal cord and in the paracentral thalamic nucleus, contralateral to the side of formalin injection. CPA and CGS21680 counteracted these effects induced by formalin. In conclusion the present study confirms and extends the role of A(1) and A(2a) adenosine receptors in the modulation of inflammatory pain and their interaction with the mu-opioid system, and suggests further investigation of these purinergic receptors from a therapeutic perspective.