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Immunoperoxidase staining of human polymorphonuclear cells incubated with nonimmune antiserum (A) and COOHterminal synenkephalin-recognizing antiserum (1:200) (B).
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In the hematopoietic system a pluripotent stem cell generates precursors for lymphoid and myeloid lineages. Proenkephalin-derived peptides were previously detected in differentiated lymphoid cells. We have studied whether the proenkephalin system is expressed in a typical differentiated cell of the myeloid lineage, the neutrophil. Human peripheral...
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... Another study found END in monocytes/macrophages in the blood and colonic mucosa in healthy controls and IBS patients [170]. END and Met-ENK were detected in healthy human blood neutrophils and monocytes [138,139,141,171], and in neutrophils and macrophages accumulating in subcutaneous surgical wounds in patients undergoing abdominal surgery [172]. Neutrophils were predominant cells expressing POMC and PENK mRNAs, as well as END and Met-ENK in wound fluid from patients undergoing cardiac surgery. ...
... The latter effect was mediated by PLC-IP3 receptor-intracellular Ca 2+ , PI3K, and p38 MAPK pathways, as in rat neutrophils [138,141] (Fig. 1C). These cells also secreted Met-ENK in response to mycobacteria and formyl peptide involving formyl peptide receptors, intracellular Ca 2+ , and PI3K [139,171]. END was released from human blood mononuclear cells following stimulation of β-adrenergic receptors [177] (Fig. 1A). ...
Pathological pain is regulated by a balance between pro-algesic and analgesic mechanisms. Interactions between opioid peptide-producing immune cells and peripheral sensory neurons expressing opioid receptors represent a powerful intrinsic pain control in animal models and in humans. Therefore, treatments based on general suppression of immune responses have been mostly unsuccessful. It is highly desirable to develop strategies that specifically promote neuro-immune communication mediated by opioids. Promising examples include vaccination-based recruitment of opioid-containing leukocytes to painful tissue and the local reprogramming of pro-algesic immune cells into analgesic cells producing and secreting high amounts of opioid peptides. Such approaches have the potential to inhibit pain at its origin and be devoid of central and systemic side effects of classical analgesics. In support of these concepts, in this article, we describe the functioning of peripheral opioid receptors, migration of opioid-producing immune cells to inflamed tissue, opioid peptide release, and the consequent pain relief. Conclusively, we provide clinical evidence and discuss therapeutic opportunities and challenges associated with immune cell-mediated peripheral opioid analgesia.
... Subsequently, cattle enkelytin, the C-terminal fragment corresponding to residues 209-237 of proenkephalin-A was also found to be antibacterial [117]. A similar proenkephalin system exists in humans although the processing machinery can differ [118]. Thus, these neuropeptides also play a communication role between neuroendocrine and the immune system [119]. ...
As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between -3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized medicine to combat drug-resistant superbugs, fungi, viruses, parasites, or cancer. Alternatively, multiple factors (e.g., albumin, arginine, butyrate, calcium, cyclic AMP, isoleucine, short-chain fatty acids, UV B light, vitamin D, and zinc) are able to induce the expression of antimicrobial peptides, opening new avenues to the development of anti-infectious drugs.
... Rat peritoneal neutrophils were obtained 4 h after intraperitoneal injection of 1% oyster glycogen (Sigma-Aldrich Chemie) [17,72]. For determination of opioid peptide release, 5610 7 neutrophils or 1610 7 CD14 + monocytes were stimulated with fMLP (1– 1000 nM) or Mycobacterium butyricum (0.06–0.66 mg/ml) after preincubation with cytochalasin B (5 mg/ml) for 5 min in Hank's balanced salt solution containing the proteinase inhibitors bestatin (5 mg/ml), aprotinin (40 mg/ml) and thiorphan (100 mM, all Sigma-Aldrich Chemie) [17,73,74] . Doses of fMLP and Mycobacterium butyricum were based on pilot experiments and the literature [74,75]. ...
... For determination of opioid peptide release, 5610 7 neutrophils or 1610 7 CD14 + monocytes were stimulated with fMLP (1– 1000 nM) or Mycobacterium butyricum (0.06–0.66 mg/ml) after preincubation with cytochalasin B (5 mg/ml) for 5 min in Hank's balanced salt solution containing the proteinase inhibitors bestatin (5 mg/ml), aprotinin (40 mg/ml) and thiorphan (100 mM, all Sigma-Aldrich Chemie) [17,73,74] . Doses of fMLP and Mycobacterium butyricum were based on pilot experiments and the literature [74,75]. In some experiments, cells were concomitantly incubated with inhibitors as described in the results section. ...
In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR agonists to boost endogenous analgesia.
... Various agents including formyl peptides (e.g. fMLP) (Vindrola et al., 1990) trigger opioid peptide release from neutrophils in vitro via intracellular calcium mobilization as well as activation of phosphoinositol-3-kinase and p38 MAPK and induce antinociception in experimental models of inflammatory pain (Brack et al., 2004a;Rittner et al., , 2006a. ...
... For wash out experiments neutrophils were incubated with 200 mmol NaCl for 5 min, washed and resuspended in Hanks balanced salt solution for 5 or 60 min before stimulation of opioid peptide release using fMLP. Doses and time points were chosen based on pilot experiments and on reports from the literature (Junger et al., 1998;Rittner et al., 2006a;Vindrola et al., 1990). Release was terminated by rapid cooling, centrifugation and harvesting of the supernatant. ...
Inflammatory pain can be controlled by intraplantar opioid injection or by secretion of endogenous opioid peptides from leukocytes in inflamed rat paws. Antinociception requires binding of opioid peptides to opioid receptors on peripheral sensory nerve terminals. In the absence of inflammation, hydrophilic opioid peptides do not penetrate the perineurial barrier and, thus, do not elicit antinociception. This study was designed to examine the conditions under which endogenous, neutrophil-derived hydrophilic opioid peptides (i.e. Met-Enkephalin and beta-endorphin) can raise nociceptive thresholds in noninflamed tissue in rats. Intraplantar injection of the chemokine CXCL2/3 (macrophage inflammatory protein-2) induced selective neutrophil recruitment without overt signs of inflammation or changes in mechanical nociceptive thresholds (paw pressure threshold). Following intraplantar injection of hypertonic saline, the perineurial barrier was permeable for hours and intraplantar injection of opioid peptides increased mechanical nociceptive thresholds. While formyl-Met-Leu-Phe (fMLP) triggered opioid peptide release from neutrophils in vitro, nociceptive thresholds were unchanged in vivo. In vitro, hypertonicity interfered with fMLP-induced p38 mitogen activated kinase (MAPK) phosphorylation and opioid peptide release from neutrophils. These inhibitory effects were fully reversible by washout. In vivo, return to normotonicity occurred within 30min while the perineurium remained permeable for hours. Under these conditions, fMLP triggered MAPK phosphorylation and induced opioid peptide-mediated increases in nociceptive thresholds in the noninflamed paw. Taken together, antinociception mediated by endogenous opioids in noninflamed tissue has two important requirements: (i) opening of the perineurial barrier for opioid peptide access and (ii) opioid peptide release from neutrophils involving p38 MAPK.
... Polymorphonuclear leukocytes (PMN) 1 contain opioid peptides such as Met-enkephalin (ENK) and b-endorphin (END) (Vindrola et al., 1990;Mousa et al., 2004). In inflamed subcutaneous tissue these opioid peptides can be released, bind to l (MOR) and d (DOR) opioid receptors on peripheral sensory nerves and locally inhibit pain (reviewed in Stein et al., 2003). ...
... PMN constitutively produce opioid peptides and store them in vesicular structures (Vindrola et al., 1990;Mousa et al., 2004), but the exact nature of the granules is unknown. Four distinct types of cytoplasmic granules have been described in human PMN: azurophil (primary), specific (secondary) and gelatinase (tertiary) granules as well as secretory vesicles (Borregaard and Cowland, 1997;Gullberg et al., 1997). ...
... For measurement of opioid peptide release 5 · 10 7 human PMN were incubated in HBSS containing the proteinase inhibitors bestatin (5 lg/ml), aprotinin (40 lg/ml) and thiorphan (100 lM) (all Sigma-Aldrich Chemie) as described (Vindrola et al., 1990;Cabot et al., 1997;Rittner et al., 2006a). In selected experiments PMN were preincubated with 5 lg/ml cytochalasin B for 5 min to facilitate degranulation of primary and secondary granules (Vindrola et al., 1990;Mocsai et al., 2000). ...
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.
... For measurement of opioid peptide release 5 ϫ 10 7 , PMN were first preincubated with 5 g/ml cytochalasin B for 5 min in HBSS containing the proteinase inhibitors 5 g/ml bestatin, 40 g/ml aprotinin, and 100 M thiorphan (all Sigma-Aldrich Chemie), as described (7,33). In some experiments, cells were concomitantly incubated with inhibitors, as described in the Results section. ...
Leukocytes counteract inflammatory pain by releasing opioid peptides, which bind to opioid receptors on peripheral sensory neurons. In the early phase of inflammation, polymorphonuclear cells (PMN) are the major source of opioids. Their recruitment is governed by ligands at the chemokine receptor CXCR2. Here, we examined whether chemokines can also induce opioid peptide secretion from PMN and thus inhibit inflammatory pain. In rats with hindpaw inflammation, intraplantar injection of CXCL2/3, but not of the CXCR4 ligand CXCL12, elicited naloxone-reversible (i.e., opioid receptor mediated) mechanical and thermal analgesia, which was abolished by systemic PMN depletion. Both CXCR1/2- and CXCR4-ligands induced PMN chemotaxis, but only CXCR1/2 ligands triggered opioid release from human and rat PMN in vitro. This release was unaltered by extracellular Ca2+ chelation, was mimicked by thapsigargin and was blocked by inhibitors of the inositol 1,4,5-triphosphate receptor (IP3) and by intracellular Ca2+ chelation, indicating that it required Ca2+ from intracellular but not extracellular sources. Furthermore, release was partially reduced by phosphoinositol-3-kinase (PI3K) inhibitors. Adoptive transfer of allogenic PMN into PMN-depleted rats reconstituted CXCL2/3-induced analgesia, which was inhibited by prior ex vivo chelation of intracellular Ca2+. These findings demonstrate that, beyond cell recruitment, CXCR2 ligands induce Ca2+-regulated opioid release from PMN and thereby inhibit inflammatory pain in vivo.
... Several immune cells produce preproenkephalin as the endogenous neuroendocrine hormone and cytokine. Such activity was evidenced for phagocytic cells (Vindrola et al., 1990), T-cells (Hook et al., 1999) and B-lymphocytes (Rosen et al., 1989). The importance of this neuroendocrine hormone for T-cell activation and proliferation was recently established by Hook et al. (2003). ...
The central nervous system (CNS) and the immune system were for many years considered as two autonomous systems. Now, the reciprocal connections between them are generally recognized and very well documented. The links are realized mainly by various immuno- and neuropeptides. In the review the influence of the following immunopeptides on CNS is presented: tuftsin, thymulin, thymopoietin and thymopentin, thymosins, and thymic humoral factor. On the other side, the activity in the immune system of such neuropeptides as substance P, neurotensin, some neurokinins, enkephalins, and endorphins is discussed.
... Several naturally occurring endogenous ligands for opioid receptors have been described: b-endorphin (END), Leu-and Met-enkephalin (ENK), dynorphin (DYN), and the more recently discovered endomorphin-1 and -2. All immune cell subpopulations including lymphocytes, monocytes and granulocytes contain opioid peptides in the peripheral blood as well as in inflamed and non-inflamed lymph nodes (Cabot et al., 1997;Mousa et al., 2002;Vindrola et al., 1990). Inflammation increases the expression of opioid peptides both in vitro and in vivo (Smith, 2003) and all opioid peptides can be detected at the site of an experimentally induced inflammation . ...
... Both IL-1 and CRF can activate their respective receptors to induce opioid peptide release from lymphocytes and macrophages (Cabot et al., 1997). Opioid peptide release from granulocytes has only been demonstrated after nonspecific activation (Vindrola et al., 1990). ...
Tissue destruction is accompanied by an inflammatory reaction. The inflammatory reaction leads to activation of nociceptors and the sensation of pain. Several mediators are responsible for pain and hyperalgesia in inflammation including cytokines, chemokines, nerve growth factor as well as bradykinin, prostaglandins and ATP. Simulatenously however, analgesic mediators are secreted: opioid peptides, somatostatin, endocannabinoids and certain cytokines. Opioid peptides secreted from immune cells are so far the best studied peptides in peripheral inflammatory pain control. This system is hampered for example by anti-adhesion molecule treatment. Novel immunosuppressive drugs for treatment of autoimmune disease targetting cytokines, chemokines or adhesion molecules should therefore be evaluated for potential harmful effects on pain.
... Several naturally occurring endogenous ligands for opioid receptors have been described: b-endorphin, Leu-and Met-enkephalin, dynorphin, and the more recently discovered endomorphins-1 and -2. All immune cell subpopulations including lymphocytes, monocytes and granulocytes contain opioid peptides in the peripheral blood as well as in inflamed and non-inflamed lymph nodes [58][59][60][61][62]. Inflammation increases the expression of opioid peptides both in vitro and in vivo [63] and all opioid peptides can be detected at the site of an experimentally induced inflammation [61,64]. ...
... Both interleukin-1 and CRF can induce opioid peptide release from lymphocytes and macrophages [58,59]. Opioid peptide release from granulocytes has only been demonstrated after nonspecific activation [62]. ...
When tissue is destroyed, pain arises. Tissue destruction is associated with an inflammatory reaction. This leads to activation of nociceptors. The following review will concentrate on pro-algesic and analgesic mediators, which arise from immune cells or resident cells in the periphery or the circulation during inflammation.
In early inflammation endogenous hyperalgesic mediators are produced, including cytokines, chemokines, nerve growth factor as well as bradykinin, prostaglandins and ATP. Simultaneously, analgesic mediators are secreted: opioid peptides, somatostatin, endocannabinoids and certain cytokines. Inflammation increases the expression of peripheral opioid receptors on sensory nerve terminals and enhances their signal transduction, as well as the amount of opioid peptides in infiltrating immune cells. Interference with the recruitment of opioid-containing immune cells into inflamed tissue by blockade of adhesion molecules or by intrathecal morphine injection reduces endogenous analgesia.
Inflammatory pain is the result of the interplay between pro-algesic and analgesic mediators. To avoid central side effects, future analgesic therapy should be targeted at either selectively blocking novel pro-algesic mediators or at enhancing endogenous peripheral analgesic effects.
... It has been found that cells of the immune system are the source of different neuropeptides, including opioid peptides 16,27,30,42 . VINDROLA et al. 40 have demonstrated that human peripheral polymorphonuclear cells contain and release proenkephalin-derived peptides. Receptors for the naturally occurring opioids have been found and characterized on human leukocytes 2,3,10,24,34 . ...
Polymorphonuclear leukocyte (neutrophil) apoptosis is an important mechanism regulating the life span and some functions of neutrophils at inflamed sites. Opioid peptides are present in the peripheral circulation and their concentrations rapidly increase as a result of stress and inflammation. The effect of opioid peptides such as met-enkephalin (M-ENK) and beta-endorphin (beta-END) on tumor necrosis factor (TNF)-alpha-induced apoptosis in human neutrophils in vitro was investigated. Neutrophils isolated from peripheral blood were cultured in the absence or presence of 10(-6)-10(-10) M of opioid peptides for 8, 12 and 18 h. Features of apoptotic neutrophils were measured by a flow cytometric method based on analysis of the apoptotic nuclei (DNA content). We found that M-ENK and beta-END enhanced both uninduced and TNF-alpha-induced neutrophil apoptosis in vitro in a dose-dependent manner. The effect of opioid peptides on the modulation of neutrophil apoptosis was not reversed by the opioid-receptor antagonist naloxone. The results suggest that M-ENK and beta-END can regulate neutrophil life span via apoptosis and in this way may participate in the resolution of inflammation.
