Touch and mechanical pain are first detected at our largest sensory surface, the skin. The cell bodies of sensory neurons that detect such stimuli are located in the dorsal root ganglia, and subtypes of these neurons are specialized to detect specific modalities of mechanical stimuli. Molecules have been identified that are necessary for mechanosensation in invertebrates but so far not in mammals. In Caenorhabditis elegans, mec-2 is one of several genes identified in a screen for touch insensitivity and encodes an integral membrane protein with a stomatin homology domain. Here we show that about 35% of skin mechanoreceptors do not respond to mechanical stimuli in mice with a mutation in stomatin-like protein 3 (SLP3, also called Stoml3), a mammalian mec-2 homologue that is expressed in sensory neurons. In addition, mechanosensitive ion channels found in many sensory neurons do not function without SLP3. Tactile-driven behaviours are also impaired in SLP3 mutant mice, including touch-evoked pain caused by neuropathic injury. SLP3 is therefore indispensable for the function of a subset of cutaneous mechanoreceptors, and our data support the idea that this protein is an essential subunit of a mammalian mechanotransducer.

Indiscriminate activation of opioid receptors provides pain relief but also severe central and intestinal side effects. We hypothesized that exploiting pathological (rather than physiological) conformation dynamics of opioid receptor-ligand interactions might yield ligands without adverse actions. By computer simulations at low pH, a hallmark of injured tissue, we designed an agonist that, because of its low acid dissociation constant, selectively activates peripheral μ-opioid receptors at the source of pain generation. Unlike the conventional opioid fentanyl, this agonist showed pH-sensitive binding, heterotrimeric guanine nucleotide-binding protein (G protein) subunit dissociation by fluorescence resonance energy transfer, and adenosine 3',5'-monophosphate inhibition in vitro It produced injury-restricted analgesia in rats with different types of inflammatory pain without exhibiting respiratory depression, sedation, constipation, or addiction potential.

Cold allodynia is a common feature of neuropathic pain however the underlying mechanisms of this enhanced sensitivity to cold are not known. Recently the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified and proposed to be molecular sensors for cold. Here we have investigated the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG) and examined the cold sensitivity of peripheral sensory neurons in the chronic construction injury (CCI) model of neuropathic pain in mice.In behavioral experiments, chronic constriction injury (CCI) of the sciatic nerve induced a hypersensitivity to both cold and the TRPM8 agonist menthol that developed 2 days post injury and remained stable for at least 2 weeks. Using quantitative RT-PCR and in situ hybridization we examined the expression of TRPM8 and TRPA1 in DRG. Both channels displayed significantly reduced expression levels after injury with no change in their distribution pattern in identified neuronal subpopulations. Furthermore, in calcium imaging experiments, we detected no alterations in the number of cold or menthol responsive neurons in the DRG, or in the functional properties of cold transduction following injury. Intriguingly however, responses to the TRPA1 agonist mustard oil were strongly reduced.Our results indicate that injured sensory neurons do not develop abnormal cold sensitivity after chronic constriction injury and that alterations in the expression of TRPM8 and TRPA1 are unlikely to contribute directly to the pathogenesis of cold allodynia in this neuropathic pain model.

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.

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 β-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. IntroductionWithin inflamed tissues, a plethora of molecules such as protons, adenosine triphosphate, glutamate, neuropeptides (e.g., calcitonin gene-related peptide [CGRP], substance P), prostaglandins, bradykinin, cytokines, and chemokines can induce pain (1, 2). Concurrently, however, endogenous counterregulatory mechanisms are mounted. It has been established that somatic inflammatory (e.g., postoperative and arthritic) pain can be effectively controlled by the immune system, in both animals and humans (3,4). This is mediated by extravasating leukocytes, which produce and liberate opioid peptides in inflamed tissues. The released opioids bind to opioid receptors on peripheral sensory neurons, resulting in the inhibition of noxious impulse propagation (5-17). Such effects are particularly interesting because they occur directly in peripheral tissues and, therefore, are free of side effects such as nausea, depression of breathing, cognitive impairment, dependence, and addiction mediated by opioid receptors in the CNS (3).Neuropathic pain is a common consequence of nerve injuries caused by trauma such as amputation, entrapment, or compression. It is characterized by persistent burning or shooting sensations and heightened responses to normally noxious (hyperalgesia) and innocuous stimuli (allodynia). Despite increasing efforts, such pain remains poorly controlled, severely impacting patients ' well-being (18-20), which makes new therapeutic approaches highly desirable. Research over the last decade has provided evidence on the association of traumatic peripheral nerve injuries with inflammatory reactions mobilizing the immune system (1,

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, β-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.

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