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,

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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.