The presence of morphine-like and codeinelike substances was demonstrated in the pedal ganglia, hemolymph, and mantle tissues of the mollusc Mytilus edulis. The pharmacological activities of the endogenous morphine-like material resemble those of authentic morphine. Both substances were found to counteract, in a dose-dependent manner, the stimulatory effect of tumor necrosis factor ax or interleukin la on human monocytes and Mytilus immunocytes, when added simultaneously to the incubation medium. The immunosuppressive effect of this opiate material expresses itself in a lowering of chemotactic activity, cellular velocity, and adherence. Codeine mimics the activity of authentic morphine, but only at much higher concentrations. Specific high-affinity receptor sites (jx3) for morphine have been identified on human monocytes and Mytilus immunocytes. In Mytilus recovering from experimentally induced stress, the return of "alerted" immunocytes to a more inactive state appears to be due to a significant rise in the content of morphine-like material in the pedal ganglia and hemolymph at this time. Thus, morphine may have a role in calming or terminating the state of immune alertness.The occurrence throughout the animal kingdom of messenger molecules identical with, or closely related to, those known in mammals has been amply documented by biochemical identification, functional analysis, and the demonstration of specific receptor sites (1-6). This applies in particular to classical neurotransmitters, neuropeptides, and cytokines. The demonstration by Spector and coworkers (9-11) and others (7, 8) added to the sample in a 6:10 (vol/vol) ratio (HCl/sample) to a final strength of 10%]. The samples were then heated at 100°C for 30 min, cooled, and centrifuged at 10,000 x g for 20 min. The resulting precipitate was discarded and the supernatant was extracted with 5 vol of 10% (vol/vol) 1-butanol in chloroform. The supernatant was adjusted to pH 8.8-9.0. The organic phase was back-extracted into 2 vol of 0.01 M HCl. Morphine recovery was 50-70%. The aqueous phase was evaporated to dryness and dissolved in 10 ml of phosphate-buffered saline (PBS, pH 7.4) and then the pH was adjusted to 8.5-9.0. Samples were passed through a Sep-Pak C18 cartridge. Morphine and codeine were eluted from the cartridge with 7 ml of 0.1 M pyridine in acetic acid (pH 6.2) containing 2.5% (vol/vol) 1-propanol. The eluate was evaporated to dryness and dissolved in 250 ,ul of 1 mM HCl. An aliquot of 200 ,ul was injected onto a C18 reverse-phase HPLC column (LiChrosorb, 0.4 x 25 cm, Merck). The samples were eluted with 0.1 M pyridine in acetic acid (pH 6.2) followed by a linear gradient of 0-25% 1-propanol/0.1 M pyridine in acetic acid at a flow rate of 1.5 ml/min. Four 1-min fractions were collected during morphine and codeine elution as determined by known standards. These fractions were then evaporated, dissolved, and assayed for morphine and codeine by a sensitive radioimmunoassay (RIA) described by . Polyclonal antibodies generated against 3...

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The antigen CD10 (common acute lymphoblastic leukaemia antigen), which is the zinc metalloprotease, neutral endopeptidase 24.11 (also known as NEP or 'enkephalinase'), is expressed by acute lymphoblastic leukaemias, normal lymphoid progenitors, mature polymorphonuclear leukocytes and certain nonhaematopoietic cells. CD10/NEP hydrolyses several naturally occurring peptides, including the endogenous opioid pentapeptides Met- and Leu-enkephalin. In invertebrate organisms such as the mollusc Mytilus edulis, Met-enkephalin triggers inflammatory responses by inducing morphological changes, directed migration and aggregation of haemocytes. We report here that a structure related to CD10/NEP is expressed by M. edulis haemocytes and that abrogation of CD10/NEP enzymatic activity reduces the amount of Met-enkephalin required for haemocyte activation by five orders of magnitude. Similar results are obtained with CD10+ human polymorphonuclear leukocytes, indicating that CD10/NEP related structures regulate enkephalin-mediated inflammatory responses in organisms whose ancestors diverged approximately 500 million years ago.

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Evidence for the participation of opioid neuropeptides in immunoregulatory activities, especially cellular adherence and migration, has been obtained in representatives of two phyla ofinvertebrates, the mollusc Mylilus edulis and the insect Leucophaea maderae. The injection of a synthetic analog of Met5]enkephalinamide, DAMA; 10-6 M) had a stimulatory, naloxone-reversible effect on the directed migration of immunocompetent hemocytes. Incubation of hemolymph in the presence of exogenous or endogenous opioid material significantly enhanced the adherence of hemocytes on albumin-coated slides as demonstrated by use of indirect Zeiss-Zonax reflectance computer analysis. Conversely, hemocyte adherence was markedly reduced by the addition of naloxone (10-8 M) to the incubation medium, either alone or in combination with DAMA. The antagonistic effects of naloxone on the stimulatory activities of opioids indicate that, like those previously reported in mammals, they are receptor-mediated. The presence of an endogenous [Metlenkephalin-like The most common immunocompetent hemocytes of invertebrates are the granulocytes (amebocytes), which ultrastructurally resemble the polymorphonuclear leukocytes of vertebrates. In lower invertebrates they are the only type of immunocyte identified thus far. These cells appear to be capable of carrying out several functions attributed to different cell types in the mammalian immune system-i.e., B and T lymphocytes and granular leukocytes (14). In Mytilus, the granular immunocytes (average diameter, 18 um) display a special fluorescence indicative of serotonin content (15). Although the functional role of this material remains to be determined, its presence serves as a convenient marker in tracing the movement of these cells.The functional versatility of the granular hemocytes of Leucophaea and related insect species is reflected in the complexity of their ultrastructural organization and an apparent capacity for cellular modulation (16). The present study focuses on the participation of opioid neuropeptides in two immunoregulatory activities, cellular adherence and migration. MATERIALS AND METHODS 626The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.