here is growing interest in documenting the presence and significance of neuropeptides in the immune system. There is also renewed interest in examining the evo- lutionary significance of various signaling molecules, and their functions and involve- ment in a variety of organisms. This report focuses on the role of opioid neuropeptides in immunology and autoimmunoregulation 1 using information gained from invertebrates and human immunocytes. Opioid peptides and immunoactivation Opioid pentapeptides stimulate cytokine release and immunocyte chemotaxis, and induce conformational changes in immuno- cytes indicative of activation 1 . In particular, [Met]enkephalin and DAMA (D'Ala 2 -Met 5 -enkephalinamide) are potent mediators of im- munocyte conformational changes in both invertebrates and mam- mals 1 . In addition, immunocytes contain a novel  2 -opioid receptor that expresses a preference for naturally occurring -opioid sub- stances 1 . It is important to note that these activities are phylogen- etically ancient and occur both in invertebrates and in mammals – animals that are 500 million years divergent in evolution 1 . The similarities even extend to the regulation of immunocyte activation by neutral endopeptidase 2 . Therefore, it must be concluded that these opioid peptide immunoregulatory similarities first evolved in more primitive organisms. Previously, it has been difficult to accept this hypothesis because of the lack of evidence of opioid precursors in invertebrate tissues. However, numerous studies have now found and sequenced mam- malian-like opioid precursor fragments, including [Met]- and [Leu]enkephalin and [Met]enkephalin-Arg-Phe, in various inverte- brates 3–9 . In addition, opioid receptors have been demonstrated in various invertebrate tissues 1 . These comparative data suggest that opioid precursors are indeed present in simple animals. Invertebrate opioids After more than 23 years of study on invertebrate opioid mecha- nisms, the opioid precursors have now been isolated and se- quenced 10–12 . The mammalian prodynorphin-like molecule 10 of 119 amino acids contains -neoendorphin-, dynorphin-A- and dynorphin-B-like pep- tides, which exhibit strong sequence hom- ology (100%, 50% and 76.8%, respectively) with their mammalian counterparts, and has an identical number of [Leu]enkephalin se- quences. In addition, study of the immune tissues of the leech Theromyzon tessulatum by reverse transcriptase-polymerase chain reac- tion (RT-PRC) using degenerate oligonu- cleotide primers 12 , indicated the presence of a mammalian-like pro-opiomelanocortin (POMC) molecule, and six of its peptides, in- cluding adrenocorticotropin (ACTH) and - melanocyte-stimulating hormone (-MSH). Of the six peptides, three showed high se- quence similarity to their vertebrate counter- parts [Met]enkephalin, -MSH and ACTH (100%, 84.6% and 70%, respectively). Immunocytes from T. tessulatum and the marine mussel Mytilus edulis contain a mammalian-like proenkephalin molecule 11 . The structure of the leech proenkephalin shows considerable amino acid sequence similarity to amphibian proenkephalin (25.4% with Xenopus laevis) but is smaller (15 kDa versus 30 kDa). By contrast, M. edulis proenkephalin is not only larger (26 kDa) but exhibits a higher sequence identity with guinea pig proenkephalin (50%; Fig. 1). Both of the invertebrate proenkephalins possess [Met]- enkephalin and [Leu]enkephalin in a ratio of 3:1 for M. edulis and 1:2 for T. tessulatum. They also contain sequences that are flanked by dibasic amino acid residues, demonstrating cleavage sites. Finally, the proenkephalins from both the above invertebrates 11 contain the antibacterial peptide enkelytin, which was first isolated by Metz-Boutique and colleagues 13 . The sequence of enkelytin exhibits 98% sequence identity with mammalian enkelytin 13 (Fig. 1). Bactericidal activity There are many similarities between enkephalins and their precur- sors in invertebrates and vertebrates, for instance, in both animal types, neural and immune tissues contain enkephalins, their pre- cursors and proenkephalin mRNA (Refs 1, 11, 12). The fact that inver- tebrate proenkephalin contains enkelytin 11 supports the hypothesis that these molecules first evolved in more primitive animals. Indeed, the presence of enkelytin, with its strong antibacterial activity 13 , strengthens the association of opioid peptides with VIEWPOINT IMMUNOLOGY TODAY Vol.19 No.6 265 Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0167-5699/98/$19.00 JUNE 1998 Enkelytin and opioid peptide association in invertebrates and vertebrates: immune activation and pain George B. Stefano, Beatrice Salzet and Gregory L. Fricchione Invertebrates contain an opioid precursor, proenkephalin. Enkelytin, an antibacterial peptide, is found in invertebrate proenkephalin, exhibiting 98% sequence identity with mammalian enkephalin. Here, George Stefano and colleagues surmise that the function of enkelytin is to attack bacteria and allow time for the immunocyte- stimulating capabilities of the opioid peptides to emerge. Furthermore, they propose that pain itself is a component of this response. T PII: S0167-5699(98)01268-7