Cellular and Molecular Neurobiology, Vol. 12, No. 5, 1992
Review
Invertebrate and Vertebrate Neuroimmune and Autoimmunoregulatory Commonalties Involving Opioid Peptides George B. Stefano 1 Received February 1, 1992; accepted March 20, 1992 KEY WORDS: invertebrate; vertebrate; neuroimmunology; autoimmunoregulation; opioid peptides.
SUMMARY 1. Evidence for bidirectional interrelationships between the nervous system and immune systems of vertebrates and invertebrates involving opioid peptides is briefly discussed. 2. The involvement of opioid peptides in autoimmunoregulatory communication also is discussed. 3. The presence of mammalian interleukin-like (1 & 6) and tumor necrosis factor-like molecules in invertebrates is reviewed as well as an apparent cascading system for these signal molecules. 4. The significance of ACTH and MSH in cellular immunosuppression and autoimmunoregulation is discussed in the context of a potential role in schistosomiasis and human immunodeficiency virus actions. 5. The review concludes with the hypothesis that the mammalian immune system has its origin in the invertebrate immune/defense system given the many similarities noted in the review based on new knowledge about the more "primitive" system. INTRODUCTION The present review provides evidence to support the hypothesis that the highly regulated vertebrate immune system probably had its origins in the invertebrate immune-defense phenomenon. This view is based on recent evidence that in both animal groups the immune and nervous systems appear to utilize similar intercellular signal molecules. Furthermore, this suggests that, as in mammals, this system in invertebrates has the potential for bidirectional communication. i Multidisciplinary Center for the Study of Aging, State University of New York, College at Old Westbury, Old Westbury, New York, 11568-0210. 357 0272-43~)/92/1000-0357506.50/0© 1992PlenumPublishingCorporation
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Indeed, the evolvement of the immune system from an earlier beginning can be said to parallel the evolvement of other mammalian systems, i.e., nervous system. Past studies on neuronal mechanisms in insects and mollusks have revealed remarkable structural, functional, and biochemical parallelisms with those in vertebrates (Scharrer, 1978, 1987; Stefano, 1982; Leung and Stefano, 1987). For example, the first experimental demonstration of a neurohormone on record was that of the "pupation hormone" of insects, reported by Kopec (1917, 1922). In some reports in the scientific literature, when reference is made to invertebrates, they are often described as "primitive" or "simple." However, their life cycle, encompassing embryonic and postembryonic development, reproductive activity, and changing metabolic and behavioral patterns, requires sophisticated signal mechanisms. These episodic events are known to be programmed with great precision and coordination by the organism's neuroendocrine control system. Numerous studies in a variety of invertebrate species have identified neuropeptides in parts of the neuroendocrine and nervous system apparatus, which is analogous to the hypothalamic-hypophysial system of vertebrates (see Scharrer, 1987). The list includes substances closely resembling the following vertebrate neuropeptides: oxytocin, vasopressin, adrenocorticotropin (ACTH); alpha-melanocyte stimulating hormone (oc-MSH); somatostatin, substance P, neurotensin, hypothalamic growth hormone releasing factor, insulin, glucagon, gastrin/cholecystokinin, vasoactive intestinal peptide (VIP), pancreatic polypeptide (PP), secretin, luteinizing hormone releasing factor (LHRF), and several endogenous opioids (see Stefano, 1986, 1988, 1991). This review concentrates on opioid mechanisms in both animal groups. Additionally, the review is divided into two major areas (nervous and immune systems) so that an argument based on parallelisms can be highlighted. This approach also enables the reader to gain insights into "comparative" neuroimmunology as well as autoimmunoregulation involving neuropeptides, given the field's recent emergence. NERVOUS SYSTEM
Since the discovery of endogenous opiate-like substances, numerous and ever-increasing numbers of studies have been performed which highlight the importance of these neuropeptides not only in neurobiology but in all the biological sciences. These opioid substances have been shown to have diverse functions that transcend their original proposed role in nociceptive/analgesic systems. The importance of this family of compounds is highlighted even more with their discovery in invertebrates (see Leung and Stefano, 1987; Luschen et al., 1991). In mammals these neuropeptides have been shown to originate from three gene products which, upon enzymatic cleavage, liberate active opioid substances along with other substances. Preliminary evidence, with regard to a larger precursor liberating active smaller opioid substances in invertebrates, has been reported (Leung and Stefano, 1987). However, at present, little is known regarding the nature of this gene product. With regard to the various gene products in mammals the higher molecular
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weight bioactive opioids in general contain one of the two enkephalin sequences at their N terminals. All known opioid peptides may be classified into three families: the endorphin, the enkephalin, and the dynorphin families. Although there is no direct evidence, the tremendous sequence homology among the precursors does strongly suggest a common origin. In addition, there is considerable similarity among the genes of the precursors in the arrangements of the introns and exons (Horikawa et al., 1983). The evolutionary and physiological significance of the structural association between fl-endorphin and the other opioid peptides remains unclear at this point. However, as POMC (proopiomelanocortin) appears to be processed to different final products in the anterior and neurointermediate lobes of the pituitary gland (Zakarian and Smyth, 1982), it is possible that POMC may serve different functions in different tissues. Both ACTH and fl-endorphin have been detected in invertebrates by immunocytochemical studies (see Stefano, 1988; Smith et al., 1990, 1991). Therefore, it is likely that macromolecules similar to POMC also may exist in the invertebrates. The analyses of a number of intermediate-size peptides in bovine adrenal chromaffin cells led to the complete identification of the macromolecular precursor proenkephalin. The identification of the enkephalins and the heptapeptide Met-enkephalin-Arg6-Phe7 in mussel pedal ganglia (Leung and Stefano, 1984; Stefano and Leung, 1984) strongly suggest the presence of an enkephalin precursor in invertebrates which is similar to proenkephalin. Prodynorphin contains three copies of Leu-enkephalin sequence but no Met-enkephalin sequence. The detection of c~neoendorphin and dynorphin in invertebrates left no doubt that proenkephalin is not the only source of Leu-enkephalin in invertebrate neural tissue (see Stefano, 1988). This also provides very strong evidence for the presence of a precursor molecule similar to prodynorphin in invertebrates. Furthermore, opiate receptor sites present in the nervous tissue of Mytilus edulis have properties very similar to those of opiate receptors in the mammalian nervous system (Stefano, 1982). In the first detailed account of opiate binding in invertebrates (Stefano et al., 1980), the binding of FK 33 824, an opiate agonist, and naloxone, an antagonist, was shown to be stereospecific, saturable, reversible, and of a high affinity. In contrast, specific binding to nonneural tissues was negligible, suggesting that the opiate sites are restricted to nervous tissues. In addition, the binding of FK 33 824 was inhibited by sodium, a process that was reversed by manganese. The binding of naloxone was enhanced twofold in the presence of a high sodium concentration and was relatively unaffected by manganese. These differential effects of ions of agonist and antagonist binding were similar to those observed in mammalian brain homogenates (Simon et al., 1975). In a subsequent study (Kream et al., 1980) the binding profiles obtained for various opiate ligands to membrane suspensions of pedal ganglia revealed the presence of both high- and low-affinity binding sites. FK 33 824 bound noncooperatively to a class of high-affinity sites (Kd = 1-3 nM) and cooperatively to a class of low-affinity sites (Kd = 6-11 nM). Hill analysis of the cooperative sites revealed Hill coefficients of n 2.6-3.7, indicating markedly positive homotropic cooperativity. The total density of binding sites for all ligands was
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approximately 160pmol/g of protein, whereby the high-affinity component comprised approximately 34% of the total. Kinetic analysis of the binding data obtained in M. edulis revealed values similar to those obtained for mammalian binding analysis; it also substantiated the cooperative nature of binding to the low-affinity site. The relative potencies of a series of opiates in displacing FK 33 824 enkephalin binding to membrane suspensions of pedal ganglia were very similar to those determined for rat brain homogenates (Kream and Zukin, 1979). The results also suggest that the high-affinity opiate binding sites which mediate alteration in dopamine levels are on presynaptic dopaminergic terminals (Stefano et al., 1981, 1982). The localization of opiate receptors on presynaptic nerve terminals has been documented for several areas of the mammalian nervous system (review by Leung and Stefano, 1987). Therefore, the opioid-dopamine interaction in M. edulis appears to be quite complex and analogous to the mechanisms existing in mammalian neural tissues. In Mytilus immunoreactive materials corresponding to enkephalin and a~-MSH were localized in separate and specific neurons (Stefano and Martin, 1983). The immunoreactivity is highly localized in the posterior central portion of the pedal ganglion. This area also is rich in dopamine-containing nerve cell bodies and fibers. In mammals, opioid receptors occur, along with enkephalinergic neurons, at high concentrations in the forebrain regions that are also rich in dopamine (review by Leung and Stefano, 1987). IMMUNE SYSTEM
Specific immune cells are capable not only of responding to neuropeptides, but also of synthesizing them (Zurawski et al., 1986). There is sufficient evidence that these substances, in addition to their interaction with the neuroendocrine apparatus, play a role in autoregulatory immune processes. More specifically, chemotactic effects of endogenous opioid peptides on polymorphonuclear leukocytes, monocytes, and lymphocytes have been demonstrated (Brown et al., 1986; Falke and Fischer, 1986; Fischer and Falke, 1984, 1986). The formation of active T-cell rosettes can be induced by Met-enkephalin (Miller et al., 1983, 1984), indicating an influence of such substances on cellular adherence (Stefano et al., 1989a). Moreover, Stefano et al. (1989a,b) have demonstrated that opioids, by stereoselect mechanisms, are involved in invertebrate autoimmunoregulatory mechanisms. This makes these animals promising models for the study of opioid functions (review by Stefano, 1989, 1991). The hemocytes of Mytilus and Leucophaea (a blattarian insect) appear to be of two major types, namely, cells with and without cytoplasmic granules (Renrantz, 1989; Stefano et al., 1989b). Histofluorescence studies on Mytilus hemocytes revealed that a subpopulation appears to contain serotonin. These serotonin cells were encountered wandering in all of the tissues examined and are presumed to be immunoactive (Stefano et al., 1989c). The view that invertebrate immunocytes exhibit considerable complexity is supported by several additional observations. Renwrantz and Stahmer (1983) demonstrated that the hemolymph of Mytilus contains an
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agglutinin with opsonizing properties. Additionally, immunocytes were found that had high-affinity dopamine binding sites (Stefano et al., 1989c). Interestingly, a subpopulation of granulocytes, immunocytes, from Mytilus edulis and Leucophaea maderae has the ability to respond to low opioid concentrations by adhering and clumping (Stefano et al., 1989a). The adherence promoting role of D A M A (D-Ala2-MetS-enkephalin) and its blockage by naloxone, in a dose-response manner, were clearly evident. In contrast, exogenous Met-enkephalin at the same low concentration of D A M A did not increase cellular adherence above control levels, due to the presence of proteolytic enzymes in the hemolymph (Shipp et al., 1990; Leung et al., 1992). Subsequent studies demonstrated that indeed neutral endopeptidase 24.11 (CD10, "enkephalinase") was present on both human and invertebrate immunocytes (Shipp et al., 1990), where it serves to modulate neuropeptide activation of the respective cells (Shipp et al., 1991; Stefano et al., 1991a). Recently we demonstrated that cytokine stimulation results in up-regulating neutral endopeptidase activity, which in turn down-regulates the cells' responsiveness to various neuropeptide substrates of this enzyme (Shipp et al., 1991; Stefano et al., 1992). Clearly, given this type of complex regulation, the importance of autoimmunoregulation is enhanced. In order to elicit an immune response in Mytilus, a specific nerve was severed (Stefano et al., 1989a). Nerve severance evoked a cellular immune response, as judged by the directional migration of yellow-fluorescent immunocytes to the lesioned area (Stefano et al., 1989a). The concentration of these cells accumulating and adhering to the lesioned tissue gradually increased, a response presumed to be due to a concentration gradient of antigenic or recognition factors. An injection of DAMA, placed in the vicinity of a severed nerve, showed that, after a period of 2 hr, the concentration gradient established by the injected material had taken precedence over that provided by putative endogenous antigenic messengers dispatched at the site of lesion. A possible explanation for this differential response is a critical difference in the concentrations of endogenous and injected ligands competing for opioid receptors. Subsequently, it was demonstrated in in vitro tests that the stimulation of locomotor behavior of invertebrate immunocytes by opioids is accompanied by distinctive conformational changes. Such changes (flattening, increase in surface area), resembling those reported in mammals (Falke and Fischer, 1986; Stefano et al., 1989c), also occur in unstimulated preparations, but at a lower frequency. The in vivo tests in Mytilus referred to above indicate that the administration of exogenous opioid material may elicit a directed movement of immunocytes. Similarly, cellular stimulation by various opioid drugs in slide tests reveals directed as well as random locomotion. While unstimulated immunocytes showed some random movements, clumping occurred only in the presence of opioids. This may be taken as evidence for the occurrence of chemotactic as well as chemokinetic activities of opioids. However, the participation of a second signal molecule, giving direction to randomly migrating cells (Hughes et al., 1990), cannot be ruled out. The reason for this possibility seems to be that stimulated immunocytes tend to move preferentially toward larger, rather than smaller, accumulations of
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the same cells. Since it may be assumed that the distribution of the administered drug is uniform throughout the slide, this substance alone could not account for the selective migratory behavior observed (Stefano et al., 1989a,b). Alternately, the larger clumps may secrete higher levels of endogenous opioid-like material. Thus, endogeneous opioids may serve to initiate locomotor behavior. It is of further interest to note that immunocytes activated by opioids seem to reach a point when they no longer respond to these drugs and to the antagonistic effect of naloxone (Stefano et al., 1989b), which may now be explained by neutral endopeptidase activation noted above and/or a cascading array of signal molecules whereby the initial step involves opioid signals, and once passed, the next signals may be of a cytokine nature (Hughes et al., 1990). Evidence for the presence of opioid receptors in the immunocytes of Mytilus and Leucophaea studied was obtained by determining the effects of naloxone on the cellular activities under consideration. Naloxone injections into the area of nerve severance of Mytilus noted above counteracted the cellular immune reaction observed in the absence of this drug (Stefano et al., 1989a,b). Regarding the presence of opioid peptides in various immunocytes, in a T-helper cell line from a concanavalin A-stimulated mouse, an open reading frame was discovered that contained 93% homology with rat brain preproenkephalin mRNA (Zurawski et al., 1986). Additionally, the cells that produced this mRNA appeared to secrete immunoreactive Met-enkephalin. These results are consistent with those reported by Monstein et al. (1986) using leukocytes from leukemia patients. Again, the use of a cDNA probe revealed an ACTH and /3-endorphin message with a final expression, following extensive verification, of a substance with/3-endorphin properties along with N-acetylated /3-endorphin (Lolait et al., 1984, 1986). Smith et al. (1986) found immunoreactive /3-endorphin in supernatants of human peripheral blood lymphocytes following corticotropin releasing factor treatments. The presence of opioid peptides in cell-free hemolymph and hemocytes, respectively, of Mytilus was demonstrated by Stefano et al. (1989a). MONOKINES
Previous reports have demonstrated the presence in starfish and tunicates of factors with IL-l-like effects (Prendergast et al., 1983; Donnelly et al., 1983; Beck and Habicht, 1986; Beck et al., 1989). Other "lymphokine-like" activities have been described in invertebrates (Ratcliffe, 1985). Since Mytilus immunocytes most resemble monocyte/macrophages, the effects of the human monokines, tumor necrosis factor (TNF) and interleukin 1 (IL-1), were determined on these cells (Hughes et al., 1990). It was demonstrated that Mytilus immunocytes respond to these substances both in vitro and in vivo in a fashion similar to human granulocytes. TNF, in a dose-dependent fashion, increases the relative reflectance of the cells and also causes the cells to flatten as indicated by the measured increase in their area and their perimeter. Recombinant human IL-1 initiates responses similar to those to TNF in Mytilus immunocytes. In addition, it appears that the immunocytes respond to IL-1 at least in part through TNF production. Finally, immunoreactive TNF and IL-1 were detected in Mytilus
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hemolymph. In the present issue the reports by Sawada and co-workers (1992) and Szfics and co-workers (1992) demonstrate that cytokines can effect invertebrate neurons. In another report in this issue Paeman and co-workers (1992) find immunoreactive IL-1 in glial cells present in the ganglia of Mytilus and Neries (a marine worm). This finding in Mytilus corroborates a previous studing demonstrating that D A M A can stimulate the secretion of an IL-l-like molecule from Mytilus pedal ganglia (Stefano et al., 1991b). Additionally, with regard to the presence of cytokine-like molecules in invertebrate ganglia, the specific anatomical localization of these cytokine-like molecules in the posterior central portion of Mytilus pedal ganglia provides the foundation for a neuroimmune connection reported in another work strongly suggesting this interaction following electrial stress (Stefano et al., 1991c). Thus, evidence is accumulating supporting not only the concept of neuroimmunology developing in invertebrates but the concept that the invertebrate immune system shares autoimmunoregulatory characteristics with mammals. In summary, the use of invertebrate models in this and other areas of biomedical research has received increasing attention in recent years (see Committee on Models for Biomedical Research, 1985). In fact, the rewards gained from experimentation with these and additional invertebrates reach beyond the elucidation of commonalties between two animal phyla. The results provide information on the evolutionary history of basic biological phenomena and, on occasion, point the way to important insights applicable also to higher organisms including mammals. This has recently been brought out in two reports demonstrating the significance of autoimmunoregulation in both human immunodeficiency virus infection and schistosomiasis (Smith et al., 1992a; DuvauxMiret et al., 1992). These reports serve to document the significance of various mammalian-like peptides also found in invertebrates, such as ACTH (Ottaviani et al., 1990; Smith et al., 1990), MSH (Stefano et al., 1991d), and Corticotropin Releasing Factor (CRF) (Smith et al., 1992b). These peptides appear to play a critical role in cellular autoim munoregulatory immunosuppression in both animal groups, and this activity depends on the activity of neutral endopeptidase. Thus, these commonalties of signal molecules, activities, and regulatory mechanisms must be viewed as demonstrating a continuity of information during the development of the immune system during evolution rather than the appearance of "chance" similarities. One is therefore left to conclude only that the invertebrate immune-defense system developed many of the strategies for antibiosenescence well before mammals evolved (Stefano, 1991).
ACKNOWLEDGMENTS The author wishes to thank the many collaborators and students that worked on the various projects outlined in this review. I especially want to thank Dr. Berta Scharrer for many discussions concerning this topic. During the preparation of the manuscript the author was partially supported by A D A M H A - M A R C MH 17138, DA 47392, NSF INT-8803664, and the State University of New York Research Foundation.
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Stefano, G. B., and Leung, M. K., (1984). Presence of met-enkephalin-Arg-Phe in molluscan neural tissues. Brain Res. 298:362-365. Stefano, G. B., and Martin, R. (1983). Enkephalin-like immunoreactivity in the pedal ganglion of Mytilus edulis (bivalvia) and its proximity to dopamine containing structures. Cell Tissue Res. 230:147-153. Stefano, G. B., Kream, R. M. and Zukin, R. S. (1980). Demonstration of stereospecific opiate binding in the nervous tissue of the marine mollusc Mytilus edulis. Brain Res. 181:445-450. Stefano, G. B., Hall, B., Makman, M. H., and Dvorkin, B., (1981). Opioids inhibit potassiumstimulated dopamine release in the marine mussel Mytilus edulis and in the cephalopod, Octopus bimaculatus. Science 213:928-930. Stefano, G. B., Zukin, R. S., and Kream, R. M. (1982). Evidence for the presynaptic localization of a high affinity opiate binding site on dopamine neurons in the pedal ganglia of Mytilus edulis (Bivaivia). J. Pharmacol. Exp. Ther. 222:759-764. Stefano, G. B., Leung, M. K., Zhao, X., and Scharrer, B. (1989a). Evidence for the involvement of opioid neuropeptides in the adherence and migration of immunocompetent invertebrate hemocytes. Proc. Natl. Acad. Sci. USA 86:626-630. Stefano, G. B., Cadet, P., and Scharrer, B. (1989b). Stimulatory effects of opioid neuropeptides on locomotory activity and conformational changes in invertebrate and human immunocytes: Evidence for a subtype of delta receptor. Proc. Natl. Acad. Sci. USA 86:6307-6311. Stefano, G. B., Zhao, X., Bailey, D., Metlay, M., and Leung, M. K. (1989c). High affinity dopamine binding to mouse thymocytes and Mytilus edulis (Bivalvia) hemocytes. J. Neuroimrnunol. 21:67-74. Stefano, G. B., Shipp, M. A., and Scharrer, B. (1991a). A possible immunoregulatory function for Met.-enkephalin-Arg6-Phe7 involving human and invertebrate granulocytes. J. Neuroimmunol. 31:97-103. Stefano, G. B., Smith, E. R., and Hughes, T. K. (1991b). Opioid induction of immunoreactive interleukin-1 in Mytilus edulis and human immunocytes: An interleukin-l-like substance in invertebrate neural tissue. J. Neuroimmunol. 32:29-34. Stefano, G. B., Cadet, P., Dokun A., and Scharrer, B. (1991c). A neuroimmunoregulatory-like mechanism responding to electrical shock in the marine bivalve Mytilus edulis. Brain Behav. lmmun. 4:323-329. Stefano, G. B., Smith, D. M., Smith, E. M., and Hughes, T. K. (1991d). MSH can deactivate both TNF stimulated and spontaneously active immunocytes. In Molluscan Neurobiology (K. S. Kits, H. H. Boer and J. Joosse, Eds.), North-Holland, Amsterdam, pp. 206-209. Stefano, G. B., Paemen, L. R., and Hughes, T. K. (1992). Autoimmunoregulation: Differential modulation of CD10/Neutral endopeptidase 24.11 by tumor necrosis factor and neuropeptides. J. Neuroimmunol. in press. Szfics, A., Stefano, G.B., Hughes, T. K., and S.-R6zsa, K. (1992). Modulation of voltage-activated ion currents on identified neurons of Helix pomatia L. by intedeukin-1. Cell. Mol. Neurobiol. (in press). Zakarian, S., and Smyth, D. G. (1982). B-Endorphin is processed differently in specific regions of rat pituitary and brain. Nature (London) 296:250-252. Zurawski, G., Benedik, M., Kamb, B. J., Abrams, J. S., Zurawski, S. M., and Lee, F. D. (1986). Activation of mouse T-helper ceils induces abundant preproenkephalin mRNA synthesis. Science 232:772-775.
Review
Invertebrate and Vertebrate Neuroimmune and Autoimmunoregulatory Commonalties Involving Opioid Peptides George B. Stefano 1 Received February 1, 1992; accepted March 20, 1992 KEY WORDS: invertebrate; vertebrate; neuroimmunology; autoimmunoregulation; opioid peptides.
SUMMARY 1. Evidence for bidirectional interrelationships between the nervous system and immune systems of vertebrates and invertebrates involving opioid peptides is briefly discussed. 2. The involvement of opioid peptides in autoimmunoregulatory communication also is discussed. 3. The presence of mammalian interleukin-like (1 & 6) and tumor necrosis factor-like molecules in invertebrates is reviewed as well as an apparent cascading system for these signal molecules. 4. The significance of ACTH and MSH in cellular immunosuppression and autoimmunoregulation is discussed in the context of a potential role in schistosomiasis and human immunodeficiency virus actions. 5. The review concludes with the hypothesis that the mammalian immune system has its origin in the invertebrate immune/defense system given the many similarities noted in the review based on new knowledge about the more "primitive" system. INTRODUCTION The present review provides evidence to support the hypothesis that the highly regulated vertebrate immune system probably had its origins in the invertebrate immune-defense phenomenon. This view is based on recent evidence that in both animal groups the immune and nervous systems appear to utilize similar intercellular signal molecules. Furthermore, this suggests that, as in mammals, this system in invertebrates has the potential for bidirectional communication. i Multidisciplinary Center for the Study of Aging, State University of New York, College at Old Westbury, Old Westbury, New York, 11568-0210. 357 0272-43~)/92/1000-0357506.50/0© 1992PlenumPublishingCorporation
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Indeed, the evolvement of the immune system from an earlier beginning can be said to parallel the evolvement of other mammalian systems, i.e., nervous system. Past studies on neuronal mechanisms in insects and mollusks have revealed remarkable structural, functional, and biochemical parallelisms with those in vertebrates (Scharrer, 1978, 1987; Stefano, 1982; Leung and Stefano, 1987). For example, the first experimental demonstration of a neurohormone on record was that of the "pupation hormone" of insects, reported by Kopec (1917, 1922). In some reports in the scientific literature, when reference is made to invertebrates, they are often described as "primitive" or "simple." However, their life cycle, encompassing embryonic and postembryonic development, reproductive activity, and changing metabolic and behavioral patterns, requires sophisticated signal mechanisms. These episodic events are known to be programmed with great precision and coordination by the organism's neuroendocrine control system. Numerous studies in a variety of invertebrate species have identified neuropeptides in parts of the neuroendocrine and nervous system apparatus, which is analogous to the hypothalamic-hypophysial system of vertebrates (see Scharrer, 1987). The list includes substances closely resembling the following vertebrate neuropeptides: oxytocin, vasopressin, adrenocorticotropin (ACTH); alpha-melanocyte stimulating hormone (oc-MSH); somatostatin, substance P, neurotensin, hypothalamic growth hormone releasing factor, insulin, glucagon, gastrin/cholecystokinin, vasoactive intestinal peptide (VIP), pancreatic polypeptide (PP), secretin, luteinizing hormone releasing factor (LHRF), and several endogenous opioids (see Stefano, 1986, 1988, 1991). This review concentrates on opioid mechanisms in both animal groups. Additionally, the review is divided into two major areas (nervous and immune systems) so that an argument based on parallelisms can be highlighted. This approach also enables the reader to gain insights into "comparative" neuroimmunology as well as autoimmunoregulation involving neuropeptides, given the field's recent emergence. NERVOUS SYSTEM
Since the discovery of endogenous opiate-like substances, numerous and ever-increasing numbers of studies have been performed which highlight the importance of these neuropeptides not only in neurobiology but in all the biological sciences. These opioid substances have been shown to have diverse functions that transcend their original proposed role in nociceptive/analgesic systems. The importance of this family of compounds is highlighted even more with their discovery in invertebrates (see Leung and Stefano, 1987; Luschen et al., 1991). In mammals these neuropeptides have been shown to originate from three gene products which, upon enzymatic cleavage, liberate active opioid substances along with other substances. Preliminary evidence, with regard to a larger precursor liberating active smaller opioid substances in invertebrates, has been reported (Leung and Stefano, 1987). However, at present, little is known regarding the nature of this gene product. With regard to the various gene products in mammals the higher molecular
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weight bioactive opioids in general contain one of the two enkephalin sequences at their N terminals. All known opioid peptides may be classified into three families: the endorphin, the enkephalin, and the dynorphin families. Although there is no direct evidence, the tremendous sequence homology among the precursors does strongly suggest a common origin. In addition, there is considerable similarity among the genes of the precursors in the arrangements of the introns and exons (Horikawa et al., 1983). The evolutionary and physiological significance of the structural association between fl-endorphin and the other opioid peptides remains unclear at this point. However, as POMC (proopiomelanocortin) appears to be processed to different final products in the anterior and neurointermediate lobes of the pituitary gland (Zakarian and Smyth, 1982), it is possible that POMC may serve different functions in different tissues. Both ACTH and fl-endorphin have been detected in invertebrates by immunocytochemical studies (see Stefano, 1988; Smith et al., 1990, 1991). Therefore, it is likely that macromolecules similar to POMC also may exist in the invertebrates. The analyses of a number of intermediate-size peptides in bovine adrenal chromaffin cells led to the complete identification of the macromolecular precursor proenkephalin. The identification of the enkephalins and the heptapeptide Met-enkephalin-Arg6-Phe7 in mussel pedal ganglia (Leung and Stefano, 1984; Stefano and Leung, 1984) strongly suggest the presence of an enkephalin precursor in invertebrates which is similar to proenkephalin. Prodynorphin contains three copies of Leu-enkephalin sequence but no Met-enkephalin sequence. The detection of c~neoendorphin and dynorphin in invertebrates left no doubt that proenkephalin is not the only source of Leu-enkephalin in invertebrate neural tissue (see Stefano, 1988). This also provides very strong evidence for the presence of a precursor molecule similar to prodynorphin in invertebrates. Furthermore, opiate receptor sites present in the nervous tissue of Mytilus edulis have properties very similar to those of opiate receptors in the mammalian nervous system (Stefano, 1982). In the first detailed account of opiate binding in invertebrates (Stefano et al., 1980), the binding of FK 33 824, an opiate agonist, and naloxone, an antagonist, was shown to be stereospecific, saturable, reversible, and of a high affinity. In contrast, specific binding to nonneural tissues was negligible, suggesting that the opiate sites are restricted to nervous tissues. In addition, the binding of FK 33 824 was inhibited by sodium, a process that was reversed by manganese. The binding of naloxone was enhanced twofold in the presence of a high sodium concentration and was relatively unaffected by manganese. These differential effects of ions of agonist and antagonist binding were similar to those observed in mammalian brain homogenates (Simon et al., 1975). In a subsequent study (Kream et al., 1980) the binding profiles obtained for various opiate ligands to membrane suspensions of pedal ganglia revealed the presence of both high- and low-affinity binding sites. FK 33 824 bound noncooperatively to a class of high-affinity sites (Kd = 1-3 nM) and cooperatively to a class of low-affinity sites (Kd = 6-11 nM). Hill analysis of the cooperative sites revealed Hill coefficients of n 2.6-3.7, indicating markedly positive homotropic cooperativity. The total density of binding sites for all ligands was
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approximately 160pmol/g of protein, whereby the high-affinity component comprised approximately 34% of the total. Kinetic analysis of the binding data obtained in M. edulis revealed values similar to those obtained for mammalian binding analysis; it also substantiated the cooperative nature of binding to the low-affinity site. The relative potencies of a series of opiates in displacing FK 33 824 enkephalin binding to membrane suspensions of pedal ganglia were very similar to those determined for rat brain homogenates (Kream and Zukin, 1979). The results also suggest that the high-affinity opiate binding sites which mediate alteration in dopamine levels are on presynaptic dopaminergic terminals (Stefano et al., 1981, 1982). The localization of opiate receptors on presynaptic nerve terminals has been documented for several areas of the mammalian nervous system (review by Leung and Stefano, 1987). Therefore, the opioid-dopamine interaction in M. edulis appears to be quite complex and analogous to the mechanisms existing in mammalian neural tissues. In Mytilus immunoreactive materials corresponding to enkephalin and a~-MSH were localized in separate and specific neurons (Stefano and Martin, 1983). The immunoreactivity is highly localized in the posterior central portion of the pedal ganglion. This area also is rich in dopamine-containing nerve cell bodies and fibers. In mammals, opioid receptors occur, along with enkephalinergic neurons, at high concentrations in the forebrain regions that are also rich in dopamine (review by Leung and Stefano, 1987). IMMUNE SYSTEM
Specific immune cells are capable not only of responding to neuropeptides, but also of synthesizing them (Zurawski et al., 1986). There is sufficient evidence that these substances, in addition to their interaction with the neuroendocrine apparatus, play a role in autoregulatory immune processes. More specifically, chemotactic effects of endogenous opioid peptides on polymorphonuclear leukocytes, monocytes, and lymphocytes have been demonstrated (Brown et al., 1986; Falke and Fischer, 1986; Fischer and Falke, 1984, 1986). The formation of active T-cell rosettes can be induced by Met-enkephalin (Miller et al., 1983, 1984), indicating an influence of such substances on cellular adherence (Stefano et al., 1989a). Moreover, Stefano et al. (1989a,b) have demonstrated that opioids, by stereoselect mechanisms, are involved in invertebrate autoimmunoregulatory mechanisms. This makes these animals promising models for the study of opioid functions (review by Stefano, 1989, 1991). The hemocytes of Mytilus and Leucophaea (a blattarian insect) appear to be of two major types, namely, cells with and without cytoplasmic granules (Renrantz, 1989; Stefano et al., 1989b). Histofluorescence studies on Mytilus hemocytes revealed that a subpopulation appears to contain serotonin. These serotonin cells were encountered wandering in all of the tissues examined and are presumed to be immunoactive (Stefano et al., 1989c). The view that invertebrate immunocytes exhibit considerable complexity is supported by several additional observations. Renwrantz and Stahmer (1983) demonstrated that the hemolymph of Mytilus contains an
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agglutinin with opsonizing properties. Additionally, immunocytes were found that had high-affinity dopamine binding sites (Stefano et al., 1989c). Interestingly, a subpopulation of granulocytes, immunocytes, from Mytilus edulis and Leucophaea maderae has the ability to respond to low opioid concentrations by adhering and clumping (Stefano et al., 1989a). The adherence promoting role of D A M A (D-Ala2-MetS-enkephalin) and its blockage by naloxone, in a dose-response manner, were clearly evident. In contrast, exogenous Met-enkephalin at the same low concentration of D A M A did not increase cellular adherence above control levels, due to the presence of proteolytic enzymes in the hemolymph (Shipp et al., 1990; Leung et al., 1992). Subsequent studies demonstrated that indeed neutral endopeptidase 24.11 (CD10, "enkephalinase") was present on both human and invertebrate immunocytes (Shipp et al., 1990), where it serves to modulate neuropeptide activation of the respective cells (Shipp et al., 1991; Stefano et al., 1991a). Recently we demonstrated that cytokine stimulation results in up-regulating neutral endopeptidase activity, which in turn down-regulates the cells' responsiveness to various neuropeptide substrates of this enzyme (Shipp et al., 1991; Stefano et al., 1992). Clearly, given this type of complex regulation, the importance of autoimmunoregulation is enhanced. In order to elicit an immune response in Mytilus, a specific nerve was severed (Stefano et al., 1989a). Nerve severance evoked a cellular immune response, as judged by the directional migration of yellow-fluorescent immunocytes to the lesioned area (Stefano et al., 1989a). The concentration of these cells accumulating and adhering to the lesioned tissue gradually increased, a response presumed to be due to a concentration gradient of antigenic or recognition factors. An injection of DAMA, placed in the vicinity of a severed nerve, showed that, after a period of 2 hr, the concentration gradient established by the injected material had taken precedence over that provided by putative endogenous antigenic messengers dispatched at the site of lesion. A possible explanation for this differential response is a critical difference in the concentrations of endogenous and injected ligands competing for opioid receptors. Subsequently, it was demonstrated in in vitro tests that the stimulation of locomotor behavior of invertebrate immunocytes by opioids is accompanied by distinctive conformational changes. Such changes (flattening, increase in surface area), resembling those reported in mammals (Falke and Fischer, 1986; Stefano et al., 1989c), also occur in unstimulated preparations, but at a lower frequency. The in vivo tests in Mytilus referred to above indicate that the administration of exogenous opioid material may elicit a directed movement of immunocytes. Similarly, cellular stimulation by various opioid drugs in slide tests reveals directed as well as random locomotion. While unstimulated immunocytes showed some random movements, clumping occurred only in the presence of opioids. This may be taken as evidence for the occurrence of chemotactic as well as chemokinetic activities of opioids. However, the participation of a second signal molecule, giving direction to randomly migrating cells (Hughes et al., 1990), cannot be ruled out. The reason for this possibility seems to be that stimulated immunocytes tend to move preferentially toward larger, rather than smaller, accumulations of
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the same cells. Since it may be assumed that the distribution of the administered drug is uniform throughout the slide, this substance alone could not account for the selective migratory behavior observed (Stefano et al., 1989a,b). Alternately, the larger clumps may secrete higher levels of endogenous opioid-like material. Thus, endogeneous opioids may serve to initiate locomotor behavior. It is of further interest to note that immunocytes activated by opioids seem to reach a point when they no longer respond to these drugs and to the antagonistic effect of naloxone (Stefano et al., 1989b), which may now be explained by neutral endopeptidase activation noted above and/or a cascading array of signal molecules whereby the initial step involves opioid signals, and once passed, the next signals may be of a cytokine nature (Hughes et al., 1990). Evidence for the presence of opioid receptors in the immunocytes of Mytilus and Leucophaea studied was obtained by determining the effects of naloxone on the cellular activities under consideration. Naloxone injections into the area of nerve severance of Mytilus noted above counteracted the cellular immune reaction observed in the absence of this drug (Stefano et al., 1989a,b). Regarding the presence of opioid peptides in various immunocytes, in a T-helper cell line from a concanavalin A-stimulated mouse, an open reading frame was discovered that contained 93% homology with rat brain preproenkephalin mRNA (Zurawski et al., 1986). Additionally, the cells that produced this mRNA appeared to secrete immunoreactive Met-enkephalin. These results are consistent with those reported by Monstein et al. (1986) using leukocytes from leukemia patients. Again, the use of a cDNA probe revealed an ACTH and /3-endorphin message with a final expression, following extensive verification, of a substance with/3-endorphin properties along with N-acetylated /3-endorphin (Lolait et al., 1984, 1986). Smith et al. (1986) found immunoreactive /3-endorphin in supernatants of human peripheral blood lymphocytes following corticotropin releasing factor treatments. The presence of opioid peptides in cell-free hemolymph and hemocytes, respectively, of Mytilus was demonstrated by Stefano et al. (1989a). MONOKINES
Previous reports have demonstrated the presence in starfish and tunicates of factors with IL-l-like effects (Prendergast et al., 1983; Donnelly et al., 1983; Beck and Habicht, 1986; Beck et al., 1989). Other "lymphokine-like" activities have been described in invertebrates (Ratcliffe, 1985). Since Mytilus immunocytes most resemble monocyte/macrophages, the effects of the human monokines, tumor necrosis factor (TNF) and interleukin 1 (IL-1), were determined on these cells (Hughes et al., 1990). It was demonstrated that Mytilus immunocytes respond to these substances both in vitro and in vivo in a fashion similar to human granulocytes. TNF, in a dose-dependent fashion, increases the relative reflectance of the cells and also causes the cells to flatten as indicated by the measured increase in their area and their perimeter. Recombinant human IL-1 initiates responses similar to those to TNF in Mytilus immunocytes. In addition, it appears that the immunocytes respond to IL-1 at least in part through TNF production. Finally, immunoreactive TNF and IL-1 were detected in Mytilus
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hemolymph. In the present issue the reports by Sawada and co-workers (1992) and Szfics and co-workers (1992) demonstrate that cytokines can effect invertebrate neurons. In another report in this issue Paeman and co-workers (1992) find immunoreactive IL-1 in glial cells present in the ganglia of Mytilus and Neries (a marine worm). This finding in Mytilus corroborates a previous studing demonstrating that D A M A can stimulate the secretion of an IL-l-like molecule from Mytilus pedal ganglia (Stefano et al., 1991b). Additionally, with regard to the presence of cytokine-like molecules in invertebrate ganglia, the specific anatomical localization of these cytokine-like molecules in the posterior central portion of Mytilus pedal ganglia provides the foundation for a neuroimmune connection reported in another work strongly suggesting this interaction following electrial stress (Stefano et al., 1991c). Thus, evidence is accumulating supporting not only the concept of neuroimmunology developing in invertebrates but the concept that the invertebrate immune system shares autoimmunoregulatory characteristics with mammals. In summary, the use of invertebrate models in this and other areas of biomedical research has received increasing attention in recent years (see Committee on Models for Biomedical Research, 1985). In fact, the rewards gained from experimentation with these and additional invertebrates reach beyond the elucidation of commonalties between two animal phyla. The results provide information on the evolutionary history of basic biological phenomena and, on occasion, point the way to important insights applicable also to higher organisms including mammals. This has recently been brought out in two reports demonstrating the significance of autoimmunoregulation in both human immunodeficiency virus infection and schistosomiasis (Smith et al., 1992a; DuvauxMiret et al., 1992). These reports serve to document the significance of various mammalian-like peptides also found in invertebrates, such as ACTH (Ottaviani et al., 1990; Smith et al., 1990), MSH (Stefano et al., 1991d), and Corticotropin Releasing Factor (CRF) (Smith et al., 1992b). These peptides appear to play a critical role in cellular autoim munoregulatory immunosuppression in both animal groups, and this activity depends on the activity of neutral endopeptidase. Thus, these commonalties of signal molecules, activities, and regulatory mechanisms must be viewed as demonstrating a continuity of information during the development of the immune system during evolution rather than the appearance of "chance" similarities. One is therefore left to conclude only that the invertebrate immune-defense system developed many of the strategies for antibiosenescence well before mammals evolved (Stefano, 1991).
ACKNOWLEDGMENTS The author wishes to thank the many collaborators and students that worked on the various projects outlined in this review. I especially want to thank Dr. Berta Scharrer for many discussions concerning this topic. During the preparation of the manuscript the author was partially supported by A D A M H A - M A R C MH 17138, DA 47392, NSF INT-8803664, and the State University of New York Research Foundation.
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