ZOOLOGICAL SCIENCE 9: 1097-1100(1992) © 1992Zoological SocietyofJapan REVIEW Recent Progress in Comparative Neuroimmunology Berta Scharrer Department ofAnatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA tween the modulatory effects of opioid peptides INTRODUCTION and those of certain other ligands [3]. Like their Duringthepastdecade, broadlybasedcompara- counterparts in vertebrates, these cells respond to tive studies on cell-mediated immunoregulatory immunomodulatory stimuli by characteristic con- processes have revealed remarkable parallelisms formational changes indicative of cellular activity. between vertebrates and higher invertebrates (see For these light microscopic observations immuno- Scharrer [1]). One distinctive feature these two cytes were incubated togetherwith various ligands groups have in common is the use of the same or at 37°C for human, and at 23°C for Mytilus and closely related chemical messenger substances in Leucophaea. Either a Zeiss Axiophot Microscope internalimmunoregulatoryprocessesaswellasthe and the Zeiss Videoplan/Vidas Image Analysis bidirectionalexchange ofinformation between the System [4] or the system designed by American immune system and the neuroendocrine system Innovision, Inc. were used. Changes in cellular [2]. Neuropeptides, formerly considered to be conformation were determined by measurements confined to the neuroendocrine apparatus, are of cellular area and perimeter, mathematically now known to be produced by and released from expressed by use of the Zeiss Form-Factor (FF) immune cells. By the same token, cytokines, i.e., forumula [4]. Additional factors, introduced by regulatory molecules characteristic of the immune Schon et al. [5], permitted a distinction between system, have been shown to be manufactured also types of cellular change, i.e., those obtained in byneuralelements, includingastrocytes. Informa- different animal species by the same signal mole- tion on the identities of these signal molecules in cules and those effected by different substances in invertebrates as well as vertebrates has been the same species. obtained by use of biochemical and immunocy- Neuropeptides tochemical methods, and by in vitro and in vivo tests on the effects ofvarious exogenous analogs. The search for multiple functional roles of Theimmunoactivecellsoftheinvertebratesthat neuropeptidesinsignalingwithin aswellasoutside have been primarily used in these studies, the of the nervous system has taken a number of mollusc Mytilus edulis and the insect Leucophaea unexpected turns since their discovery in special maderae, aresubpopulationsoftheanimals' hemo- neurosecretory neurons some 70 years ago. Cur- cytes. Theyshareanumberofpropertieswithcells rently available evidence indicates that members of the mammalian granulocyte-monocyte-macro- of this large family of compounds, especially phage lineage. Evidence for the existence of opioid peptides, may have dose-dependent stim- different subsets ofinvertebrate immunocytes was ulatory as well as suppressive effects on various obtained by the observation of differences be- immunoregulatory processes. The substances acting in these capacities are furnished by im- Received August 10, 1992 munoactive cells as well as cells ofthe neuroendo- 1098 B. SCHARRER crine system. This information was initially obtained by tests The capacity of invertebrate immunocytes to with the synthetic analog of Met-enkephalin, produce and release a Met-enkephalin-like mate- DAMA, which is resistant to the effect of the rial was demonstrated by high pressure liquid naturally occurring neutral endopeptidase 24.11 chromatogaphy and radioimmunoassay. This ma- (NEP). The same high potency values (10-11 M) terial was found to be present in cell-free were accomplished by the administration of Met- hemolymph as well as hemocytes of Mytilus [4]. enkephalin or of the heptapeptide Met- Evidence for its presence in Leucophaea hemo- enkephalin-Arg6-Phe7 with the addition of the lymph is more tentative (see Scharrer et al. [6]). special enzyme inhibitor phosphoramidon [9-11]. Met-and Leu-enkephalin was alsoidentified in the In eight additional drugs examined, including the pedal ganglia of Mytilus [7]. closely related Leu-enkephalin, the required con- Immunocytochemical methods have revealed centration for comparable stimulatory effects was substancesantigenicallyrelatedto Met-enkephalin in the area of 10~9M. Beta-endorphin was active _10M andnumerousotherneuropeptidesintheneuroen- at 10 [8]. docrine system of Leucophaea and other insects The search for stereoselective mechanisms (see Scharrer et al. [6]). mediating the immunostimulatory effects of opioids and nonopioid substances has provided 1. Stimulatory activities pharmacological and bindingevidence forthe pre- Immunocytes of invertebrates and vertebrates sence of receptors on immunoresponsive cells. respondtostimulationwithappropriateconcentra- The results of Scatchard analysis indicate that tions of opioid and other peptides by increased these binding sites on human granulocytes and adherenceto albumin-coated slidesand the forma- Mytilus immunocytes are monophasic, stereospe- tion of clumps. Prior to becoming mobile, these cificandofhighefficiency. Inbothvertebtrate and cellsflatten andchangetheirstructurefromround- invertebrate tests the complexity of ligand- ed to ameboid {Mytilus) or elongated {Leuco- receptorbindingobserved turned outtobe greater phaea, human). The addition ofthe opioid antag- than anticipated. Multiple opioid receptors, in- onist naloxone to the preparation, concurrentwith cluding delta-, mu-, kappa-, and presumably epsi- the cells' exposure to exogenous opioids, blocks lon-receptors are preferentially used by different their activation. Activated immunocytes of Myti- messenger molecules performing selective func- lus have been observed to move in the direction of tions [8]. cell clusters of the same type [4]. The high potency of Met-enkephalin demon- Moreover, in vivoexperimentswith this mollusc strated in in vitro tests with Mytilus and human or mammalian laboratory animals showed that granulocytessuggests that itplays a distinctive role chemotactic movements ofimmunoactive cells are in immunoregulation. On the basis ofmammalian guided by concentration gradients of certain spe- in vivo experiments and of preliminary clinical cific recognition factors. Thus an immune-type tests, Jancovic et al. [12] called attention to the response, experimentallyinducedinMytilusbythe potential value ofMet-enkephalin in thetreatment severance of a nerve, resulted in the migration of ofcertainimmunediseases. The proposal madeby immunocytes and their gradual accumulation in Stefano et al. [8] that, in its immunoregulatory DAMA the lesioned area. After the injection of role, Met-enkephalin interacts with a special sub- (D-Ala -Met -enkephalin) into an intact animal, type ofopioid receptor(delta2) hasbeen substanti- but not that of a number ofcontrol substance, the ated by recent experiments with the equally potent cells accumulated at the site of injection [4]. opioid (D-Ala2) deltorphin I [13]. Preincubation An important feature observed by Stefano etal. ofhumangranulocyte membraneswith DALCE (a [8] was that the stimulatory effect of Met- select detla-opioid antagonist) revealed a binding enkephalin on cellular conformation and locomo- capacity of this opioid, as well as that of DAMA, tor activity is significantly higher than that of distinctly different from that of other opioids. related neuropeptides or nonpeptide substances. In comparing the modulatory effects of various Comparative Neuroimmunology 1099 opioids on certain growth processes, Zagon et al. TNF. Moreover, IL-1 and TNF were demon- [14] likewise found Met-enkephalin to be more strated to be present in the hemolymph of potent than the rest. This pentapeptide had a Leucophaea (T. K. Hughes, Jr., Personal com- strong inhibitory effect on the growth of neuro- munication). blastoma cells in tissue culture. The special recep- A molecule with the biochemical and biological tor proposed to be selectively involved was found characteristicsofmammalian interleukin 1 hasalso in abundance in the cerebellum ofinfants and in a been reported to be produced by an echinoderm, human brain tumor, but not in the cerebellum of Asteriasforbesi [16]. normal adults. Modeofoperationofimmunoregulatorysubstances 2. Immunosuppressive effects Neuropeptides, cytokines and additional factors In contrast to most of the neuropeptides tested (including special enzymes) interact to form an thus far two, derived from pro-opiomelanocortin, efficient immunoregulatory network. Depending i.e., ACTH and MSH, are immunosuppressive. on their concentration and additional determining When tested in appropriate concentrations, they factors present, certain messenger molecules may have been shown to inactivate immune cells that be either immunostimulatory or -inhibitory. In are either spontaneously active (5-10% in un- addition to its immunosuppressive function, men- treated preparations) or have been experimentally tionedearlier, ACTH may stimulate the activityof activated by other signal molecules known to have B-lymphocytes and the phagocytotic capacity of a stimulatory effect [11]. Part of this activity of molluscan immunocytes [17]. Its own production, ACTH seems to be indirect, i.e., by its conversion as well as that ofendorphins, by immune cells can to MSH under the influence of the special en- be inducedby anotherneuropeptide, corticotropin dopeptidase (NEP) mentioned earlier. These releasing hormone [18]. Conversely, the synthetic DAMA observations have been made in experiments with opioid has been shown to stimulate the vertebrate and invertebrate immune cells. formation of an IL-1-like molecule in human and Information on additional immunoregulatory Mytilus immune cells [11]. effects brought about by various neuropeptides is One way in which the degree of cellular activa- primarily based on mammalian studies. Such tion is kept within appropriate limits is by enzy- effects include antibody production, histamine re- matic degradation ofsome ofthe signal molecules lease from mast cells, cytokine activity, vasodila- involved [10]. The observation that the local tionininflammatoryarea, proliferationoflympho- concentration of Met-enkephalin (and of the re- cytes, and phagocytotic activity. lated heptapeptide Met-enkephalin-Arg6-Phe7) is downregulated by NEP also applies to the inverte- Cytokines brate Mytilus [11]. Tests with two major com- In the searchforbasicimmunoregulatoryprinci- pounds resulting from the hydrolysis of the hep- ples shared by invertebrates and vertebrates, the tapeptide showed that they antagonize its effect, resultsofpilot studies on the presence and activity presumably by competing for part of the same ofcytokines in molluscs and echinoderms promise receptor sites as those used by the heptapeptide. tobe asrewardingasthoseon neuropeptides. The A balancing mechanism is thus provided by the hemolymph of Mytilus was found to contain im- enzymatic generation of antagonistic fragments munoactive interleukins (IL-1 and IL-6) as well as from the agonist's molecule. tumor necrosis factor (TNF-a). Its immunocytes Interference with the normal operation of the respond to the respective mammalian cytokines in immunoregulatory network may occur in various vitro andin vivoin amannercomparable tothatof ways and may have important biomedical conse- human granulocytes [15]. Like in mammals, IL-1 quences. In schistosomiasis, immunosuppression, appears to participate in the internal regulation of attributable to the release by an invertebrate para- the immune system of Mytilus in part indirectly, site of immunosuppressive signal molecules re- i.e., by its stimulatory effect on the formation of sembling those of the host, appears to influence 1100 B. SCHARRER the course of the disease [19]. Similarly, the (1988) Cell. Mol. Neurobiol., 8: 269-284. survival of the human immunodeficiency virus in 7 Leung, M. and Stefano, G. B. (1984) Proc. Natl. patients seems to be supported by its ability to Acad. Sci. USA, 81: 955-958. stimulate the production of ACTH by the host 8 Stefano, G. B., Cadet, P. and Scharrer, B. (1989) Proc. Natl. Acad. Sci. USA, 86: 6307-6311. [20]. 9 Shipp, M. A., Stefano, G. B.. D'Adamio, L., An involvement of neuropeptides in the re- Switzer, S. N., Howard, F. D., Sinisterra,J., Schar- sponse to stressful conditions has been demon- rer, B. and Reinherz, E. (1990) Nature, 347: 394- strated not only in vertebrates, but also in the 396. mollusc Mytilus [21]. The animal's immune/de- 10' Shipp, M. A., Stefano, G. B., Switzer, S. N., fense system can be alerted, for example, by Griffin,J. D. andReinherz,E. L. (1991) Blood,78: 1834-1841. mechanical interference with the valves of the 11 Stefano, G. B., Shipp, M. A. and Scharrer, B. in-currentsyphon. Thisleadstoasignificantrisein (1991) J. Neuroimmunol., 31: 97-103. the number of "activated" immunocytes, pre- 12 Jankovic, B. D., Levic, Z., Nikolic, J. and Strojisa- sumed to be due to the release of an endogenous vijevic, N. (1989) Int. J. Neurosci., 48: 155. opioid-like material from the brain. 13 Stefano, G. B., Melchiorri, P., Negri, L., Hughes, In conclusion, the rewards gained from abroad- T. K. andScharrer,B. (1992) Proc. Natl. Acad.Sci. 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