Neuroimmunology: the importance and role of a comparative approach

Berta Scharrer

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA

Introduction

The intent of this overview is to provide a framework for the more detailed contri- butions to be presented in various areas of comparative neuroimmunology. A rich body of information has been obtained from stud- ies in invertebrate animals. By comparison, results obtained in lower vertebrates are still relatively scarce.

What the studies in invertebrates have revealed is a remarkable degree of paral- lelism with the immunoregulatory pheno- mena known in mammals. In fact, some of the recent invertebrate work has substan- tiated general principles in neuroimmuno- modulation and has opened new vistas. The heuristic value of a broadly based compara- tive approach has proved to be equally rewarding in other areas of research, e. g., that concerned with neuroendocrine interac- tions.

It was the discovery of the phenomenon of neurosecretion by Ernst Scharrer in 1928 which introduced us to the existence of a class of blood-borne neuronal messenger substances (neuropeptides) capable of com- municating with the endocrine system "in its own language" (for review see Scharrer, 1989a, b). The importance of this material

in establishing a hormonal link between the two systems of integration became increas- ingly apparent. The recognition of its role in both vertebrates and invertebrates was crucial in the evolvement of the discipline of neuroendocrinology (Scharrer and Scharrer, 1963). These studies foreshadowed and stim- ulated further developments outside of the confines of neuroendocrinology. One impor- tant outgrowth was the demonstration that comparable interactions occur between the nervous and the immune systems.

In studies dealing with neuroimmunology, we ask the same questions as in those con- cerned with neuroendocrine phenomena. (a) What do the two systems have in common, and (b) how do they interact with each other? What the partners have in common is that they control important functions in the organism. Both make use of chemi- cal messenger substances in autoregulatory activities, as well as the dispatch of signals to other organ systems. Moreover, the ac- tivities of the nervous and the immune systems are coordinated with each other by a bidirectional exchange of information and directives.

An interesting insight gained is that the chemical messenger substances produced by, and eliciting responses in, cells of either

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system are the same or at least closely related to each other. Neuropeptides, formerly con- sidered to be characteristic of the neuroen- docrine apparatus, are now known to be manufactured and dispatched by immunore- gulatory cells as well. By the same token, cytokines, which we primarily assign to the immune system, have been identified as products of neural, especially astroglial ele- ments. Recent developments (see Hughes et al., 1990) have shown that this equally applies to vertebrates and invertebrates. Since we have to add neurotransmitters and various hormones other than neuropeptides to the list of participating substances, this adds up to a rich repertoire of signal mole- cules. To date, most attention has been given to neuropeptides in their immunoregu- latory roles in invertebrates as well as ver- tebrates. The exchange of neuropeptide sig- nals among various organs is accomplished by the presence of stereospecific receptors (see Scharrer et al., 1988).

Immunoact ive invertebrate cells

Before a discussion of recent insights gained in experiments with invertebrates in com- parative terms, a brief characterization of their immunoactive cells seems to be in order. In efforts to identify these cellular elements we are guided by structural as well as functional features they share with those of vertebrate immunocytes. What we can state with reasonable confidence is that immunoactive invertebrate cells represent a subpopulation of the animals' hemocytes. However, it is not yet possible to match specific immunoregulatory activities observ- ed with ultrastructurally distinctive classes of hemocytes.

As to the ultrastructural features of in- vertebrate hemocytes we refer primarily to a comprehensive study of the blood elements of five species of insects, with special em- phasis on Leucophaea maderae, the species used in our immunological studies (Scharrer,

1972). These hemocytes appear in a variety of forms. Many of them contain conspicu- ous secretory granules and other organelles resembling those of vertebrate granulocytes, in different combinations. The existence of many transitional forms makes classification according to cellular lineage difficult. In fact, different cell types observed in the hemolymph may represent different stages in an individual cell's life cycle. Its capacity to undergo structural transformations may reflect its ability to adjust its functional performance to physiological demands.

In any event, the rich ultrastructural arma- mentarium of insect hemocytes is in keeping with their functional versatility. In addition to those concerned with immunoregulation, the hemocytes are engaged in activities such as synthesis, uptake and transport of certain materials, removal of breakdown products and foreign objects, tissue repair, coagu- lation of hemolymph, and maintenance of extracellular connective tissue. There is some evidence that these different func- tions are carried out by a division of labor. Even within the population of immunoactive hemocytes, there may be groups capable of responding to different signal molecules depending on the presence of specific recep- tors.

Ultrastructurally, the hemocytes of molluscs such as Mytilus edulis resemble those de- scribed for insects. Like those of Leucophaea, the immunoactive hemocytes of this bivalve are capable of undergoing striking structural changes, as demonstrated by time-lapse video- microscopy (Stefano et al., 1989b). The de- gree of these conformational alterations is a valuable indicator of cellular activation.

To date, the premise that certain inverte- brate hemocytes are immunoactive is based primarily on experimental evidence. They respond in the same way to stimulation by the same chemical messengers as those effec- tive in the vertebrate immune system. They produce the same signal molecules, and pos- sess specific receptors for them (Stefano et

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al., 1989a, b). They are, therefore, referred to as immunocytes.

Bidirectional exchange of information

Most of our knowledge on bidirectional signals between the nervous and the im- mune systems comes from mammalian stud- ies (see Blalock, 1989; Stefano, 1989). We know that neural messages are sent to the immune system, for example under the conditions of stress. These directives may reach immunoactive structures directly or by way of non-neural hormones dis- patched by endocrine cells that are un- der neurohormonal control. Direct neuronal messages to the immune system may be blood-borne or by innervation. For example, the spleen and thymus receive synaptic input from fibers that may contain substances re- sembling Met enkephalin, neuropeptide Y, cholecystokinin, neurotensin, etc. (Felten et al., 1985).

Conversely, there is increasing evidence (including clinical data) demonstrating that signals dispatched by immunoactive cells reach the nervous system, and thus pro- vide part of the information recorded and processed by the neuroendocrine regulatory apparatus. For example, microiontophoretic application of gamma-interferon to various parts of the rat brain combined with simul- taneous sir/gle-neuron recordings, revealed alterations in cellular activity in all brain structures examined. Moreover, systemic administration of gamma-interferon resulted in certain behavioral responses (Dafny et al., 1985).

An indication that endogenous neuron- derived peptides deliver signals to the im- mune system of an invertebrate was ob- tained in recent experiments with Mytilus (Stefano et al., 1990a, b). Stressful stimuli (electrical shock to neural structures, and mechanical interference with the valves of the in-current syphon) with which the ani- mal could not cope, brought about signs

of activation of its immune/defense system. The hemolymph of animals subjected to this treatment consistently yielded numbers of "activated" hemocytes that by far exceeded those in untreated Mytilus. This response is comparable to that brought about by the ad- ministration of exogenous opioid peptides to intact animals, to be discussed below. In oth- er words, these cells show the characteristic conformational changes (increase in cellular surface, formation of pseudopodia) prior to mobilization associated with the state of activation in both vertebrates (Falke and Fischer, 1985) and invertebrates (Stefano et al., 1989b). Moreover, these preparations of Mytilus cells no longer appear to respond to the administration of exogenous opioids. This suggests that the group of hemocytes activated in the stress experiment is the same as that responding to exogenous opioids in the intact animal. This relationship is sub- stantiated by the fact that the administration of naloxone prior to that of stressful stimuli yielded hemolymph preparations in which few hemocytes appeared to be activated. By contrast, once activated, as a consequence of induced stress, immunocytes no longer re- sponded to the administration of this opioid antagonist, i.e., ameboid cells did not return to the rounded inactive form. This indicates that in unstressed opioid-activated as well as stress-activated immunocytes naloxone seems to be able to block the process of mobilization, but once it has occurred it can- not reverse it. The conclusion drawn from this experiment is that the immune/defense system of Mytilus can be alerted by an endogenous opioid-like material presumed to be released by the brain.

Autoregulatory processes

In addition to its communication with the nervous system, the immune system uses the same signal molecules to ensure coordin- ation of its activities within its various parts. It is in this area of autoregulatory activities

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that major contributions have been made in recent years by work in invertebrates, i. e., molluscs and insects.

Rather comprehensive studies in Mytilus and Leucophaea have demonstrated that, like in vertebrates, internal cell mediated immunoregulatory processes are carried out with the participation of enkephalin-like and additional neuropeptides. Various tests were carried out with exogenous drugs as well as endogenous Met-enkephalin-like material extracted from cell-free hemolymph and hemocytes of Mytilus (Stefano et al. 1989a, b).

Two processes, adherence and migration of immunoactive cells have been shown to be markedly enhanced in the presence of DAMA (D-AIa 2 -Met 5 enkephalin amide) a stable synthetic analogue of Met-enke- phalin. The same is true for other exogenous and endogenous opioids examined. In slide tests, the addition of such substances to the hemolymph resulted in a significant increase in cellular adherence and clumping. The operation of stereospecific receptors in this process was demonstrated by the fact that the addition to the incubation medium of naloxone, in a concentration of 10-8 M, markedly reduced these effects.

An additional result of the examination of these hemolymph preparations with Nomar- ski optics was that in the presence of opioid substances a stimulation of the cells' locomo- tory behavior is preceded by distinctive con- formational changes. As mentioned earlier, these structural and behavioral responses are indicative of the process of immunocyte act- ivation. In Mytilus, the presence of DAMA shows these effects in approximately 35% of the total cell population within a period of 15 minutes.

The responses of immunocytes of Leuco- phaea generally parallel those in Mytilus, but they show certain characteristics (Stefano et al., 1989a). The unstimulated hemocytes tend to be somewhat elongated rather than round- ed, and after activation they are less mobile

than those of Mytilus, probably because of the high viscosity of the hemolymph fluid of this insect.

The formation of immunocyte clusters ob- served in opioid-stimulated hemolymph prep- arations of Mytilus indicates that single ame- boid cells are chemotactically attracted by cells of the same kind. The question as to whether this oriented locomotory activity is due to an opioid or to another endogenous messenger molecule is still undecided. However, the first possibility is supported by an in vivo experiment (Stefano et al., 1989a) showing directed migration of Mytilus immunocytes to the site of injection of DAMA. The possibility that this in vivo response was due to non- specific irritation by the puncture was ruled out by appropriate controls. Therefore, the result obtained indicates that the locomotory activity of opioid-stimulated immunocytes was guided by a gradient in the concentration of the exogenous opioid.

Another item of interest addressed in the work with Mytilus was the comparison of the stimulatory effects of DAMA on cel- lular conformation and locomotory activity with those observed in the presence of re- lated neuropeptides (Stefano et al., 1989b). Eight additional drugs tested yielded cellu- lar responses comparable to those observed with DAMA, but all of these proved to be less potent. This focuses attention on the question of the possible occurrence of multiple types of opioid receptors on inver- tebrate immunocytes. Whereas heterogeneity of neuropeptide-receptor subtypes is well es- tablished in mammals, no definite information on this problem had been obtained thus far in invertebrates. The only previously available evidence pointed to the presence of delta-like binding sites in molluscs (Stefano, 1982). Three compounds indicative of the presence of delta receptors in these hemocytes, name- ly [D-Pen 2, D-Pen 5] enkephalin (Stefanc et al., 1989b), DAMA and [D-Ala2-Leu 5] enkephalin, revealed activity at the same peak concentrations (10-9M) as the mu-type

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ligands and the kappa type ligands tested. Only beta-endorphin showed a higher peak activity (10-1°M), but DAMA proved to be the most potent drug used with the lowest peak concentrations of 10-11M observed. This result sets DAMA apart from related drugs and suggests the possible existence of a subtype of delta-receptors, specifically responding to DAMA.

Discussion

In their immunoregulatory activities inverte- brate and vertebrate animals make use of neuropeptides and additional messenger substances (lymphokines, serotonin, etc.) released by neuroendocrine centers as well as immunoregulatory cells. The latter ad- dress neural structures (afferent limb of the bidirectional communications system) and other immunocytes (autoregulatory pro- cesses). Opioid stimuli have been shown to enhance chemokinetic as well as chemotactic migratory behavior of immunocytes (review, Stefano, 1989). In both groups of animals the enhancement of locomotory activity of immunocytes is accompanied by characteris- tic changes in cellular structure, first report- ed in mammals by Falke and Fischer (1985). Moreover, evidence has been obtained that invertebrate immunocytes possess the same high affinity receptors for opioid-like signal molecules than those of mammals (see Stefano, 1989).

Mytilus immunocytes are now known to respond not only to a variety of opioids but also to certain non-opioid signal molecules (Stefano et al., 1989b; Hughes et al., 1990) and there is strong evidence for the existence of delta-, mu-, and kappa-receptors, as well as a special delta-receptor for Met-enke- phalin-like material. This raises the question as to whether subtypes of receptors for spe- cific ligands are located in separate groups of immunocompetent hemocytes, or whether a given cell can have more than one type of receptors. There are some indications that

a certain type of Mytilus cells can respond to opioid as well as nonopioid ligands (T.K. Hughes, personal communication).

Conclusions

Recent progress in the elucidation of cell- mediated immunoregulatory processes in in- vertebrates has shown them to be more highly developed than previously thought. Remarkable structural and functional paral- lelisms between vertebrate and nonverte- brate immune systems make information gained from invertebrate models generally significant. Insights gained from this broad comparative approach, especially from work with Mytilus edulis, have gone beyond the detection of commonalities and have stimu- lated new approaches in mammalian studies. For example, the concept of a special role of Met-enkephalin in immunoregulation, in contradistinction to that of the closely re- lated Leu- enkephalin, is based on the de- tection of its high potency in Mytilus. The efficiency of the network connecting the neuroendocrine and the immune systems seems to have been developed in the course of a long evolutionary history (see Stefano, article in this issue). Further exploration of these relationships on a comparative basis promises to be highly rewarding.

Acknowledgements

The author's research referred to in this article was supported by Grant NIH NS 22344-02.

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