Phylogeny of the neuroendocrine-immune system: Fish and shellfish as model systems for social interaction stress research in humans

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0959.8030193 $24.00 + .OO 1993 Pergamon Press Ltd.

PHYLOGENY OF THE NEUROENDOCRINE-IMMUNE SYSTEM: FISH AND SHELLFISH AS MODEL SYSTEMS FOR SOCIAL INTERACTION STRESS RESEARCH IN HUMANS Francesco Chiappelli, * Claudio Franceschi, ** Enzo Ottaviani, t Mario Farm?, $ and Mohamed Faisall *Department of Anatomy & Cell Biology, Psychoneuroimmunology Program, and Brain Research Institute, University of California at Los Angeles; and Laboratory of Human Immunology & Psychoneuroimmunology, West Los Angeles Veterans’ Administration, Los Angeles, California 90024, USA, **Institute of General Pathology, tDepartment of Animal Biology, University of Modena, Italy, XDepartment of Psychology, University of Bologna, Italy, IDepartment of Environmental Sciences, Virginia Institute of Marine Science, School of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062, USA

Abstract. Significant interactions among various physiological systems and between an organism’s physiological and psychological structures are evident throughout phylogeny. Environmental stimuli induce neuroendocrine responses that include the activation of the sympathetic nervous system and of the hypothalamic-pituitary-adrenal (HPA) axis, and that modulate cellular and humoral host defense mechanisms from the invertebrates to humans. Because increased levels of HPA products are among the most consistent physiological responses to stress, and because cell-mediated immune (CMI) mechanisms are crucial to the initiation, propagation, and regulation of antigen-specific immune responses, this review focuses on the phylogeny of HPA-CM1 interaction under basal conditions and following stressful stimuli. Research has characterized several paradigms for the study of the physiological outcomes to a variety of stressors. In recent decades, a substantial literature has emerged that describes the neuroendocrine-immune response to social confrontation in invertebrates and in vertebrates. The social confrontation paradigm provides an ideal model for the elucidation of the phylogeny of the HPACM1 interactive system to a specific stressful stimulus. We have characterized the neuroendocrineimmune outcomes of social confrontation in fish. Our data help to explain physiological and pathological mechanisms in fish. Implications of this body of knowledge to clinical medicine and aging are discussed. Keywords. Endogenous

Psychoneuroimmunology. opioid system

Social confrontation,

INTRODUCTION

strates

Psycho-neuroendocrine-immune interactions

tenable:

The various the organism sidered interact.

physiological and that

to be independent Recent

systems from

experimental

each

been conother

evidence’

HPA-CM1

that the Cartesian

interactions,

mind/body

dualism

is un-

of the body is interdependent and intimately intertwined with, and cannot be separated from the well-being of the mind (cf. the clas-

that constitute

have traditionally

Phylogeny,

health

indeed demon-

roendocrine-immune system, and of its relevance in the context of social conflict research. The reader interested in a broader view of the field is urged to consult other sources, including recent textbooks and textbook chapters in the field, e.g. Chiappelli, F. (in press). Neuroendocrine modulation of the immune system. In: Greger, R., Koepchen, H.-P., Mommaerts, W., Winhorst, U. (eds.) Human Physiology: From Cellular Mechanisms to Integration. Section L, Chapter 90. Springer-Verlag, New York (written 1993).

*Address all correspondence to Francesco Chiappelli, Department of Anatomy&Cell Biology, School of Medicine, University of California at Los Angeles, Los Angeles, California 90024-1763, USA. ‘This review does not attempt to be exhaustive and all-inclusive. It focuses on certain areas of investigation that were selected to describe the phylogeny of the neu327

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Chiappelli et al.

sic dictum: Mens sana in corpore sano, and other related perspectives2) (l-8). The interactions among the various physiological systems, and between the organism’s physiological and psychological structures, have profound implications for fundamental and biomedical research. The central nervous system (CNS) regulates and modulates behavioral, hormonal, and immune responses. We are now at the threshold of an era when many of the mechanisms underlying these interactions will be characterized, and when this information will be efficiently translated and incorporated into new and successful intervention modalities in veterinary and clinical medicine. This will help us understand the etiology and required modes of treatment for a variety of fish diseases, and will directly benefit the aquatic environment. These interactions are physiologically significant and clinically relevant in humans as well (l-8). Environmental stimuli induce neuroendocrine responses that often include the activation of the sympathetic nervous system and of the hypothalamic-pituitary-adrenal (HPA) axis. This can be demonstrated experimentally by an elevation of plasma levels of catecholamines, of the anterior %alen’s and Hippocrates’s view of health was shared by several ancient cultures, some that remain current even today: (a) in his Codex Florentiniis, Bernardino de Sahagun indicates that the Aztecs believed that three principal forces (or “souls”) regulated the body, and therefore health: “tonalli,” located in the head; “teyolia,” located in the heart; and “ihiyotl,” located in the liver. Health, for the Aztecs, depended upon the relative amounts of each “soul” at a given time, and upon the maintenance of balance among them. (b) Hindu medicine is based on Aytir Veda (“knowledge of longevity”), and defends that health depends upon the harmonious balance among three forces: “Vayo” (air, wind [=breadth]), “Pittam” (sun, bile), and “Kapham” (moon, phlegm). (c)The basis of Taoism and of Chinese medicine is the “Nei Ching,” written (it is believed) by Huang Ti, the Yellow Emperor of China (26982598, B.C.). In this medical paradigm, disease results from the failed harmonization between two forces: “Ying” and “Yang.” Ying refers to the earth and the moon, the absence of light, cold and wet. Yang refers to the sun and the sky, the presence of light, heat and dry. Thus, ensue contrasting polarities: light-dark, heat-cold, wet-dry. Sickness arises when the balance between these forces is deranged. Environmental forces derive from the five elements (wood, fire, earth, metal, and water), which correspond to five seasons (spring, summer, late summer, autumn, and winter), five winds (east, south, center, west, and north), and five humors (tears, sweat, saliva, sputum, and urine). In addition, the elements correspond to five Ying organs (liver, heart, spleen, lungs, and kidneys) and five Yang organs (gall bladder, small intestine, stomach, large intestine, and bladder) (Wong, K.C., Lien-teh, W. 119361 History ofChineseMe&ine. 2nd ed. Shanghai National Quarantine Service).

pituitary proopiomelanocortin (POMC) gene products (e.g. @-endorphin [@El and adrenocorticotropin hormone [ACTH]), and of adrenocortical glucocorticoid (CC) steroids (e.g. cortisol in man, corticosterone in rodents and in fish). Environmental stimuli also induce significant changes in the population and function of immune cells in peripheral blood and lymphoid organs. Significant alterations in cell-mediated immunity (CMI) are modulated3 by neuroendocrine products (e.g. catecholamines, HPA axis peptides, and hormones). Neuroendocrine-immune interactions are intimately interwoven and constitute a physiologically significant “neuroendocrine-immune” system. In humans, the psychological makeup of the individual appears to determine the extent of the catecholaminergic, hormonal, and immune responses to given stimuli (psychosocial, physical, physiological, antigenic) (7-12). One goal of this review is to critically examine the evolution of certain elements of the neuroendocrine-immune system and of its ability to respond to stressful stimuli. Several research paradigms have been developed for the study and characterization of HPA-CM1 responses to stressful stimuli. Socially conflictual interactions occur across the entire animal kingdom, from invertebrate organisms such as lobsters and ants, to vertebrates from fish to humans (13,14). No other experimental stress research paradigm now available is more relevant and applicable to the human situation than is social confrontation, and the resulting establishment of dominance and submissiveness. To begin to define and to characterize the neuroendocrine-immune outcomes of social confrontation and their underlying molecular mechanisms, it is important to develop appropriate and reliable laboratory model systems. The use of fish and shellfish in this context is invaluable. Aquatic animals and neuroendocrine-immune interactions

The aquatic ecosystem and its component organisms are continually exposed to both natural and anthropogenic stressors. This is often detrimental to 31t is important to note the following semantic distinction: while cytokines and other immune products mediate immune responses they modulate neuroendocrine and other physiological responses. Conversely, neuropeptides and hormones, which mediate neuroendocrine responses, modulate immune responses. In this context, Rabin and his colleagues have proposed that the term “immunopeptide” should be used to refer to products of immune cells and organs that mediate immune responses as well as products from other physiological systems that modulate these responses.

Phylogeny of the neuroendocrine-immune system

the survival and reproduction of indigenous species because of the interactive nature of these stressors: multiple anthropogenic factors often act in an integrated manner in continuously changing environments. Hundreds of thousands of species of aquatic animals exist, ranging from unicellular organisms to mammals, and the physiopathological responses to these stimuli have been investigated only in a few of these species. For instance, teleosts undergo certain physiological responses (e.g. rise in plasma levels of GC and catecholamines following stressful stimuli), which closely resemble those observed in mammals. At low to moderate levels of environmental stress (handling, subordination, toxicity), behavioral responses (avoidance), acclimation (physiological response), and compensation occur. Acclimation and compensation allow the fish to function normally while exposed to a stressor, but growth is blunted. Adaptive processes to stressful stimuli produce measurable changes in the fish ecosystem, population size, and overall physiological state and processes. When the stressful environmental condition exceeds the organisms’ tolerable limit, it can induce a series of pathological processes that lead to severe disease conditions. The severity of these outcomes depends upon the amplitude of the environmental change. If too severe, it can be lethal. Phylogeny of neuroendocrine-immune interactions One initial concern of the use of fish as biological model systems for neuroendocrine-immunology research is their validity from an evolutionary perspective. Evidence demonstrates that neuroendocrine-immune interactions exist in fish and appear earlier than fish in phylogeny. Therefore, this review aims to underscore the relevance of fish research as a reliable and economical animal model for human neuroendocrine-immune and social interactive research. To achieve this goal, this review outlines the physiological relevance of the neuroendocrine-immune system across phylogeny, and describes the behavioral, neuroendocrine, and immune characteristics of the response of fish to social stress. NEUROENDOCRINE-IMMUNE INTERACTIONS IN INVERTEBRATES Evidence shows that invertebrates are capable of host defense and neuroendocrine responses. They can distinguish between self and non-self: they accept autografts and reject allo- and xenografts. The mollusc PIanorbarius corneus rejects allografts (e.g.

329

ganglia from other P. corneus), and xenografts (e.g. ganglia from the snail Helix lucorum), but fails to reject autografts (e.g. tentacles removed from the specimens being transplanted). Allo- and xenografts elicit an initial inflammatory reaction followed by the encapsulation of the foreign tissue. Round hemocytes (RH) and spreading hemocytes (SH) participate in these host defense events. The first cell type exhibits several morphological and functional characteristics of vertebrate lymphocytes; the second cell type resembles mammalian macrophages both morphologically and functionally (15 18). Invertebrates are also capable of recognizing foreign stimuli and of mounting integrated antigeninduced defense responses. Allo- and xeno-antigens (see above) and stressful stimuli trigger overlapping responses in RH and SH cells, which concomitantly act to neutralize the stimuli by perturbing body homeostasis. These interactive phenomena are mediated by a variety of molecules, which are recognized by monoclonal or polyclonal antibody species directed against a myriad of neuropeptides and cytokines produced in mammals (19-24). These properties appear to be early HPA-CM1 interactive mechanisms, and suggest that neuroendocrineimmune interactive mechanisms are quite ancestral, present as early as the earlier forms of the animal kingdom. The hemolymph of P. corneus contains immunoreactive materials similar to ACTH and @E (irACTH, ir+E). Immunocytochemical and flow cytometric data confirm the presence of these peptides in SH cells. Concentrations of ir-ACTH (47 pg/103 cells) and ir-PE (39 pg/103 cells) are detected by radioimmunoassay in these phagocytic cells. Avidin-biotin-peroxidase immunocytostaining demonstrates that ir-ACTH localizes primarily in the cytoplasm and the plasma membrane. Our studies also tested whether ir-ACTH is bound by specialized receptors on the cell membrane and then internalized, or whether it is produced and secreted by these cells. The data show that these cells produce and secrete ir-ACTH, ir+E, as well as immunoreactive corticotropin-releasing hormone (ir-CRH; 21 pg/103 cells) (20). Rodent and human lymphocytes also express the POMC gene and its products (10,ll). Chemotaxis is a process of nonrandom cellular locomotion crucial for allowing cells to move toward the chemo-attractant. This process permits them to localize and approach foreign material for its eventual phagocytosis. This function is important for the organism’s survival and is well preserved and conserved throughout evolution.

330

F.Chiappelli et al.

Chemotactic cells with phagocytic capability in several invertebrates, including P. corneus, Viviparus ater, and Lymnaea stagnalis, have detectable levels of ir-ACTH, ir-PE, and ir-CRH (19-21,23). Indeed, POMC products are chemotactic to both vertebrate macrophages and invertebrate phagocytic SH cells. Host defense-acting cells show flattening, elongation, and formation of pseudopodia in presence of opioids in the freshwater snail model we have developed, in the mollusc model Mytilus edulis and in the insect model Leucophaea maderae (17,25-27). ACTH, but not BE, increases the capability of SH cells to phagocytose Staphylococcus aureus bacteria (21,22). These observations confirm that the specificity of modulation of chemotaxis and phagocytosis by POMC gene products is ancestral, and thus probably crucial for species survival. Cytotoxicity is another ancient and important cellular immune function, performed by several myeloid- and lymphoid-derived immune cells. Cytotoxic cells kill, destroy, and eliminate foreign cells, invading parasites, and tumor cells. Cytotoxicity is found in a wide variety of invertebrates, from sponges and coelenterates, through sipunculids and annelids to the mollusks, arthropods, echinoderms, and protochordates (29), as well as in all vertebrate species from fish to mammals. Natural (MHC-unrestricted) killer-like activity exists in the invertebrate P. corneus, and is mediated by the RH cell population. As indicated above, this cell type has a distinct morphological resemblance to the vertebrate lymphocyte and participates in allo- and xenograft rejection. The cytotoxic activity of RH cells can be modulated by human recombinant interleukin-2 (IL-2), as that of vertebrate lymphocytes. RH cells express membrane markers recognized by the mouse anti-human monoclonal antibodies directed against the cluster designations CD16 and CD56, characteristic of mammalian cells endowed with natural killer (NK) activity. The type of cytotoxic events induced by molluscan effector cells to K.562 target cells is similar to that induced by vertebrate effector cells on the same target cells (30,31). To determine the evolutionary pathway of the modulation of cytotoxic responses by neuroendocrine products of the stress response, we supplemented the hemolymph of P. corneas with supraphysiological concentrations (IO-‘M) of ACTH and CRH. We observed a significant production of biogenic amines (epinephrine, norepinephrine, and dopamine) by the hemocytes. The release of amines was maximal 15 min after stimulation and lasted approximately 45 min. By contrast, BE, present in hemocytes and serum of P. corneus, failed to stimulate

further secretion of biogenic amines by these hemocytes (22,32). These findings are important in the context of the evolution of neuroendocrine-immune interactions because NK-mediated cytotoxic activity is modulated by HPA, catecholaminergic, and other neuroendocrine responses in mammals as well (7,8,33,34). In summary, several lines of evidence converge to indicate that cell types of the lymphoid and the myeloid lineage participate in the neuroendocrineimmune orchestra as early in phylogeny as the invertebrates. A leading role in this context is played by CRH and POMC gene products. Furthermore, cells obtained from a variety of species of invertebrates, Pisces, amphibia, reptilia, aves, and mammals contain significant levels of ir-ACTH and ir$E (10,11,28). These studies underscore that basic interactive mechanisms are maintained as the neuroendocrine-immune system increases in complexity during phylogeny. Host defense responses become more sophisticated in Pisces and mammals compared to invertebrates. Because no other feature better distinguishes the progression of immune responses in primitive fish than the appearance of anticipatory immunity and of relatively well characterized lymphocytes, and because data show that ir-ACTH, found only in phagocytic cells in the invertebrates, become detectable in lymphocytes from anuran amphibians onwards (35), we conclude that evolution of the neuroendocrine-immune interactive network must occur concomitantly with the evolution of immune responses (Table 1). SOCIAL

CONFRONTATION

IN INVERTEBRATES

Social conflict is a universal behavior exhibited by all members of the animal kingdom (13,14,3640). Over three decades ago, the establishment of dominance and submissiveness in ants was described as a model of insect social behavior (36). More recent studies have described social interactive paradigms in other invertebrate species, including crustaceans such as the lobster (13). These studies do not, however, engage in the types of neuroendocrine-immune research questions we now can address. An open field of research therefore remains to be explored to characterize the neuroendocrine-immune system and conflictual social interactions in invertebrates. NEUROENDOCRINE-IMMUNE INTERACTION

IN FISH

Fish leukocytes can mediate a variety of immune responses, including:

Phylogeny of the neuroendocrine-immune

Table 1. Neuroendocrine-immune

331

system

interactions in phylogeny Vertebrates

Invertebratesa

Fishb

Rodent’

Humand

Yes ? ? ?

Yes ? Yes Yes

Yes Yes Yes Yes

Yes Yes Yes Yes

POMC gene products produced by immune cells POMC gene product receptors on immune cells GC modulation of immune cells CC receptor on immune cells “References bReferences ‘References dReferences

(15-28,30-36). (37,38,41-44,47,49,50,66,73). (4,10,11,48,51-53,83,89,92,95,101-109). (4,10,51,57-77,79-82,84-88,90-94,96-100,111,121)

target cell cytotoxicity (37,38), phagocytosis (41), . antibody production (42), and ?? response to classical T and B cell mitogens

??

??

(43).

In fact, all the innate and acquired host defense mechanisms and endpoints found in mammals are observed in bony fish (44). These functional similarities exist despite the several anatomical differences that distinguish the fish from the mammal immune system, such as the absence of bone marrow and lymph nodes. The molecular basis of the fish immune response specificity also resembles that of mammals. Similar genes encode for cell receptors in fish and in mammals. The mechanisms that serve to diversify the products of these genes (e.g. gene rearrangements) are also similar in fish and in mammals (45). Immune responses are modulated by neuroendocrine mediators in fish under stressful conditions in a physiological manner similar to that which has been characterized in mammals. Activation of the HPA axis during responses to stressful stimuli are common to both fish and mammals (46-49). In fish, hypothalamic CRH stimulates the expression of the POMC gene-derived proopiocortin, from the anterior pituitary. Proopiocortin contains the sequence for ACTH and the endogenous opiate alkaloid, PE, within it. The amino acid sequence of ACTH is similar in bony fish and humans (50). Ir/3E occurs in the intermediate pituitary of the holostean fish, Amia culva, and of the Australian lungfish, Neoceratodus forsteri (49). ACTH stimulates the interrenal tissue of the fish (homologous to the mammalian adrenal cortex) to secrete GC. The parallel activation of the sympathetic nervous system (also mediated through the hypothalamus) leads to an increased production of catecholamines (epinephrine and norepinephrine) from the chro-

maffin cells (homologous to the mammalian adrenal medulla). Noradrenergic (NA) innervation of lymphoid organs, a neuroendocrine-immune hallmark in mammals (see below, 5 l-53) has been observed in certain fish. NA innervation of Coho salmon (Oncorhynthus kisutch) spleen is evident particularly in loci rich in leukocytes. Innervation appears to be functionally important because significant increase in splenic plaque forming-cell response to sheep red blood cells conjugated to trinitrophenol follows pharmacological denervation (6-hydroxydopamine) in Coho salmon (54). The endogenous opioid system contributes to the regulation of fish social behavior such as schooling (55), and of a variety of stress responses. Animals across phylogeny, from mollusks to humans, exhibit increased production and secretion of endogenous opioids in response to stressful stimuli. These conditions often produce some form of analgesia and cross-tolerance with morphine and other opiates. Stress-induced analgesia in mammals is reversed by the classical opioid antagonists, naloxone, or naltrexone (48,56). As elaborated further below, social confrontation between aggressive fish (e.g. 7Xapiusp.) leads to mobilization of endogenous opioids, and to suppression of several immunological parameters in the subordinate fish. Both natural cytotoxic activity and the proliferative response of pronephric leukocytes to a variety of mitogens are blunted in the submissive fish. The opioid antagonist, naltrexone, largely blocks this immunosuppression. This indicates the involvement of opioids in the mechanisms leading to immune suppression. Treatment of pronephric leukocytes with @E in vitro, or with the serum from the subordinate fish produces a similar degree of suppression, that is also blocked by naltrexone (37,38).

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F. Chiappelli et al.

These studies are significant from an evolutionary perspective because several studies have described the effects of /3E, ACTH, and other POMC products upon cytotoxic and proliferative responses in mammals (8,10,57-65). Fish immune cells, which are modulated in vivo and in vitro by POMC products, can therefore provide a useful model system to begin to understand the mechanisms involved in the POMC-mediated modulation of immune responses in humans. Endogenous opioids and other POMC-derived products generated during a stress response are often, though not always, immunocompromising in mammals. For instance, adult F344 female rats subjected to a stress paradigm that produces a typical analgesic response and cross-tolerance with morphine (i.e. intermittent footshock) show reduced median survival times and percent survival following intraperitoneal injections of mammary ascites adenocarcinoma tumor cells. The tumor enhancing effect of this form of uncontrollable stress is opioid-mediated since it can be blocked by naltrexone and mimicked by morphine (48,56). However, under given experimental conditions and for given species and strains, these peptides may also enhance immune competence in mammals (8,57,61,65). Research must now determine if these mixed effects of opioids upon immune cell functions are also to be observed in fish. GCs are secreted down-stream of POMCderived products, and modulate several physiological processes (7,66-71). Hig serum CC levels characterize fish exposed to polluted waters, to pesticides, or to heavy metals. Stressed and subordinate fish also have eIevated serum GC. Cortisol suppresses the fish immune responses, including in vitro induction of B cell activation (66,72,73). Steroid effects are principally receptor mediated (7,67-69). However, the hydrophobic nature of steroids renders them capable of penetrating the plasma membrane, thus potentially interfering as well with the metabolism of phospholipids and the kinase cascade, as our data obtained with human lymphocytes indicate (see below). Immune cells from vertebrates express functional GC receptors (Table 1). In fish, GC receptors are expressed in leukocytes, hemopoietic organs, and gills, as shown by the studies of juvenile coho salmon (74). To determine whether or not the number and/or affinity of the leukocytic GC receptors are altered during the observed changes in immune function following stress or cortisol treatment, juvenile coho salmon were either acutely or chronically stressed, or fed a single meal containing cortisol. The binding affinity consistently de-

creased in all tissues tested from chronically stressed fish, compared to tissues from unstressed controls. Acute stress also increased the number of GC receptors in splenic and anterior kidney leukocytes, suggesting a physiologically important heterogeneity of peripheral GC receptor regulation to stress in fish (74). Because GCs contribute to the regulation of migration of leukocytes in rodents and in humans (7,67), the hypothesis that the outcome observed in salmon also results from a selective migration of leukocyte subpopulations characterized by different densities of GC receptors, should now be tested by a variety of convergent biochemical and flow cytometry studies. SOCIAL CONFRONTATION IN FISH Stressful social situations affect the neuroendocrine-immune system of fish. An in vivo paradigm was developed and characterized, which involves social conflict between individuals of certain aggressive fish (e.g. rainbow trout, European eels, tilapia) during the process of the establishment of hierarchy (63,75). The paradigm can be briefly described as follows: two healthy fish chosen at random from a large group are held separately in a confined area of an aquarium for a period of 4-7 days; ?? upon removal of the partition, the two fish exhibit a characteristic sequence of interactions that involves vigorous physical combat, including tail beating, ramming, chasing, mouth fighting, and biting. The frequency of mouth bites can be up to 12-13 bites/minute; ?? the outcome of this combat is that one fish emerges as dominant (alpha) and the other as subordinate or submissive (beta). The subordinate fish suffers the continuous threatening behavior of the dominant fish. When the aggressive behavior of the dominant partner is continuous and unabated, the subordinate fish cannot withstand it and soon dies. ??

The subordinate animal manifests several significant physiological alterations and a significant loss of resistance to disease and immune competence (Table 2) (76) (Fig. 1). Subordinate fish (e.g. eels) show significant biophysiological changes that underscore the severity of the disturbances in several metabolic processes and electrolyte balance. Blood glucose and lactate concentrations of subordinate fish increase significantly compared to the dominant fish. Liver glycogen content and levels of Na+ and K+ ions fall sharply in the subordinate fish.

Phylogeny

of the nemoendocrine-immune

Table 2. Spreading of Aeromonas hydrophilia in rainbow trout, Oncorhynchus mykiss after 11 h of social interaction Dominant

Subordinate

b

s

1

k

b

s

1

k

Infection via water Low cont.’ (n = 6) High cont.’ (n = 6)

2 6

0 2

0 0

0 0

4 6

0 6

0 6

0 3

Intramuscular infection Low conc.3 (n = 6) High conc.4 (n = 6)

1300 4311

4 5

4 5

0 3

0 3

Values are number of fish from which bacteria reisolated. b = blood; s = spleen; 1 = liver; k = kidney. ‘1.3-1.7 x 10s 24.9-5.6 x IO5 31.7 x lo2 ‘2.3 x lo4 (Reprinted with permission from [76].)

333

present in the subordinate fish’s hemopoietic organs. The number of immunocompetent cells in the peripheral blood of subordinate eels, rainbow trout, and tilapia is lower compared to that of their dominant counterpart (Fig. 2). Lymphopenia and granulocytosis are also evident and are accompanied by vascular degeneration of all leucocyte types in the hemopoietic organs in spleen or anterior kidney (73,78-80). Despite the relatively short period of social interaction (i.e. lo-12 h), subordinate fish also exhibit marked histoanatomical changes:

could be

leukocyte counts of subordinate fish decrease while their leukocrit values rise significantly compared to the dominant fish. This outcome is attributed to the greater number of large granulocytes

The total

system

??

subordinate eels have a shrunken stomach, whose mucus membrane folds, flattens, or disappears. The mucous epithelium atrophies, gastric glands degenerate, and the submucosal blood vessels contract (81). This pattern is strikingly similar to the sequence of events leading to the formation of gastric ulcers in rodents exposed to stress (82), and may suggest important contributions of the mucosal immune system (83) to the modulation of the neuroendocrine-immune response following social confrontation;

Fig. 1. Social subordination paradigm. Encounter between dominant (alpha) and subordinate (beta) fish, the resulting physiological changes, and how these modulate the immune system (reprinted with permission from Cooper, E.L., Faisal, M. J. Experimental Zoology Supplement 4: 46-52, copyright 0 1990 Wiley-Liss, a division of John Wiley and Sons, Inc. [77]).

334

F. Chiappelli et al.

cells

I

1031

cl lymphocytes

a Fig. 2. Effect of social subordination on the white blood cells of eels. C, Control; alpha, dominant; beta, subordinate. (Reprinted with permission from [73].)

the gill respiratory epithelium of beta-eels shows severe vascular degeneration. The number of chloride cells increase, and these cells exhibit considerable necrosis (74); ?? the cells of the interrenal tissue become enlarged and show a greater structural heterogeneity in subordinate compared to control or dominant eels (73). These latter pathoanatomical observations are consistent with the observed hypercortisolemia in beta fish. Plasma CC levels of the dominant eels are similar to that of the control fish not engaged in social confrontation, but plasma CC levels of subordinate eels are significantly higher than those of either control or dominant fish (84). In very exhausted beta-fish, CC levels drop, indicating adrenocortical insufficiency following over-stimulation. Subordination is also accompanied by a rise in the serum levels of endogenous opioid (37,38), which confirms HPA activation during social confrontation. ??

Increased susceptibility of fish to pathogenic organisms results from stress-mediated immunosuppression (85). Experiments, which have monitored subordinate rainbow trout infected (via water or intramuscular injection) with a moderately virulent strain of the gram-negative bacterium, Aeromonus hydrophila (causative of motile aeromonas septicemia in fish), demonstrate that the bacterium more

easily invades and multiplies in the blood and internal organs of the subordinate fish compared to the dominant fish (Table 2, 76). To explain the increased susceptibility of subordinate fish to bacterial invasion, studies have examined the function and morphology of leukocyte subsets. Histological and ultrastructural assessments of hemopoietic pronephric tissue have determined that potential phagocytes (histiocytes, reticulum cells, endothelial cells, and neutrophils) become activated in subordinate rainbow trout. Phagocytes show marked hypertrophy, increased pseudopod formation, and enhanced auto-phagocytosis. In vivo and in v&-o tests using yeast cells also indicate an increase of the phagocytosis rate in the subordinate fish (86). Ongoing data demonstrate that a considerable percentage of the phagocytes show signs of degeneration and form necrotic tissue areas as they disintegrate (Faisal et al., unpublished observation). Natural cytotoxic cell activity by effector leukocytes in fish is equivalent to NK cell activity against tumor targets in mammals. A reduction in this function correlates with the presence of chemically induced hepatocellular carcinoma (87). A significant suppression in this activity occurs in beta-tilapias, compared to dominant fish following a 5 h conflict (Fig. 3) (37,38). The beta-fish’s cytotoxic cells cannot recognize, attach to, and therefore form conjugates

Phylogeny of the neuroendocrine-immune

25

Cylotoaicity

20 15 8 IO 3

5 300 250

TO200

Q

Saline

carrlw

;I50 ?5 100 50 Y P

0

Z

250-1

0

Con A

CQfWOl

system

335

To test whether or not the neuroendocrineimmune system is functionally important in fish, we tested the role of endogenous opioids in the observed immunosuppression. Alpha and beta-fish were injected intramuscularly with the endogenous opioid antagonist naltrexone 60 min before the encounter. This intervention blocked about 50% (depending upon the immune measure tested) of the observed immunosuppression. The suppression to the response to LPS was not altered by naltrexone, indicating that endogenous opioids are, at least in part, responsible for the blunting of cytotoxic and T cell mediated, but not B cell mediated, immune responses following social conflict (Fig. 3) (37,38). Recent studies with adult male Wistar rats indicate that corticosterone production following chronic (four weeks) social interaction stress is not the sine qua non physiological trigger leading to depression in cellular immunity (89). These data confirm the existence of complex neuroendocrineimmune modulatory responses to social confrontations in vertebrates. They also indicate that fish can provide important animal models for neuroendocrine-immune research (Fig. 4). These models should be developed and characterized further, and the underlying molecular mechanisms directing the observed outcomes elucidated.

Dominont Subordmota

Fig. 3. Effects of in viva naltrexone treatment on cytotoxicity (LU& and proliferative response to mitogen (PHA, ConA and LPS, specific CPMs) of pronephric leukocytes of dominant and subordinate tilapia following 5 h social confrontation, and of control fish. (Reprinted with permission from [37].)

with tumor target cells (88). Taken together, these observations suggest that the modulation of cell adhesion molecules by the neuroendocrine products in fish may be similar to that of mammals (7). Suppressed proliferative responses to mitogens are also evident in beta-fish (Fig. 3) (37,38). Despite controversy as to whether or not mitogenic responses in vitro are representative of lymphocyte activation following antigen stimulation in vivo, these measurements are defacto used routinely in clinical immunology laboratories. The stimulation of pronephric leukocytes from beta-fish in response to phytohemagglutinin (PHA), concanavalin A and lipopolysaccharide (LPS) derived from E. coli is blunted compared to the response from cells from the dominant fish (37,38). These data correlate with the previously described loss of in vivo immune competence in beta-fish.

NEUROENDOCRINE-IMMUNE INTERACTION IN MAMMALS Research has characterized the complex interaction between the nervous, neuroendocrine, and immune systems in adult and in developing mammals. Experimental and clinical data indicate that psychosocial and neuroendocrine signals regulate immune responses, and that immune products regulate neuroendocrine and psychosocial processes (1,3,4,7,8, 10,11,90-96). Specifically in the context of this review, the interaction between the pituitary-adrenocortical (PA) and the cell-mediated immunity (CMI) systems, and certain psychosocial elements (e.g. perceived person/environment [PE] fit), as they pertain to the social conflict paradigm and the function and regulation of PA/CMI, are discussed. Parvocellular neurons in the paraventricular nucleus of the hypothalamus secrete CRH into the hypothalamic-hypophyseal tract. In this manner, and in concert with vasopressin, catecholamines, and other neuropeptides (e.g. angiotensin 11, enkephalins, cholecystokinin), CRH stimulates membrane transduction (production of cyclic adenosine monophosphate) in the corticotropin cells of the anterior pituitary, and leads to the synthesis and release of POMC gene products. ACTH acts on the ronafas-

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Subordmation

Fear d predators Disease cousmq orqamsms (wruses. bacterta, poraslter)

Fig. 4. Interactions of the brain with components of the environment and the immune system. (Reprinted with permission from Cooper, E.L., Faisal, M. J. Experimental Zoology Supplement 4: 46-52, copyright 0 1990 Wiley-Liss, a division of John Wiley and Sons, Inc. [77].)

ciculata intermediary cell lining of the adrenal cortex, and stimulates production and secretion of CC. A fine physiological regulatory system drives elevated circulating levels of CC to feedback to the hippocampus (blocking the neuroendocrine stimulation of CRH neurons), the hypothalamus, and the pituitary, lowering CRH, ACTH, and ultimately GC secretion. This feedback loop regulates PA output and is mediated largely by the steroid receptors (94,97,98). Two types of steroid receptors exist in mammals. The type I mineralocorticoid-preferring receptor is found in high density, particularly in the hippocampus. The type II GC-preferring receptor is widely distributed in the CNS. Due to their distinct affinity for GC, type I receptors are almost completely occupied at relatively low GC concentrations, but occupation of type II receptors occurs only when GC levels are elevated following hyperactive HPA responses. Type I steroid receptors mediate the reg-

ulation of circadian variations in HPA activity and circulating GC levels, and type II receptors control HPA output when GC concentrations become abnormally elevated during and following stressful stimuli (99). Peripheral mammalian tissues also express steroid receptors. The mineralocorticoid receptor present in the kidney is analogous to the type I hippocampal steroid receptor. The liver and other peripheral tissues sensitive to GC-mediated modulation (e.g. lymphoid organs such as thymus and spleen) express type II steroid receptors. Little or no type I steroid receptors can be detected in lymphoid organs (100). Additional steroid receptor-independent processes participate in the regulation of the “fast” ACTH pulse by GC. We have shown that GC can interfere with membrane transduction events, and phosphoinositol metabolism is significantly decreased by the synthetic GC, dexamethasone (DEX),

Phylogeny of the neuroendocrine-immune

in human peripheral lymphocytes stimulated in vitro with PHA (Baseline: 1027+190 mean CPM*SD;

PHA: 2038+146, p < 0.05 vs. Baseline; PHA + DEX: 13275278, NS vs. Baseline, p < 0.05 vs. PHA). GCs also act to modulate HPA-CM1 activity indirectly via lipocortins.4 Catecholaminergic stimulation plays an important role in the physiological regulation of the HPACM1 system: serotonergic innervation stimulates the release of CRH and POMC gene products (97,98); ?? dopaminergic and fl-adrenergic innervation is predominantly inhibitory. Both adrenergic and noradrenergic pathways regulate visceral sensory information from the brainstem to CRH neurons. A complex limbic circuitry contributes to regulate CRH release: the bed nucleus of the stria terminalis receives input from the amygdala and the hippocampus, thus integrating sensory and neocortical information and influencing CRH output by hypothalamic neurons. The circadian variations in PA output and the regional periodicity of catecholamines in the CNS are correlated (97998); ?? ganglionectomy, vagotomy, and retrograde immu??

nochemistry studies indicate that NA innervation to the spleen of young adult (200-250 g) Sprague

Dawley rats is supplied largely by the superior mesenteric-celiac ganglia. This innervation may represent the second neuron in the classical twoneuron sympathetic chain, and suggests the existence of non-NA innervation of the spleen as well, although no afferent supply to the spleen has yet been detected, despite the use of the sensitive fluoro-gold retrograde immunostaining technique (53); . In adult rodents, as observed in salmon (see above), NA fibers in the spleen are not randomly distributed but show close association with specific loci rich in leukocytes, including the perivascular lymphatic sheath, and local surgical denervation 4Physiological levels of lipocortin-1, a member of the calcium-binding annexin superfamily, are regulated by CC. In rodents, a profound drop in lipocortin-I mRNA and product follows adrenalectomy; this response is blunted by supplementation of adrenalectomized rats with physiological concentrations of CC. The lipocortin-1 receptor is expressed primarily by phagocytic cells (KU = 500 ng/ml); because blood lipocortin-1 levels are relatively low (50 ng/ml) compared to the dissociation constant of the receptor, there is low occupancy of the receptors in circulating phagocytes, and the modulation of the inflammatory response is localized at the sites of inflammation where lipocortin-1 concentrations are considerably higher.

system

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of the spleen of adult (2-3 month old) female Holtzman rats, or general chemical peripheral sympathectomy of newborn rats with 6-hydroxydopamine combined with adrenalectomy leads to an enhancement in the number of plaque forming cell in response to immunization with sheep red blood cells (95). In fact, an inverse relationship exists between the magnitude of the immune response and splenic norepinephrine levels: high responder rats, but not low responders (as measured by plaque-forming cells) demonstrate continuous reduction in splenic norepinephrine content 3 days following immunization (95); ?? during an immune response to sheep red blood cells, decreased levels of norepinephrine occur centrally in the hypothalamus of Holtzman rats (92,95,101); . decreases in peripheral and central norepinephrine levels are associated with, and up-regulate selected components of the immune response. These effects are interdependent since intraperitoneal injections of IL-l alter norepinephrine metabolism in the CNS (102); products of stimulated immune cells regulate sympathetic output, also largely at the level of the hypothalamus (103-106); stimulation of isolated rat splenocytes and human peripheral blood mononuclear cells results in the production of a factor which, when injected intraperitoneally into young adult female Holtzman rats, is capable of raising circulating levels of corticosterone. This factor, the “glucocorticoid increasing factor,” is inactive in hypophysectomized or DEX-treated animals (107); intraventricular injections of IL-l are associated with a rise in circulating ACTH that can be blocked by anti-CRH antibody (108,109); intraperitoneal injections of human recombinant IL- 1, but not tumor necrosis factor, in mice or rats also raise ACTH and corticosterone levels by about 5 fold 2 h after administration (92,110); administration of a booster shot of the recall Tetanus toxoid antigen triggers the expected immune recall response in human subjects, and also generates a significant rise in plasma cortisol within a few hours, well before the appearance of antitetanus antibodies (96). This protocol, used to assess HPA functions in patients with acquired immune deficiency syndrome (AIDS), showed that these patients mounted a blunted hormonal response to the antigenic stimulation, suggesting significant disruption in neuroendocrine-immune interaction in human immunodeficiency virus disease (111);

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An essential product of immune activation, IL-1 also plays a significant role in physiological regu-

lation. Besides the stimulation of CC release and related outcomes, it induces a state of hypoglycemia - presumably resulting from glucose uptake by cells and tissues (92). The implications of these observations to clinical and veterinary medicine are

51nthe late 196Os,the seminal work by Calve and colleagues determined that the bone marrow is innervated. NA fibers, sprouting from the appropriate branches of spinal nerves, penetrate the bone tissue with blood vessels. Some fibers remain within vascular plexi in the parenchyma, others penetrate the bone marrow. This process initiates prenatally, about the time of the onset of hematopoiesis, and is sustained throughout the life span. NA innervation of the thymus occurs postnatally in the rodent: fibers begin to penetrate the thymic parenchyma by the end of the first week and considerably increase in number and in complexity of arborization by the second week, at which time NA innervation of the thymus resembles that observed in young adults. In the adult, NA fibers originate from post-ganglionic cell bodies (superior cervical and stellate ganglia) of the upper para-ventral ganglia of the sympathetic nervous system and penetrate the thymic gland alongside blood vessels. The initial entry point is the thymic capsule, beyond which NA fibers distribute to the capsular and septal systems. These fibers can remain in association with blood vessels, or penetrate these connective tissues independently. NA fibers progress from the thymic capsule and septum to its parenchyma, and end in the cortex. Most of the NA fibers distribute in the thymic cortical region, with the highest density at the cortico-medullary region. Few NA fibers are also found in the thymic medulla, but these fibers are generally associated with arterial or venous vessels. It is not known whether this developmental process is identical in all mammals. Indeed, this may not be the case since the development of the CNS is not uniform across species (e.g. myelinization is complete in primates neonatally, but this process takes place during the first two weeks of postnatal life in the rodent). It is likely that the time sequence of innervation of the human thymus is somewhat different than that of mouse or rat. It is possible that it may be complete by birth. This may have profound implications for neonatal neuroendocrine-immunology, particularly in the case of perinatal HIV transmission because the presence of NA fibers are crucial for the development of thymus’s functions in T cell development. The thymus involutes with aging, but NA fibers are retained and are confined within the compartment of thymic tissue that remains functional. Thus, aged thymus appears to be hyperinnervated with NA fibers at certain sites. Thus, the distribution of NA fibers in the thymus is retained in aging. These observations may contribute to explain the apparent thymic functionality in the elderly (i.e. continued generation, albeit at a reduced rate of naive T cells) despite its profound morphological involution. Development of splenic innervation is a postnatal event in rodents. As for the thymus, it is not known when splenic innervation occurs in humans. This information could also be critical in the design and development of successful modes of intervention for neonatal infectious diseases, such as pediatric AIDS. In rodents, innervation occurspan’ passu with the development and compartmentalization of the spleen (i.e. few fibers in the developing white pulp during postnatal week 1, increasing NA innervation between weeks 1 and 2 as the marginal zone develops, substantial

increase of the NA plexus about the central arteriole during development of the periarteriolar lymphatic sheath early in week 3) during the initial 28 days of postnatal life. The adult pattern of NA innervation of the spleen is fully developed by the end of the first month of life in the rat, and it persists through 12-15 months of age. NA fibers, which sprout from the celiac-superior mesenteric ganglionic complex, are also associated with vasculature, and penetrate the spleen at the hilar region. Afferent and efferent lymphatics are found at the splenic hilus. The fibers branch off where the splenic artery branches off, thus forming complex plexi that distribute within the white pulp in close association with the central splenic arterioles and their respective branches. Smaller plexi are found in association with the venous vasculature within the trabeculae and the capsule, or just outside it. Within the white pulp, NA fibers may remain associated mainly with the arterial vasculature, or they may freely distribute deeply into the tissue along the inner regions of the marginal and parafollicular zones. Occasional fibers are found in the follicles and the red pulp. NA terminals in the spleen often occur in close, direct contact with lymphocyte in the synapse-type junction discussed above, whose distance has been estimated to be in the order of 5-7 nm. In older rats, progressive loss of T lymphocytes and of a family of specialized macrophages (identified by the expression of the ED-3 marker) coincide with progressive loss of NA innervation of the spleen. Whether or not this diminution of splenic innervation plays a role in the immunocompromise associated with aging remains to be established. In rodents, NA fibers enter lymph nodes at the concave side of the node (hilts), in association with the arterial and venous systems. The lymph is carried to the lymph nodes by the afferent lymphatics (rich in naive T cells), which penetrate the nodal capsule at its convex side; lymph leaves the node in the efferent lymphatics (rich in memory T cells), which exit the node at the hilus. Fibers distribute in the capsular/subcapsular compartments and in the septum, and eventually arborize in the paracortical regions rich in T lymphocytes, as well as in parafollicular regions. Germinal centers and nodules are devoid of NA innervation, which suggests the absence of direct neural modulation of the immune events at these sites. NA fibers sprout in most instances from the spinal nerves that innervate their respective anatomical regions. This process follows that of the spleen during ontogenesis. In the human tonsils, NA fibers form dense paravascular plexi, from which individual fibers sprout into parafollicular regions, in areas rich in T lymphocytes. Lymphoid nodules and epithelium of these organs typically show little NA innervation. NA fibers penetrate these organs relatively early in development, before their invasion by cells of the lymphoid lineage. There is extensive innervation and plexi formation in enteric mucosal tissues by NA and other fibers. Substance P and calcitonin gene related peptide fibers penetrate the lamina propria into the epithelium; submucosal and myenteric innervation of the lamina propria principally involve substance P, somatostatin, and cholescystokinin; these fibers are joined by NA and vasointestinal peptide fibers in the Peyer’s patches.

??

NA innervation to the lymphoid organs is also evident during ontogenesis (5132).

Phylogeny of the neuroendocrine-immune system

profound because chronic infections may be associated with chronic states of hypercortisolemia and hypoglycemia, which in turn may contribute to the onset of opportunistic infections and the aggressive progression of pathology. Extensive interactions between the CNS and the mucosal immune system also exist in vertebrates. The gastrointestinal tract is richly innervated by largely autonomous fibers integrated with sympathetic, parasympathetic, and sensory afferent systems, which ramify from myenteric and submucous plexi extensively throughout the wall of the intestine. Adrenergic efferent innervation of the stomach arises in part from celiac and mesenteric sympathetic ganglia, which innervate the spleen. These gastric networks participate in the regulation of gastric acid secretion and probably play a significant role in ulceration. Rich NA innervation is evident in lymphoid aggregates of the mucosal system (e.g. Peyer’s patches6), and the lamina propria and gut epithelium. The gastrointestinal tract is rich in the production of and in the variety of responses to several neuropeptides (e.g. substance P, somatostatin, vasoactive intestinal peptide, cholecystokinin, opioids). These responses include lymphocyte migration and redistribution. Mucosal immune responses are finely regulated by the interaction between gut-wall structures, intraepithelial lymphocytes (B, T, and large granular lymphocytes), and the nerve endings and the neurotransmitters they contain (83). Thus, the neuroendocrine-immune system in vertebrates involves site-specific (i.e. peripheral vs. mucosal) interactions between immune and neuroendocrine components, which are similar, albeit more complex, than those evident in the invertebrates. SOCIAL

CONFLICTS

IN MAMMALS

Social confrontational situations are common in mammals. Human social interactions are no exceptions. Based on our findings that the neuroendocrine-immune system is affected by social confrontation in fish, the hypothesis that social confrontation profoundly interferes with certain neuroendocrine-immune processes in humans should be tested. Mammals, rodents in particular, have been used extensively in the characterization of the behavioral, hormonal, and immune outcomes to social interaction and aggression (39,40,46,89,112-l 14). In mice and rats, social conflicts lead to the activation of the sympathetic system and of the HPA axis. As also noted in fish (see above), the endogenous 6Tonsilla intestinalis folliculi

lymphatici

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opioid system, including /3E, is stimulated in defeated mice, which develop a form of analgesic response that is abolished by opioid receptor antagonists (e.g. naloxone or naltrexone). This form of analgesia also shows cross-tolerance with morphine (39). Intruding and subordinate male rats show a significant rise in plasma ACTH and PE (47). Furthermore, social conflict in rodents produces significant alterations in immune competence. Antibody production is generally impaired in defeated and overcrowded mice, although studies have also reported a greater antibody response, compared to the dominant animal (40,89,112,115). These mixed outcomes reiterate that variables such as the age and sex (phase of estrous, if female) of the animals, the housing protocols, and the social conflict paradigm are among the crucial variables that can significantly alter the outcome of social confrontation studies in mammals. In addition, while the T cell dependent production of antibody species is reportedly impaired following chronic and repeated exposure to a social conflict in rodents, T cell independent antibody production (e.g. to polyvinylpyrrolidone) remains mostly unaltered (114), suggesting that the T cell compartment of mammals is likely to be impaired in vivo in a social confrontation experimental paradigm. These observations may be related to the findings in fish discussed above. Furthermore, that in vitro challenge of splenocytes fails to demonstrate suppressed T cell lymphoproliferative responses in defeated animals, suggests that the migration of T lymphocytes from the spleen and other lymphoid organs to the site of antigen injection may be impaired in these animals; the inherent ability of the cells to respond to challenges and stimuli may be unaltered. This interpretation of the data confirms the potential role of POMC products, catecholamines, and other neuroendocrine outcomes of social confrontation in the modulation of lymphocyte homing and expression of adhesion molecules (7). Research in the field of social confrontation in nonhuman primates and in humans suggests similar neuroendocrine responses as those noted above; that is, activation of the catecholaminergic and of the HPA systems (116). This research is particularly complex in humans because of the sophistication of our psychological system, compared to ants, lobsters, fish, or mice. Whereas we, as other animals, experience stressful experiences and events in the external environment, such as a confrontational encounter, we have well developed abilities to think, reason, and interpret these events (i.e. cognitive information processing). We can carefully plan our defense response, and adapt it to new circumstances

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as they arise. Usually, albeit not always, we are capable of substituting word exchange to physical combat. Thus, social confrontation among humans often becomes a complex battle of verbal and nonverbal language and cues, of written statements, of alliances with others, of intellectual acuity and political ability. Much of our ability to face environmental challenges depends upon our self-efficacy; that is, our perceived sense of being able to exercise control over potential threats. Plasma epinephrine and nonepinephrine levels are relatively low in individuals with high self-efficacy during interaction with a phobic object (these individuals would be dominant in social confrontation paradigms); moderate-to-low self-efficacy, by contrast, is associated with elevated plasma catecholamines (9). These data, taken together with the lines of evidence discussed above lead us to submit that low selfefficacy, which is likely to translate to social submissiveness in humans, may correlate with lower immune competence. Indeed, a pessimistic explanatory style (the tendency to perceive events generally as negative because the challenge they present appears to be unsurmountable when their nature is viewed as stable and unchangeable, global and all-encompassing and internal [i.e. originating from the individual’s inabilities], is typically associated with poor health and increased risk of disease) (117). Recent studies in the elderly showed significant negative correlation between a composite score of negative event ratings and CD4KD8 ratio (-0.52, p < 0.05) and proliferative response to low PHA doses (-0.48, p < 0.05). The conclusion offered by the authors that “. . . a pessimistic style might be an important psychological risk factor-at least among older people-in the early course of certain immune mediated diseases” appears to be warranted (121). The phenomenology of social confrontation among humans usually results from the same types of stimuli as in fish and invertebrates. Territory, for instance, is one major trigger of confrontational encounters in all animals, although humans prefer to call it “space” (parking space, office space, closet space, etc.). Consciously or subconsciously, humans typically consider this situation from the following perspectives: “reality of the surroundings” (i.e. environment) (for example: office space in our organization is tight, but both Bob and I should qualify for this new office); “perceived environment” (for example: this new office should be assigned to me);

et al.

“reality of the internal environment” (i.e. self) (for example: I might have to improve the quality of my work in order to compete with Bob for this new office space); ?? “perceived self” (for example: I need this new office space, besides I deserve it).

??

Intense social confrontation can ensue from this type of situation if both Bob and I hold the same thought patterns. These thought patterns characterize a lack of fit between reality and perception, between the environment and the self. The lack of person/environment fit is a significant source of stress in humans (5,6). In humans, events associated with social confrontation can result in physical encounters similar to other species of the animal kingdom, but these events, as indicated above, usually take different qualities (e.g. verbal and nonverbal argumentative interactions). Regardless of the manner in which they are expressed, socially conflictual situations in humans produce a perception of loss of adaptation of the person within his/her environment. This lack of person/environment fit leads to physiological responses that include significant neuroendocrine and immune alterations. Humans have the capability of interpreting events. This ability allows them to assess, gauge, and evaluate situations and the events that lead to them. In this manner, they can add or detract to the stressful potential of given social interactions. Presently, it is believed that fish cannot perform this kind of cognitive assessment, and therefore must confront each social interaction situation at its face value. The ability of humans to evaluate social confrontational situations is directed by several psycho-cognitive and psycho-social parameters. For example, humans have distinct temperaments, personalities, and motivational structures that may make them more or less combative or, on the contrary, more submissive. From the time of Hippocrates and Galen to the 19th Century, Western medicine heavily relied upon the theoretical constructs of the four temperaments. The four basic elements (earth, wind, water, and fire) translated into the four bodily humors. Distribution of these humors in a given individual determined one of four temperaments: choleric, sanguine, phlegmatic, and melancholic.’ It is tempting to draw parallels be‘Quattuor humores in human0 corpore constant: /sanguinis cum cholera, phlegma, melancolia./Terra melancolia, aqua phlegma/a& sanguis, cholera ignis. (De Quattuor Humoribus Corporis: Flos Medicinae Salernitanis). Attributes of the individual derive from the balance

Phylogeny of the neuroendocrine-immune system

tween the latter two temperaments and today’s illness-prone “Type-C” personality (i.e. selective suppression of positive emotions; distinct from the psychiatric disorder alexithymia, the relatively nonselective suppression of all emotions), low self-efficacy, and a pessimistic explanatory style. Regardless of the validity of these theoretical constructs, it is clear that the individual’s behavior and personality styles determine his/her stress response. They are modulated by a number of factors: cultural background, socioeconomical status, social support network, the particular period during his/her life span (i.e. physiological and psychological age), and personality characteristics. For these reasons, psychosocial stressors in humans, including abrasive social interactions, can have profound immunomodulatory (mostly suppressive) effects, and can predict contemporary and subsequent psychosomatic

symptoms.

In addition,

negative

moods,

such as anxiety, pessimism, or depression may aggravate these outcomes (118-120,122-124). Certain moods and personality variables may moderate the immunosuppressive action of stressful stimuli. This was originally demonstrated by Norman Cousins in his description of his bout with the collagen disease, ankylosing spondylitis. The normal course of this disease is rapid and severe. He devised a program of laughter and positive emotions, which he then used in conjunction with all the necessary medical treatment and assistance. Together, these two modes of intervention produced additive beneficial effects, and helped him overcome his illness (125). Recent experimental data confirm that individuals with a good sense of humor have a weaker negative relationship between stressors and salivary immunoglobulin A (sIgA) than those with little or no sense of humor (126). Our research confirms the role of personality variables in the immunomodulatory outcomes of stressful stimuli. We contrasted two styles of personality, “barrier”(overt and conscious behavior aimed at facing stressful life events head on-outgoing dominant personality style) (129), and subconscious among these elements (“eucrasia”: appropriate balance, “dyscrasia”: imbalance). For example, cold is an attribute that derives principally from the earth element (melancholy) and the water element (phlegm), hot derives from the fire element (choleric) and the air element (sanguine), dry derives from fire and earth, and humid/moist from water and air. Distribution of the humors in the individual determines one of four temperamental styles: Choleric (fire, “yellowbile”), sanguine (air, blood), phlegmatic (water, phlegm), and melancholic (earth, “black bile”). Galen reported that melancholic women were more prone to develop cancer than were sanguine women.

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defense mechanism of “repression” (unexcitable, slow, and painstaking personality style), with respect to the nature of the response (objective or subjective) to stressful daily hassles. Objective responses included physiologic responses, such as immunomodulatory events (e.g. sIgA levels); while subjective measures included both emotional outbursts or other behavioral expressions, and indefinite signs of danger. The two personality styles correlated with a significantly attenuated stress response; however, while barrier was associated with the subjective response, the repression style induced changes in sIgA levels (127-129). Therefore, our experimental data confirm that responses to stressful stimuli, including socially abrasive situations (and perhaps confrontation), involve both conscious and subconscious responses in humans. This supports recent data from the neuropsychological (130) and cognitive/information processing literatures (131) which emphasize the complex dichotomous nature of the process of assimilation of human beings within their environment. SUMMARY In summary, research to date indicates that the neuroendocrine-immune system is present early in evolution. These interactive responses are well conserved from the invertebrates to man. Key mediator molecules, including CRH, ACTH, and /3E, are unchanged throughout phylogeny, and neuroendocrine-immune processes follow a similar pattern and blueprint. In the invertebrates, these neuroendocrine-immune phenomena occur in the hemocyte, which acts as an “immune-mobile brain” capable of both host defense and neuroendocrine responses (17,18). This property is conserved in mammals and human lymphocytes, which not only respond but also produce POMC products (4,10,11). Social confrontational interactions are stressful stimuli observed throughout phylogeny. The fish social subordination paradigm reveals a clear relationship between environment, behavior, hormones, and immunity. Mammals, including humans, respond to challenging environmental stimuli by much the same physiological mechanisms, although these relationships are made more complex due to the intricate nature of psychological processes in humans. IMPLICATIONS Taken together, the studies reviewed here indicate that neuroendocrine-immune responses are ancient and well-conserved, and that they are physiologically significant. That these processes are crucial to the well-being of the organisms has profound

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implications for veterinary medicine, particularly marine biology, and fish disease. These lines of evidence are also relevant to clinical medicine and gerontology, as patients and the aged often exhibit a significant degree of neuroendocrine and immune dysregulation and impairment (94,132,133). Often compounding the pathological profile of seriously ill patients is profound psychological despair, loneliness, and helplessness (134). If one conceives the encounter of an individual with a severe disease from a psychological viewpoint, one easily determines that the individual faces his/her condition with a frame of mind similar to that of a person faced with a foe in a confrontational paradigm. As the patient becomes sicker, he/she translates this progression psychologically as defeat. The psychological submissiveness that may ensue in the face of worsening conditions cannot but have deleterious effects to the patients’ mental and physical health. In addition, seriously ill patients often tend to seek psychological refuge and comfort in alcohol and or drug of abuse. These substances, in and of themselves, are deleterious to immune competence because they impair cellular immune events directly, and because they trigger neuroendocrine responses that are immunosuppressive (135). This constellation of variables may well make the difference between successful or unsuccessful medical intervention, and may be particularly crucial in several conditions characterized by their progressive nature. These considerations may be particularly important, for example, in the rate of progression of AIDS, and may contribute to determine the patients who rapidly progress to frank AIDS from the long-term survivors. Similarly, they may pertain to aging, and the “will to live” may simply reflect the unwillingness to become submissive to the multiplying ills and discomforts that accompany old age. Based on our current understanding of neuroendocrine-immune interactions and of their consequences for health, it is crucial now to go beyond traditional approaches and begin to develop new psychoneuroimmune interventions for populations at risk. The main goal of these interventions should be to integrate medical and psychological assistance and care such as to heal both the mind and the body. It is also important to continue to explore animal models of behavioral-neuroendocrine-immunology, particularly fish, because they provide simple, reliable, and economical systems to improve our understanding of human conditions. In addition, these systems generate much needed new knowledge in marine and fish biology and pathology.

Lastly, the significance of studies that describe and characterize the origin of the neuroendocrineimmune system from a developmental and an evolutionary viewpoint cannot be overemphasized because these studies provide the essential tools we need to understand who we are and how we function. work was funded in part by the UCLA-Psychoneuroimmunology Program, the Fetzer Foundation, and NIDA lR29 DA-07683.Other sources of support for the work from our laboratories cited here were listed in the original research publications. The authors are indebted to the late Norman Cousins for his considerable contribution and support to the emergence of the field of Mind/Body transaction, and to the late Professor Cabriele Peters for her significant contribution to the development of the fish social confrontation paradigm we have described herein. This is Virginia Institute of Marine Science Contribution Number 1794.

Acknowledgments-This

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