I. Introduction

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The brain and the immune system, or the "supersystems", a term recently coined by Tada (1997), are the two major adaptive systems of the body. Although the immune system has been often regarded as autonomous, the last two to three decades provided strong evidence that the central nervous system (CNS²) receives messages from the immune system and vice versa messages from the brain modulate immune functions. Thus, the brain and the immune system are involved in functionally relevant cross-talk, whose main function is to maintain homeostasis.

Two pathways link the brain and the immune system: the autonomic nervous system (ANS) via direct neural influences, and the neuroendocrine humoral outflow via the pituitary. The general immunosuppressive and anti-inflammatory effects of adrenal glucocorticoids, the end-products of the hypothalamic-pituitary-adrenal (HPA) axis, have been known for over 50 years. Evidence accumulated over the last two decades indicates, however, that the sympathetic nervous system (SNS), a major component of the ANS, innervates all lymphoid organs and that catecholamines (CAs), the end products of SNS, modulate several immune parameters. Thus, primary and secondary lymphoid organs are not only extensively hardwired by noradrenergic nerve terminals but also the immune cells, thereby the immune system is tuned by norepinephrine (NE) released locally from nonsynaptic varicosities or circulating epinephrine secreted by the adrenal medulla. Therefore, the SNS provides another major integrative and regulatory pathway between the brain and the immune system.

CAs, similar to glucocorticoids, have been often regarded as immunosuppressive. Recently, however, there has been accumulating evidence that both CAs and glucocorticoids, under physiologic conditions or at levels that can be achieved during stress, influence the immune response in a less monochromatic way. This new understanding helps explain some well known, but often contradictory, effects of the neuroendocrine or stress system on the immunity and on the onset and course of common human pathologic conditions, such as infections, autoimmune/inflammatory, allergic, and neoplastic diseases.

In the present overview, we shall attempt to provide a summary of this evidence and to review current concepts and ideas of how the SNS and the immune system influence each other, with a focus on the roles of the main sympathetic neurotransmitter NE and the main sympathoadrenal hormone epinephrine at the sympathetic-immune interface. Emphasis has been placed on physiological, functional, and pharmacological aspects of the SNS-immune communication.

Neuroscience and immunology developed independently for many years. Thus, the question how the brain communicates with the immune system remained enigmatic until comparatively recently. Evidence that lymphoid organs are innervated dates back to the end of last century when nerves, independent of blood vessels, were found to enter lymph nodes (Tonkoff, 1899). Between 1880 and 1920, J. N. Langley, in conjunction with H. K. Anderson, defined the major functional features of the sympathetic and parasympathetic systems, showing how different effector tissues were affected by segmental ventral root stimulation (cf. Janig and McLachlan, 1992a). In 1898, Otto von Fürth isolated a bioactive compound from animal tissue and called this partly purified product "Suprarenin". Three years later, Takamine and Aldrich independently isolated the responsible component in crystalline form (cf. Benschop et al., 1996). Takamine named the substance, and Aldrich found the correct formula (C₉H₁₃NO₃). Thus, about 100 years ago adrenaline (epinephrine) was the first hormone to be isolated from tissue. During experiments in 1907, a by-product in the synthesis of adrenaline was identified. This substance, which became commercially available as "Arterenol" in 1908, was in fact noradrenaline (norepinephrine), which would formally be discovered and isolated from tissue 40 years later. Since the effects of Arterenol were less pronounced than those achieved by adrenaline, production was cancelled in 1910 (cf. Benschop et al., 1996).

At the end of the last century and at the beginning of this century Ilya Metchnicoff and Paul Ehrlich, respectively, developed the concepts of cellular and humoral immunity (see Paul, 1993), while Sherrington introduced the concept of chemical neurotransmission (cf. Vizi and Labos, 1991). Loeper and Crouzon (1904) were the first to describe a pronounced leukocytosis after subcutaneous injection of the adrenomedullary hormone epinephrine in humans. Ishigami (1919) was probably the first to indicate the role of the stress-immune system interaction in alteration of the infectious process. In 1919, studying subjects suffering from chronic tuberculosis, he observed a decrease in the phagocytic activity of leukocytes during the periods of greatest psychological stress. In the 1920s Metal'nikov and Chorine (1926) showed that immune reactions could be conditioned by classical Pavlovian means. Later, anatomists used silver staining to demonstrate that the thymus gland is innervated (see Kendall and Al-Shawaf, 1991). However, at this time, the thymus was regarded as a rudimentary organ, whose function as a primary lymphoid organ of the immune system would be discovered only 30 years later. In the 1930s, Hans Selye described involution of the thymus in animals exposed to stressors and developed the concept of stress response (Selye, 1936). Also in the 1930s, physiologists like Walter Cannon called this response "fight or flight" reaction and linked the adaptive response to stress with CA secretion and actions. Cannon also emphasized the "generalized" sympathetic response, or the "wisdom of the body" that occurs during stress, contrasting this with more "discrete" functions of parasympathetic pathways (see Chrousos and Gold, 1992; Janig and McLachlan, 1992a). At about the same time, pharmacologists like Loewi and Dale, in pursuing the concept of chemical synaptic transmission, mimicked the responses of peripheral organs to autonomic nerve stimulation by applying substances that they could extract from the same or other peripheral organs.

In the 1940s, von Euler (1946) isolated from a lymphoid organ, the spleen, norepinephrine (NE) and later provided evidence that NE is the major neurotransmitter released from sympathetic nerves. However, in the next two decades the spleen was often considered as a "blood reservoir", and studies concerning the role of sympathetic innervation of the spleen focused on its role in regulation of splenic contraction (of the capsule, in rodents and certain mammals), vascular resistance, and blood flow. This led to the assumption, at this time, that NE-containing nerve fibers in the spleen have no other functions. Interestingly, in the 1950s, Dougherty and Frank (1953) noticed about a 400% increase within 10 min after subcutaneous injection of epinephrine of what they called "stress-lymphocytes". These cells had the morphology of large granular lymphocytes or natural killer (NK) cells, whose function and characteristics were described in the late seventies (see Benschop et al., 1996).

Only in the 1970s and the 1980s, however, due to the pioneering work of Hugo Besedovsky and coworkers, did it become clear that classic hormones and newly described cytokines are involved in functionally relevant cross-talk between the brain and the immune system (Besedovsky et al., 1975, 1979, 1986). They have shown that an immune response induces an increase of plasma corticosteroid levels (Besedovsky et al., 1975, 1981, 1986), alters the activity of hypothalamic noradrenergic neurons (Besedovsky et al., 1983), and drops the content of NE in the spleen (Besedovsky et al., 1979; Del Rey et al., 1982). Also in the 1970s, the first hormone receptor on lymphocytes was described functionally, when it was reported that adrenergic agents modulate lymphocyte proliferation (Hadden et al., 1970). In the 1970s and 1980s, the first comprehensive morphological studies provided evidence that both primary and secondary lymphoid organs are innervated by sympathetic/noradrenergic nerve fibers (see text below). Furthermore, altered immune function has been induced by classical behavioral conditioning (Ader and Cohen, 1982), by stressful stimuli (Keller et al., 1983; Cohen et al., 1991; Chrousos, 1995), or by lesions in specific regions of the brain (Carlson and Felten, 1989b). Finally, evidence was obtained in experimental animals that the susceptibility to autoimmune diseases is modulated by the activity of the stress system (Sternberg et al., 1989a,b; Wilder, 1995) or that stress mediators may exert both pro- and anti-inflammatory effects (Karalis et al., 1991; Chrousos, 1995). Thus, in the last two decades we witnessed an explosive growth of a new interdisciplinary research area that studies the neuroimmune communication, or simply, the physiology and pharmacology of the immune system.

	II. Anatomy and Physiology of the Autonomic Nervous System

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The ANS regulates the function of all innervated tissues and organs throughout the vertebrate body with the exception of skeletal muscle fibers. Thus, it forms the major efferent component of the peripheral nervous system, containing integrative neuronal connections and even complete reflex arcs. The ANS is largely autonomous (independent) in that its activities are not under direct conscious control. The ANS consists of three components: the sympathetic (noradrenergic) and parasympathetic (cholinergic) systems, which originate in the CNS (with cell bodies in the brainstem and spinal cord); and the enteric system, which lies within the wall of the gastrointestinal tract. The most extensive and physiologically most diverse component is the SNS, which sends axons to all parts of the body. The enteric system, which contains a similar number of neurons as the spinal cord (Furness and Costa, 1980), regulates intestinal functions; this system is modulated by projections from the sympathetic and the parasympathetic systems (Vizi et al., 1991).

The sympathetic division originates in nuclei within the brain stem and gives rise to preganglionic efferent fibers that leave the CNS through the thoracic and lumbar spinal nerves ("thoracolumbar system"). Most of the sympathetic preganglionic fibers terminate in ganglia located in the paravertebral chains that lie on either side of the spinal column. The remaining sympathetic ganglia are located in prevertebral ganglia, which lie in front of the vertebrae. From these ganglia, postganglionic sympathetic fibers run to the tissues innervated. Most postganglionic sympathetic fibers release NE; they are noradrenergic fibers; i.e., they act by releasing NE. The adrenal medulla contains chromaffin cells, embryologically and anatomically homologous to the sympathetic ganglia in that they are derived from the neural crest. The adrenal medulla, unlike the postganglionic sympathetic nerve terminals, releases mainly epinephrine, and to a lesser extent NE (the approximate ratio is 4:1); the chromaffin cells of the adrenal medulla are innervated by typical preganglionic sympathetic nerve terminals, whose neurotransmitter is acetylcholine. Thus, the principal end products of the SNS are NE and epinephrine, called CAs (Fig. 1).

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Role of the SNS and the HPA axis in the bi-directional neuro-immune communication; activation of HPA axis and SNS by certain cytokines, such as IL-1, TNF-, and IL-6 (feedback loop from the immune system). Simplified scheme of the sympathetic-immune interface (lower panel): role of different presynaptic receptors in modulation of the release of NE from the sympathetic nerve terminals in lymphoid organs; cotransmission of NE and NPY and their role in regulation of blood flow and lymphocyte traffic, cotransmission of NE and DA; role of different receptors on target immune cells in transmitting the message from CNS. Note that immune cells express mostly -adrenoreceptors, see text for the other type of receptors and conditions that determine their expression. Due to the space restraint and the number of immune cells as targets of the SNS input, different types of lymphoid cells are not depicted, see text for details. Solid lines, neuronal projections; dotted lines, hormonal influences.

CAs are synthesized from tyrosine that is transported into the noradrenergic ending or varicosity by a sodium-dependent carrier. Tyrosine is converted to dihydroxyphenylalanine (DOPA) (the rate-limiting step in the NE synthesis) by the enzyme tyrosine hydroxylase (TH) and finally to dopamine (DA), and a carrier that can be blocked by reserpine transports dopamine into the vesicle. Dopamine is converted to NE in the vesicle by dopamine--hydroxylase (DBH). In the adrenal medulla, NE is further converted to epinephrine. TH- and particularly DBH-immunostaining are often used as specific markers for noradrenergic innervation in various organs.

Living organisms survive by maintaining an immensely complex dynamic equilibrium of the internal milieu or homeostasis, a term coined by Walter Cannon. The systemic sympathetic and adrenomedullary (sympathetic) system (SNS) and the HPA axis are the peripheral limbs of the stress system, whose main function is to maintain both basal and stress-related homeostasis. At rest CAs maintain homeostasis as major regulators of fuel metabolism, heart rate, blood vessel tone, and thermogenesis. When homeostasis is disturbed or threatened by internal or external challenges, both the SNS and HPA axis become activated, resulting in increased peripheral levels of CAs and glucocorticoids that act in concert to keep the steady state of the internal milieu. In the 1930s, Hans Selye defined this reaction as general adaptation syndrome or stress response (Chrousos and Gold, 1992). Any immune challenge that threatens the stability of the internal milieu can be regarded as a stressor; i.e., under certain conditions an immune response can activate the stress system (Fig. 1). In fact, the last 15 years have provided evidence that certain cytokines, and particularly tumor necrosis factor (TNF)-, interleukin (IL)-1, and IL-6 activate both the SNS and the HPA axis (Besedovsky et al., 1986; Chrousos, 1995); (see also Fig. 1, long feedback loop between the immune system and the brain).

Centrally, the two principal components of the general adaptational response are the corticotropin-releasing hormone (CRH) and the locus ceruleus-NE (LC-NE)/autonomic (sympathetic) nervous system (Fig. 1). The CRH system is best characterized in the paraventricular nucleus (PVN) of the hypothalamus. The LC-NE/sympathetic systems are located in the brain stem. Functionally, the CRH and LC/NE/sympathetic systems seem to participate in a positive, reverberatory feedback loop so that activation of one system tends to activate the other as well (Chrousos and Gold, 1992). This includes projections of CRH-secreting neurons from the lateral PVN to the sympathetic systems in the hindbrain, and conversely, projections of catecholaminergic fibers from the LC-NE system, via the ascending noradrenergic bundle, to the PVN in the hypothalamus. Activation of the LC-NE system leads to release of NE from an extraordinarily dense network of neurons throughout the brain, resulting, centrally, in enhanced arousal and vigilance, and peripherally, in increased sympathetic output, i.e., increase of the release of NE from the varicose sympathetic nerve terminals and epinephrine from the adrenal medulla.

	III. Autonomic/Sympathetic Innervation of Lymphoid Organs: Nonsynaptic Communication

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Many organs of the body, such as heart and the gastrointestinal tract, receive both sympathetic (noradrenergic) and parasympathetic (cholinergic) innervation. It is usual, however, for one type of innervation to predominate over the other. Lymphoid organs, similar to blood vessels, receive predominantly sympathetic/noradrenergic and sympathetic/neuropeptide Y (NPY) innervation (cf. Madden et al., 1995). Histofluorescence studies done since the late 1960s have firmly established the presence of noradrenergic nerves fibers in the lymphoid organs of all species studied (Dahlström and Zetterström, 1965; Zetterstrom et al., 1973; Reilly et al., 1976, 1979; Giron et al., 1980; Williams and Felten, 1981; Bulloch and Pomerantz, 1984; Felten et al., 1985; Kendall et al., 1988). More recently, the presence of noradrenergic innervation in lymphoid tissues was confirmed using specific immunohistochemistry for TH and DBH (Felten and Olschowka, 1987; Fink and Weihe, 1988; Weihe et al., 1991; Vizi et al., 1995; Kurz et al., 1997). Most of the current knowledge about the innervation of lymphoid organs is based on studies in rodents; relatively few data are available for humans (for reviews see Felten et al., 1985, 1988; Weihe et al., 1991).

Light microscopic studies using histofluorescence techniques revealed that postganglionic sympathetic nerve fibers enter the thymus along with blood vessels and distribute to the capsular and septal system; these fibers form varicose plexuses in the subcapsular cortex and at the corticomedullary junction; they are extremely sparse in the medulla (Williams and Felten, 1981; Felten et al., 1985; Kendall and Al-Shawaf, 1991; Kranz et al., 1997; Cavallotti et al., 1999). The vast majority of nerve profiles are localized around the vasculature and limited to the cortex. Within the cortex, the densest plexuses are found in the outer cortex, where immature thymocytes reside and develop; the deep cortex, and particularly the corticomedullary junction, an area important for immigration of thymocytes from the thymus, is also richly innervated, mostly along the vasculature. Interestingly, the distribution of mast cells within the thymus parallels the distribution of noradrenergic fibers; the mast cells being usually accumulated in patches immediately adjacent to NE fibers in the perivascular zone (Williams and Felten, 1981). Using specific immunocytochemistry, both TH- (representing the catecholaminergic nature of stained nerve profiles) and DBH-immunostained (demonstrating the noradrenergic feature of stained nerve profiles) nerve fibers (studded with a large number of boutons) are seen in the thymic capsule, subcapsular region, and connective tissue septa (Vizi et al., 1995). The vast majority of immunoreactive nerve profiles are localized around the vasculature. Some TH- and DBH-stained fibers, running along the thymic connective tissue septa, separately from vessels, branch into the cortical parenchyma (Vizi et al., 1995). At the ultrastructural level noradrenergic varicosities are seen in proximity to thymocytes, mast cells, fibroblasts, and eosinophils (Novotny et al., 1990; Vizi et al., 1995). Catecholaminergic nerve fibers also run in close contact to thymic epithelial cells (TEC) that, similar to thymocytes and mast cells, also express -adrenoreceptors (Vizi et al., 1995; Kurz et al., 1997). Since TECs form the blood-thymus barrier in the outer thymic cortex, these cells may be targets for circulating epinephrine from the adrenal gland or NE released from the perivascular nerves.

The splenic nerve contains approximately 98% sympathetic nerve fibers (Klein et al., 1982), and most studies suggest that the innervation of the spleen is predominantly sympathetic. Noradrenergic postganglionic innervation originates mainly in the superior mesenteric/celiac ganglion, and the nerve fibers enter the spleen around the splenic artery, travel with the vasculature in plexuses, and continue along the trabeculae in trabecular plexuses (Williams and Felten, 1981). Fibers from both the vascular and trabecular plexuses enter the white pulp and continue mainly along the central artery and its branches. Noradrenergic varicosities radiate from these plexuses into the periarterial lymphatic sheath. The greatest density of noradrenergic fibers in the spleen is associated with the central artery of the white pulp and associated periarterial lymphatic sheath; dense linear arrays of varicosities extend away from the periarterial plexus and travel into the parenchyma (Williams and Felten, 1981; Felten et al., 1985). Sympathetic nerve fibers are present among cells in the T-dependent area; macrophages and B cells residing in the marginal zone and the marginal sinus, the site of the lymphocyte entry into the spleen, also receive NE innervation (Felten et al., 1985; Felten and Olschowka, 1987). Innervation of the B cell-containing follicles is sparse (Williams and Felten, 1981); the red pulp contains scattered fibers, primarily associated with the plexuses along trabeculae and surrounding tissues.

In lymph nodes, noradrenergic fibers enter at the hilus with the vasculature, and distribute either into a subcapsular nerve plexus or travel with blood vessels through the medullary cords. These fibers run adjacent to both vasculature and lymphatic channels in the medulla and continue with small vessels into the parenchyma of paracortical and cortical regions (Felten et al., 1984; Fink and Weihe, 1988). Within the nodes a large part of the nerves distribute in the medullary and paracortical areas independently of blood vessels (Novotny and Kliche, 1986). Noradrenergic fibers supply paracortical and cortical zones (T cell-rich regions) but are absent from nodular regions and germinal centers, the B cell-containing areas (Felten et al., 1984). In the human palatine tonsils, noradrenergic fibers distribute along the vasculature to form dense perivascular plexuses and single fibers traveling in parafollicular areas. The epithelium and lymphoid nodules are devoid of noradrenergic fibers (Yamashita et al., 1984; Bellinger et al., 1992).

Data on bone marrow innervation are rare compared with that on other lymphoid tissues. Generally nerves enter the marrow accompanying arteries, travel with the vascular plexuses deep in the marrow, arborize in surrounding parenchyma, and end among hemato- and lymphopoietic cells. Most of the nerves supply the arterial component of the marrow's circulation, but there is a substantial innervation of the sinusoidal parts and parenchymal elements where they may influence hematopoiesis and cell migration (Felten et al., 1988). Recently, immunoreactivity for TH, a marker for noradrenergic nerves was found around large vessels, with occasional TH-positive fibers extending into the bone marrow of mice. NPY immunopositive fibers were smaller and less abundant than those for TH but were found in a similar pattern (Tabarowski et al., 1996). Sensory nerves containing substance P (SP) and calcitonin gene-related peptide (CGRP) also innervate the bone marrow. Studies in rodents indicate that the development of the innervation of bone marrow occurs late in fetal life, just before the onset of hemopoietic activity (Calvo and Haas, 1969; Miller and McCuskey, 1973).

Gut-associated lymphoid tissue (GALT) and bronchus-associated lymphoid tissue (BALT) receive both sympathetic and peptidergic innervation (Felten et al., 1985; Weihe et al., 1991). The nerve network within mucosal tissues is very extensive (Cooke, 1986; Bienenstock et al., 1989). It has been calculated that the number of nerve cell bodies present in the gastrointestinal tract is equivalent to that found in the spinal cord (Furness and Costa, 1980). Neuropeptides are found in very large amounts in these tissues, particularly SP, vasoactive intestinal peptide (VIP), and somatostatin (Cooke, 1986). Although this issue is not extensively studied, the available information suggests that in GALT including Peyer's patches that represent clusters of lymphoid nodules in the intestines, and the appendix of the rabbit, varicose noradrenergic fibers arborize profusely in the interdomal region of the lamina propria. Here, fibers follow small vessels and branch freely in the parenchyma among fields of lymphoid cells, usually not in association with blood vessels (Bellinger et al., 1992). Nerves predominate in T cell zones of lymphoid aggregates, where they contain neuropeptides and the sympathetic neurotransmitter NE. Interestingly, as in other lymphoid tissues, intestinal mucosal mast cells lying immediately under the epithelium are apparently selectively associated with enteric nerves, and this is not a random finding (Cooke, 1986). In these tissues, the noradrenergic varicosities are also adjacent to serotonergic enterochromaffin cells (Felten et al., 1985). In addition, the nasal mucosa receives tonic discharges from the sympathetic nerves but not from the parasympathetic nerves. Although recent evidence suggests that sympathetic nerve stimulation may up-regulate immunoglobulin (Ig)A secretion in the submandibular glands (Carpenter et al., 1998), as a whole there is almost complete lack of information of how the sympathetic innervation and endogenous CAs may affect mucosal immunity. Interestingly, mucosal immune responses tend to bias toward T helper (Th) 2 responses (Ernst et al., 1999). Since CAs and the mast-cell product histamine mediate a Th2 shift (Elenkov et al., 1996, 1998; Wilder and Elenkov, 1999) (see Section X.) an interesting hypothesis to pursue is whether locally released biogenic amines might contribute to the dominance of the Th2 responses observed in these tissues.

Double immunofluorescence reveals the coexistence of DBH-(noradrenergic) and NPY-like immunoreactivity in sympathetic nerve fibers of lymphoid organs (Lundberg et al., 1988). Klein et al. (1982) have demonstrated that NE is also co-stored with opioid peptides in the population of large dense-cored noradrenergic vesicles of the spleen, although no evidence, so far, is available of co-release of opioids and NE from the sympathetic nerve fibers of lymphoid organs.

Sympathetic/noradrenergic and sympathetic/NPY postganglionic nerve fibers innervate both the smooth muscle of the vasculature and the parenchyma of specific compartments of primary and secondary lymphoid organs (Felten et al., 1985). Both noradrenergic and NPY nerve fibers and their varicosites do travel in plexuses that run adjacent to smooth muscle cells of the blood vessels in lymphoid organs; it is therefore possible that both NE and NPY, released from these fibers, play a role in controlling blood flow to these organs and may influence lymphocyte traffic (Fig. 1). However, noradrenergic fibers also travel with smaller vessels that are devoid of smooth muscle cells. In addition, some noradrenergic fibers are present in the parenchyma of lymphoid organ tissue that are not associated with blood vessels (Felten et al., 1985; Vizi et al., 1995). Thus, NE released from perivascular or parenchymal nerve fibers may affect lymphoid cells and exert an immunomodulatory role. Noradrenergic innervation of lymphoid tissue appears to be regional and specific; generally, zones of T cells, macrophages, and plasma cells are richly innervated, while nodular and follicular zones of developing or maturing B cells are poorly innervated (Felten et al., 1985). The main target cells of the noradrenergic innervation appear to be immature and mature thymocytes, TEC, T lymphocytes, macrophages, mast cells (Blennerhassett and Bienestock, 1998), plasma cells, and enterochromaffin cells. Noradrenergic nerve fibers, particularly in the thymus, are closely associated with mast cells in both perivascular and parenchymal zones, suggesting a possible humoral role for NE and histamine in the development of T cells in the thymus. Noradrenergic innervation is present early in the development, and their arrival generally precedes the development of the cellular compartment of the immune system suggesting a role for NE in maturation of the immune system (see Section VII.).

In addition to the autonomic/sympathetic innervation, all lymphoid organs also receive sensory peptidergic innervation that is confined mostly to the parenchyma (Weihe et al., 1991). The most abundant peptides are tachykinins (substance P, neurokinin A), CGRP, and vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI). Double immunofluorescence reveals coexistence of tachykinins with CGRP and of TH and NPY. The coexistence of peptides with peptides and of NPY with markers of the catecholamine pathway conforms to the general scheme described for the peripheral innervation of other organs. Similar to other organs, the tachykinins/CGRP fibers most likely have sensory origins. As a general pattern, and as in the case of noradrenergic innervation, a close spatial relationship between peptidergic nerve fibers and mast cells, T cells and macrophages is observed (Weihe et al., 1991). Peptidergic nerves also appear to be sparse in pure B cell regions. Neuromast cell contacts are relatively often seen in all lymphoid organs, with the exception of the spleen (Weihe et al., 1991). Mast cells bear receptors for SP that, after stimulation, trigger release of histamine and other factors such as leukotrienes; NE, however, through stimulation, respectively of - and ₂-adrenoreceptors is known to stimulate and inhibit, respectively, the release of histamine from mast cells (Kaliner et al., 1972; Tomita et al., 1974). Thus, apart from their direct immunomodulatory effects, NE released form postganglionic noradrenergic nerve terminals or SP antedromically released from sensory nerves may exert an important immunomodulatory role, indirectly, via modulation of histamine release from mast cells in the parenchyma of lymphoid organs.

Neuromast cell connections and neuromacrophage connections, as well as neuro-T cell contacts, are not restricted to the preformed lymphoid organs and tissues, but are also regularly encountered in virtually all somatic and visceral tissues (Weihe et al., 1991). T cells, macrophages, and mast cells are regularly seen in any peripheral nerve and in both sympathetic and sensory ganglia. In the skin postcapillary venules, macrophages, mast cells, and peptidergic nerves, stained for tachykinins and CGRP, form a typical quadruplet, whereas in the outer wall of larger blood vessels, TH/NPY fibers also join in to form neuromast cell and neuromacrophage interrelations. Furthermore, close interrelations but no coincidence of TH/NPY-immunoreactivity and of SP/CGRP fibers are frequently observed in perivascular regions (Weihe et al., 1991).

	IV. Nonsynaptic Release of Norepinephrine in Lymphoid Organs: Presynaptic Modulation and Effect of Drugs

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Whereas endocrine signaling depends on blood-borne access of hormones to immune cells, neurotransmitter signaling depends on local availability of the specific neurotransmitter from neural release. Neural release is achieved when the propagated electrical signal reaching the axon terminal triggers a depolarization that causes the release of neurotransmitter, provided that [Ca²⁺]_o is available (Fig. 1). Recently, several studies using blood-perfused pig spleen in vivo, or medium-perfused Atlantic cod and rat spleen in vitro, demonstrated that the sympathetic nerve terminals in the spleen are able to store, take up, and, subsequently, release NE in response to field stimulation (Lundberg et al., 1989b; Ehrenstrom and Ungell, 1990; Elenkov and Vizi, 1991). Using an in vivo microdialysis technique NE levels can be monitored in the spleen of conscious rats (Shimizu et al., 1994). Splenic content of NE has been depleted up to 95% (depending on the species and the strain) by the noradrenergic neurotoxin 6-hydroxydopamine (6-OHDA), indicating that most splenic NE is of neuronal origin (cf. (Felten and Olschowka, 1987). More recently we have demonstrated that the rat thymus takes up and releases substantial amounts of NE (Hasko et al., 1995b; Vizi et al., 1995). The release of NE in lymphoid organs, both in vitro and in vivo, is [Ca²⁺]_o- and frequency-dependent and a tetrodotoxin-sensitive process, thus indicating its neuronal origin. In addition, the inhibition of vesicular storage of CAs by reserpine results in blockade of the release of NE evoked by field stimulation (Vizi et al., 1995), suggesting that the release of NE in the thymus is of vesicular origin.

The existence of DA receptors on several cell lines of the immune system, including those found in the spleen, has been shown (Santambrogio et al., 1993), and functional studies have suggested that DA receptors on immunocytes of the spleen may have a role in the regulation of immunocompetence (Won et al., 1995). The possible source of immunoregulatory DA in the spleen has been found (Bencsics et al., 1997b). In view of the lack of dopaminergic innervation, plasma DA (Van Loon, 1983) or DOPA (Kvetnansky et al., 1992) could be a candidate (see Fig. 1).

It was shown (Bencsics et al., 1997b) that the noradrenergic axon terminals in the spleen are able to take up DA from the circulation, convert it in part into NE, and release it as both DA and NE in response to neural activity. The ratio of [³H]DA and [³H]NE in the spleen loaded with [³H]DA was found to be dependent on both temperature and time of loading, and could be modulated by various drugs such as desmethylimipramine, a NE uptake blocker, and disulfiram or fusaric acid, dopamine -hydroxylase inhibitors. This phenomenon may reveal a new mechanism by which immunocytes in the spleen can be regulated by the neuroendocrine system. Therefore, it is suggested that under physiological conditions, the source of DA being taken up by noradrenergic terminals can be circulating DA, especially during stress, since exposure of an organism to any of a variety of stressors that increase sympathetic tone is accompanied with an increase in plasma concentrations of DA (Van Loon, 1983).

Electrical stimulation of the splenic nerves induces frequency-dependent output of NPY-like immunoreactivity and NE, suggesting co-release of these neurotransmitters (Lundberg et al., 1989c) (Fig. 1). As in other organs, the classical neurotransmitter NE is preferentially released from small storage vesicles at low frequency, whereas at higher frequency, an increased proportion of large dense-cored vesicles release both NPY and NE (Lundberg et al., 1989c). Thus, during resting conditions little NPY is released, whereas in situations of high sympathetic activity, the contribution of NPY becomes more important. Although in many tissues, ATP is co-stored with NE and NPY in the large dense-cored vesicles of sympathetic nerve terminals, so far, no evidence is available to support release or co-release of ATP with NE from these terminals in lymphoid organs.

Since Sherrington's classic work in 1906 it has become a doctrine of neurophysiology that the synapse, a part of the surface of separation between neurons, is the primary site of neuronal information processing (Tansey, 1997). Thus, the chemical substances are released by depolarization from the axon terminals across the synaptic cleft (about 15-100 nm) and act on the postsynaptic membrane equipped with receptors. Now, however, it is clear that, in contrast to some regions of the CNS and particularly to the neuromuscular junction, where classical synapses are observed, postganglionic neurons in the periphery innervating blood vessels, vas deferens, and smooth muscle terminate in a network of varicose areas (boutons en passant) that lack synaptic contact with their target cells (cf. Vizi 1984a,b, 2000; Vizi and Labos, 1991). Similarly, in Auerbach' plexus of the gut, or the cerebral cortex where noradrenergic axon terminals do not make synaptic contacts with cholinergic axon terminals, NE should diffuse over relatively long distances before modulating the release of acetylcholine from the cholinergic neurons. It has been shown (cf. Vizi and Kiss, 1998; Vizi, 2000) that in the CNS the monoamines (NE, dopamine, serotonin) released from nonsynaptic varicosities into the extracellular space, diffusing far away from the release sites, make functional interactions with other neurons without making synaptic contacts. NE exerts its effect through nonsynaptic, high-affinity ₂-ARs (Vizi, 2000). Thus, many of the neurotransmitters, especially the monoamines and peptides, show a release profile that is halfway between specific synaptic neurotransmission and relatively nonspecific endocrine secretion. This release profile is referred to as "nonsynaptic" (Vizi, 1979, 1980, 1984, 1991, 2000; Vizi and Labos, 1991; Vizi and Lendvai, 1999).

Nonsynaptically, the neurotransmitter is released from free nerve endings into a large extraneuronal space, with no postjunctional specializations, and hence, the neurotransmitter diffuses a considerable distance (sometimes this could be more than 1 µm) before interacting with its receptors on target cells (Vizi, 1980, 1984a,b, 2000; Vizi and Labos, 1991; Vizi and Kiss, 1998). Conversely, in the case of classical synaptic neurotransmission, e.g., at the neuromuscular junction, the distance between release sites and the postsynaptic receptor is much shorter. The nonsynaptic neurotransmission is relatively slow and tonic, while the synaptic neurotransmission is short and phasic (Table 1).

This slow interaction should be distinguished from the very slow, hormone-mediated humoral control. In the time domain from 100 msec to several minutes such "slow" tuning effects seem very conceivable; moreover, they would appear to be useful in controlling autonomic functions and the balance between the sympathetic and parasympathetic nervous system.

Although some occasional "synaptic-like" contacts have been described in the spleen (Felten and Olschowka, 1987), most of the ultrastructural studies reveal that noradrenergic fibers in lymphoid organs are confined to the connective tissue septa and do not make "classical" synaptic contacts with target cells. Thus, for example in the thymus, some of these connective tissue septa are extremely fine, sometimes 1 µm or less. They invest thymic "microlobules" and, thus, penetrate far into the parenchyma (Novotny and Kliche, 1986; Novotny et al., 1990; Vizi et al., 1995). These septa are clearly delineated in the juvenile animals, but indistinct in the aged rats, thus creating sometimes the "spurious impression" using light microscopy that parenchymal cells receive direct innervation (Novotny et al., 1990). In fact, ultrastructural studies failed to observe classical synapses between thymocytes and neuronal elements in the rat thymus (Novotny and Kliche, 1986; Novotny et al., 1990; Saito, 1991; Vizi et al., 1995). This is substantiated by the observation that noradrenergic nerve fibers, along the thymic tissue septa, branch into the parenchyma and some of the septal NE nerve terminals are about 200 nm from the surface of thymocytes (Vizi et al., 1995).

Similarly, in the spleen, a recent ultrastructural study reveals that the innervation is confined to the connective tissue system, which includes the capsulo-trabecular, peri-vascular and reticular systems. All components of the connective tissue system are continuous with each other, and the nervous elements appearing in the reticular system are the elongated ones from other connective tissue systems, especially peri-vascular connective tissue. It has been proposed that the minute connective tissue space of the reticular system serves as a NE canal. NE is released from the noradrenergic nerve varicosities in this tissue, diffuses, and is temporary stored in this enclosed space (Saito, 1991). The reticular system in the spleen divides the parenchyma into small nonendothelial vascular spaces with its own meshwork, and free mobile immunocytes stagnate in these spaces. This stagnation of the mobile immunocytes and the presence of the noradrenergic nerves in the NE canals provide an opportunity for the immunocytes and nerves to meet each other (Saito, 1991).

The nerve-target interactions in lymphoid organs have not been studied in detail. However, as in general, in the periphery, it appears that in lymphoid organs NE is also released nonsynaptically, i.e., from varicose axon terminals, which do not make synaptic contacts. Moreover, NE released from perivascular or connective tissue septa plexuses of nerve terminals diffuse away through surrounding adventitia or collagenous fibrils, in a paracrine fashion. Thus, adrenoreceptors on immune cells are targets of remote control, and, thus, NE, may play a modulatory role in signal transmission at the sympathetic-immune interface (Vizi et al., 1995). Similar nonsynaptic transmission may operate in the blood vessel wall (between varicose nerve terminals and smooth muscle cells) of these organs, where NE and NPY might be involved, as already mentioned, in regulation of blood flow and lymphocyte traffic. This is substantiated by a recent ultrastructural observation that the distance between a naked axon and a smooth muscle cell of arterioles and muscular venules in the lymph nodes ranges from 100 to 800 nm (Villaro et al., 1987).

The release of transmitters from varicose axon terminals in response to action potentials is a very random process subject to presynaptic modulation through stimulation of receptors located on the varicose axon terminals (Vizi, 1979; Starke, 1981; Stjärne et al., 1990; Vizi et al., 1991; Vizi and Labos, 1991). These presynaptic receptors can be classified as auto-, homo-, and heteroreceptors depending on the origin of the transmitters and localization of the receptors. Autoreceptors receive messages by transmitter released from the same neuron. They are involved in negative or positive feedback modulation. Homoreceptors receive signals from the adjacent neuron, whose transmitter is the same as the neuron where the homoreceptors are located. The homoreceptors can also serve as autoreceptors. Heteroreceptors are located on axon terminals that do not manufacture transmitters capable of exerting an effect on these receptors. These receptors receive messages by transmitters from other neurons.

Whereas autoreceptors play a role in keeping the release constant, heteroreceptors are involved in interneuronal communication, in cross-talk between neurons at the presynaptic level. Since none of the transmitter/modulator substances tested have any receptor-mediated effect on the propagation of action potentials in nerve trunks, the site of their action is on the axon terminals. The activation of presynaptic receptors located on the axon terminals may interfere with processes involved in the transmitter release. The significance of the presynaptic modulation is that the input to the postsynaptic cell can be selectively modulated without changing the excitability of the effector cell as a whole (Vizi et al., 1991). In many tissues, the noradrenergic nerve terminals are equipped with auto-, homo-, and heteroreceptors sensitive to different endogenous and exogenous ligands or drugs (Table 2).

Both in the periphery and some regions of the CNS, NE released from noradrenergic axon terminals reduces its own release evoked by the subsequent stimuli (negative-feedback modulation) through stimulation of presynaptic ₂-adrenoreceptors (cf. Vizi, 1979; cf. Starke, 1981). ₂-ARs have been divided into three subtypes (_2A,_2B, and _2C) on the basis of pharmacological and molecular cloning evidence (Bylund et al., 1994; Kable et al., 2000). The negative feedback phenomenon also operates in lymphoid organs. We have demonstrated that both in the rat spleen and thymus, in an in vitro perfusion system, the application of highly selective ₂-AR antagonists and prazosin, an _2C-subtype selective AR antagonist (cf. Docherty, 1998) results in a significant increase in stimulation-evoked release of NE (Elenkov and Vizi, 1991; Vizi et al., 1995; Hasko et al., 1995b). The release is enhanced because it escapes from the negative feedback modulation exerted by NE itself. Vice versa, when a selective ₂-AR agonist is applied, the release of NE is inhibited (Elenkov and Vizi, 1991). This indicates that the noradrenergic axon terminals in the rat spleen and thymus are equipped with presynaptic _2C-ARs (Table 3) and that the release of NE from these noradrenergic nerve terminals is under tonic inhibition by the endogenously released NE.

View this table: [in this window] [in a new window]   TABLE 3 Subtype of 2-adrenoreceptor responsible for the negative feedback modulation of NE release in the periphery and the central nervous system

The noradrenergic nerve terminals of the spleen and the thymus are also equipped with presynaptic M-muscarinic-, N-nicotinic-, P₁-purinergic (A1) and PGE₂-prostaglandin E₂ heteroreceptors (Elenkov and Vizi, 1991; Hasko et al., 1995b). Stimulation of presynaptic M-muscarinic (by acetylcholine or selective drugs), P₁-(A1, adenosine-sensitive), and PGE₂ receptors reduces the release of NE, whereas the stimulation of presynaptic N-nicotinic (acetylcholine, nicotine) receptors releases NE from the varicosity (cf. Wonnacott, 1998; Vizi and Lendvai, 1999) and increases the stimulation-evoked release of NE, i.e., NE released in response to neuronal firing. Additional evidence indicates that in the spleen opioid receptors (the subtype has not been clarified), NPY2, ₂-ARs, and D2 receptors (Gaddis and Dixon, 1982; Lundberg et al., 1989a; Sato et al., 1992; Bencsics et al., 1997b) can also participate in the modulatation of the release of NE (Table 2). Through these receptors the release of NE (i.e., the message from the CNS to these lymphoid organs) can be modulated by endogenous ligands (e.g., NE, epinephrine, dopamine, NPY, opioids, prostaglandin E2, adenosine, etc.) or by drugs present in the circulation (Fig. 1).

NPY is a 36-amino acid peptide that acts as a neurotransmitter and neuromodulator in the CNS and the peripheral nervous system. NPY-positive nerve fibers are present in all lymphoid organs (Lundberg et al., 1988; Weihe et al., 1989; Weihe et al., 1991). These fibers predominantly supply the vasculature, where they mainly occur as perivascular plexuses and both NE and NPY, released from these fibers, control blood flow and may affect lymphocyte traffic. They branch off only rarely to run into the lymphoid parenchyma (Felten et al., 1985). Evidence was obtained that both NE and NPY are released together (Lundberg et al., 1989c) (Fig. 1). A few reports suggest that lymphoid cells might also express NPY receptors. Thus, in vitro, NPY suppresses human NK cell activity (Nair et al., 1993), whereas low levels of mRNA for NPY-Y1 was found in rat splenic lymphocytes (Petitto et al., 1994). Thus, overall, the precise expression and function of -ARs, DA, and NPY receptors on different lymphocyte/leukocyte subpopulations await further studies.

	V. Systemic and Local Effects of Cytokines on Sympathetic Nervous System Activity

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In the 1970s Besedovsky and coworkers provided the first evidence that the immune system and its products can signal the CNS. Thus, immunization with sheep red blood cells (SRBC), administration of lymphocyte-conditioned medium or IL-1 in animals, induced an increase of plasma corticosteroid levels, altered the activity of hypothalamic noradrenergic neurons, and dropped the content of NE in the spleen (Besedovsky et al., 1979, 1983, 1986; Del Rey et al., 1982). Later, it become clear that during an immune response certain cytokines, such as IL-1, IL-6, and TNF- can signal the brain, which through a complex CRH-dependent pathway, triggers activation of both the SNS and the HPA axis (Berkenbosch et al., 1987; Sapolsky et al. 1987; Dunn, 1988; Elenkov et al., 1992b; Kovacs and Elenkov, 1995). Thus, administration of IL-1 in the periphery increases the turnover of NE in the hypothalamus (Dunn, 1988, 1998; Zhang et al., 1998; Dunn et al., 1999) and raises peripheral plasma (Berkenbosch et al., 1989) and CNS (Dunn et al., 1999) NE metabolism and extracellular levels; intracerebroventricular (i.c.v.) and peripheral injection of interferon (IFN)- or IL-1 produces a long-lasting increase of the sympathetic activity of the splenic nerve and an increased turnover of NE in the spleen (Katafuchi et al., 1991); as a result, the release of NE in the spleen is enhanced, as indicated by a recent in vivo microdialysis study (Shimizu et al., 1994). It has been shown (Zhang et al., 1998) that i.c.v. infusion of CRF increases extracellular concentration of NE in the hippocampus and cortex as determined by in vivo voltammetry. Thus, the SNS, similar to HPA axis (Besedovsky et al., 1986), is involved in a long feedback loop between lymphoid organs and CNS. The afferent limb of this loop seems to operate by blood-borne cytokines that, via circulation or through the afferents of the vagus nerve (Maier et al., 1998), activate the central components of the stress system. The efferent loop consists of the SNS, its projections to lymphoid organs and the release of NE from the sympathetic nerve terminals in these organs. This feedback loop seems to be a regulatory one, since i.c.v. infusion of IL-1 or IFN- rapidly decreases peripheral and splenic NK cell activity and suppresses the mitogen response and the production of IL-1 and IL-2 of splenic cells (Sundar et al., 1989; Brown et al., 1991). These effects depend upon intact splenic sympathetic innervation (Sundar et al., 1990; Brown et al., 1991), whereas direct splenic nerve stimulation results in reduced NK activity (Katafuchi et al., 1993).

Distinct functional pathways exist within the ANS and SNS, i.e., ANS consists of a set of subdivisions, innervating different effectors, each of which is controlled by specific reflex mechanisms related to the function of the effector. This has been firmly established for the lumbar sympathetic nervous system to skin, skeletal muscle and viscera, and for the thoracic sympathetic outflow to the head and for several parasympathetic systems (Janig and McLachlan, 1992a,b).

The function-specific unit(s) that are involved in the activation of the SNS during an immune response, their properties and behavior, are not fully understood. However, the above-mentioned observations suggest that the inflammatory/immune response may actually activate different pathways of SNS, as compared with other stressors or stimuli. This is substantiated by the observation that the content of NE in the rat spleen drops during the peak of the immune response to immunization with SRBC; however, the content of NE in the heart remains unchanged (Besedovsky et al., 1979). Similarly, an intravenous administration of IL-1 results in a dose-dependent long-lasting increase of the sympathetic activity of the splenic and adrenal nerves; however, the activity of renal nerves shows only a transient increase, which is followed by a long-lasting suppression (Niijima et al. 1991; Elenkov, 1993). Endotoxin impedes vasoconstriction in the spleen (Rogausch et al., 1997). A recent study by Terao et al. (1994) demonstrated that intraperitoneal or i.c.v. injection of IL-1 accelerated NE turnover in the spleen, lung, diaphragm, and pancreas without appreciable effects in other organs examined. IL-6, however, did not affect NE turnover in every organ examined, in contrast to its substantial effect on plasma corticosterone levels. Thus, it appears that each immune response, similar to different stressors (see Pacak et al., 1998), may have its own specific central neurochemical and peripheral neuroendocrine "signature".

The above-mentioned data suggest that systematically administered TNF- and IL-1 trigger centrally the sympathetic output that results in an increase of NE turnover in several lymphoid and nonlymphoid organs in the periphery. In apparent contrast, the local effect of these cytokines might be different. Thus, we have shown that TNF- inhibits the stimulation-evoked release of NE from isolated rat median eminence (ME) (Elenkov et al., 1992a). The ME is a hypothalamic structure not protected by blood-brain barrier; here neurosecretory projections, such as CRH from the PVN, terminate and control hormone secretion from the anterior pituitary. Noradrenergic varicosities in the ME are not equipped with ₂-presynaptic-ARs (Vizi et al., 1985). However, this hypothalamic structure expresses high-density ₂-ARs that are exclusively located on the axon terminals of the hormone-containing neurons (Plotsky et al., 1989). Because NE released in this region might exert tonic inhibitory control on hormone release through stimulation of ₂-ARs (Vizi et al., 1985; Plotsky et al., 1989), it was suggested that TNF-, by inhibiting NE release (i.e., by disinhibition of this control), might trigger an increase of CRH release and subsequently an increase of ACTH from the anterior pituitary (Elenkov et al., 1992a). Recently, evidence was obtained that TNF- was also able to inhibit the release of NE in rat hippocampus (Ignatowski and Spengler, 1994; Ignatowski et al., 1997). The regulation of NE release in this structure by TNF- appears to be associated with an alteration of ₂-ARs responsiveness. Administration of the antidepressant desipramine to rats for 2 weeks transformed the presynaptic TNF- response. It was suggested that this mechanism might play a role in the delayed clinical effect of this drug (Ignatowski and Spengler, 1994). In contrast, local administration of IL-1 by intracerebral microdialysis technique in rats resulted in an elevation of NE concentration in the medial prefrontal cortex (Kamikawa et al., 1998). Furthermore, evidence was obtained in this study indicating that IL-1 induces a rise in NE levels by activation of the glutamatergic system and the glutamate-induced increases in prostanoids and nitric oxide (NO).

In the periphery, Hurst and Collins (1993, 1994) reported that both IL-1 and TNF- inhibited the release of NE from longitudinal muscle-myenteric plexus preparations of rat jejunum. Interestingly, both IL-1 and TNF- also inhibited the stimulation-evoked (i.e., neural) release of NE from superfused isolated atria from humans and mice; the effect of IL-1 was suggested to be mediated through formation of prostaglandins (Foucart and Abadie, 1996; Abadie et al., 1997). These effects might have important clinical implications. It appears that the heart is a TNF--producing organ; both myocardial macrophages and cardiac myocytes themselves synthesize TNF-. Accumulating evidence indicates that myocardial TNF- is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion injury, sepsis, chronic heart failure, viral myocarditis, and cardiac allograft rejection (Meldrum, 1998). Thus, the effect of TNF- on NE release in the heart might interfere with these pathologic conditions and represents a realistic goal for clinical medicine.

	VI. Expression of Adrenoreceptors on Lymphoid Cells: Signal Transduction

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