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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NE and epinephrine mediate their effects on target cells via stimulation of two principal receptors: alpha () and beta () adrenergic receptors (ARs). -ARs are now subdivided into ₁, ₂, and ₃ subtypes, whereas -ARs are subdivided into two types, ₁ and ₂, each of which are now known to comprise additional subtypes. Virtually all lymphoid cells express -ARs, with the exception of T helper (h) 2 cells (see below). Several studies, using human isolated peripheral mononuclear cells suggest that the -ARs numbers, differ between different types of lymphoid cells. The precise ordering of -AR density among T cells, B cells, and monocytes is somewhat inconsistent. One study shows that the specific order of receptor density is NK cells > CD14⁺ monocytes > T_cytotoxic (Tc) ~ B cells > Th cells (Maisel et al., 1990a), whereas another study demonstrates slightly different order NK cells > Tc ~ B cells ~ monocytes > Th cells (Maisel et al., 1989). Lymphocyte and NK cell -ARs belong to the ₂-AR subtype, and the receptor number ranges from about 4000 receptors/cell for B cells and NK cells, approximately 1800 receptors for Tc, to between 200 and 750 binding sites for Th cells (Khan et al., 1986). The discrepancy in receptor number in the above studies is most likely atttributed to the small number of subjects employed in some of them. When a larger group of 28 subjects of either sex was studied, Maisel et al. (1989) confirmed that lymphocyte subsets differ in their ₂-AR density, with NK cells having the greatest and helper T cells having the lowest number of receptors; Tc, B cells, and monocytes had an intermediate number of receptors. Importantly, a recent study in mice demonstrates ₂-AR expression on Th1 cells, but not on Th2 cells (Sanders et al., 1997). This phenomenon may provide a mechanistic basis for differential modulation of Th1 and Th2 functions by CAs, discussed below. Other lymphoid cells also express ₂-ARs, including thymocytes, TEC, neutrophils, basophils, and eosinophils (Plaut, 1987; Yukawa et al., 1990; Kurz et al., 1997). The number of ₂-ARs appears to vary during the life span (stem cell to mature activated immunocyte). For example, in mice, immature thymocytes probably have a lower number of ₂-ARs than more mature T cells (Radojcic et al., 1991).

The coupling between ₂-AR and adenylate cyclase may also differ in various lymphocyte subsets; in other words, high ₂-AR density does not necessarily mean high cAMP response after stimulation of these receptors. Thus, some subsets, such as B cells may have a high prevalence of low affinity receptors, which are weakly coupled to the adenylate cyclase. In contrast, NK cells, Tc, and monocytes possess a substantial number of ARs, probably in a high-affinity state that are very responsive to -AR stimulation with regard to cAMP response. In fact, two recent studies demonstrate that NK cells, Tc, and monocytes are very responsive to -AR stimulation, with regard to cAMP accumulation; however Th and B cells showed only a modest response (Maisel et al., 1989; Knudsen et al., 1995).

The presence of ₂-ARs on peripheral blood mononuclear cells (PBMC) that contain mainly lymphocytes and monocytes, is controversial. Since platelets express ₂-ARs, and mononuclear preparations often contain platelets, the binding of specific ₂-ligands might be due to contamination with platelets. Thus, no ₁- and ₂-ARs could be identified on platelet-depleted mononuclear cells (Casale and Kaliner, 1984), in contrast to preliminary reports showing the presence of ₂-ARs on PBMC. However, some functional studies implicate both ₁- and ₂-ARs in modulation of some immune parameters (see below). Thus, alternatively, -ARs probably are not expressed under normal conditions on PBMC (lymphocytes, monocytes); however, they may be expressed in certain lymphoid compartments, such as alveolar and peritoneal macrophages, or hematopoietic cells (see below), or under certain pathologic conditions. One such condition might be polyarticular juvenile rheumatoid arthritis (JRA). Thus, PBMC from patients with polyarticular JRA, in contrast to healthy volunteers, respond to ₁ receptor stimulation with an increased production of IL-6 (Heijnen et al., 1996).

In recent years the expression of dopamine D3, D4, and D5 receptors was demonstrated using molecular biological techniques (Takahashi et al., 1992; Nagai et al., 1993, 1996; Bondy et al., 1996). Also, recently radioligand binding studies provided additional evidence that peripheral human blood lymphocytes might express D3, D4, and D5 receptors (Ricci and Amenta, 1994; Ricci et al., 1995, 1997, 1998). Although DA receptor agonists have been reported to modulate some immune parameters, such as antibody response, T cell proliferation, and cytokine production (Pierpaoli and Maestroni, 1978; Hasko et al., 1996a), the functional role of DA in immunomodulation remains poorly understood. Nevertheless, it was found that DA taken up from the circulation by noradrenergic varicosities is released from there and may influence the cytokine production (Bencsics et al., 1997b). Furthermore, the possibility remains that some of the in vivo effects of DA might be mediated through its inhibition of the release of anterior pituitary hormones, which, alternatively, may affect immune functions.

Heterotrimeric G-proteins regulate the transduction of transmembrane signals from cell surface receptors to variety of intracellular effectors, such as adenylate cyclase (AC) and phospholipase C (PLC). G-proteins consist of three distinct classes of subunits, , , and ; in general the effector specificity is conferred by the -subunit. At least seventeen different -subunit genes have been identified in mammals and these have been divided into four major subfamilies: G_s, G_i, G_q, and G_12. The G_s family contains G_s and G_olf; these -subunits stimulate AC. The G_i family contains G_i, G_o, and G_z subunits, which can inhibit AC and modulate potassium and calcium channels. In addition, -subunits of the complex of G_i proteins have been shown to regulate -isoforms of PLC. It is important that the -subunits of the G_q subfamily (G_q, G₁₁, G₁₄, G₁₅, and G₁₆) have also been shown to activate the -isoforms of PLC. The role of G₁₂ subfamily is less well characterized (cf. Wu et al., 1995; Grant et al., 1997).

1. Cyclic Adenosine 5'-Monophosphate. NE and epinephrine transduce their biological information through stimulation of ARs. ARs directly activate G-proteins that stimulate enzymes, such as AC and PLC to induce the production of second messengers, such as cyclic adenosine 5'-monophosphate (cAMP) or intracellular inositol 1,4,5-triphospate (IP₃), diacylglycerol (DAG), and Ca²⁺, respectively. In general, the -ARs couple to G_s protein to activate AC that increases intracellular cAMP; the ₂-ARs couple to the G_i proteins to inhibit AC and subsequently the formation of cAMP, and the ₁-ARs couple to the G subunits of the G_q class to activate PLC that increases IP₃ and DAG. Subsequent to the generation of these second messengers, cAMP activates protein kinase A (PKA), whereas DAG activates protein kinase C (PKC), and IP₃ mobilizes Ca²⁺ from intracellular stores. Ca²⁺ is further linked to the Ca²⁺/calmodulin (Ca²⁺/CaM) pathway.

Recent studies indicate that the selectivity in the interactions of the ARs with different G-proteins is rather promiscuous in certain systems. Thus, recent evidence indicates that G₁₅ and G₁₆ are expressed only in certain hematopoietic cells with particularly high expression in pre-B cell lines (Grant et al., 1997

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2. Intracellular Ca²⁺. It has been suggested that [Ca²⁺]_i plays an important role in the pathophysiology of endotoxemia and sepsis (Song et al., 1993; Hotchkiss et al., 1995). LPS has been shown to increase [Ca²⁺]_i in a variety of cells including macrophages (cf. Haskó et al., 1998e). Calcium channel blockers (verapamil, diltiazem) decrease the production of proinflammatory TNF- (Hotchkiss et al., 1995; Szabó et al., 1997a) and IL-1 (Hotchkiss et al., 1995) but increases that of IL-10 (Szabó et al., 1997a). Dantrolene, a drug that inhibits the release of intracellular Ca²⁺ from its cytoplasmic stores suppresses plasma and tissue concentration of TNF- (Haskó et al., 1998e), IL-1, and IL-1 (Hotchkiss et al., 1995), increase plasma level of IL-10 and inhibits the production of IL-12 and IFN- in endotoxemic mice (Németh et al., 1998) and reduces [Ca²⁺]_i in macrophages (Haskó et al., 1998e). In this study, it was also shown that the effect of dantrolene to inhibit IL-12 production is independent of the presence of IL-10 in the plasma. In IL-10-deficient mice (C57BL/6), the IL-12 and IFN- responses to LPS were more than 70 and 3 times higher (Németh et al., 1998), but dantrolene was still effective to inhibit their production without affecting the nuclear translocation of NF-B (Haskó et al., 1998). Dantrolene is also able to prevent NO production by LPS (Haskó et al., 1998).

	VII. Role of Sympathetic Innervation in Immune System Development and Hematopoiesis

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Clear distinction should be made between the role of SNS in immune system development and the effect of CAs on immune responsiveness (described under Section X.). At days 1 to 3 postnatally a few scattered fibers are present in the capsule and the septal system of the rat thymus and along the hilar vessels of the spleen. There is progressive growth into the parenchyma of these organs by postnatal day 7 when noradrenergic fibers begin to form a loose network. By postnatal day 14 in the rat the noradrenergic fibers increase in density, displaying both vascular and parenchymal patterns of innervation comparable with that seen in young adult thymus and spleen (Ackerman et al., 1987; Bellinger et al., 1987, 1988, 1992). At this age, NE concentration in the spleen also reaches adult levels (Ackerman et al., 1987).

With normal aging the thymus progressively degenerates and becomes infiltrated with adipose tissue. Noradrenergic innervation over the course of thymic involution does not decline, but rather persists, even though one of its presumed targets, thymocytes, are lost. With the shrinkage of this organ, noradrenergic nerve fibers are confined to a smaller volume of tissue, giving the appearance of hyperinnervation (Bellinger et al., 1992). Thus, unlike secondary lymphoid organs, the microenvironment of the aged thymus is capable of maintaining noradrenergic nerve fibers.

The adult pattern of nordrenergic innervation of the rat spleen persists through 12 months of age (Bellinger et al., 1987, 1992). At this time, a loss of T-lymphocytes in the periarteriolar lymphatic sheath, and a decline in the density of macrophages in the marginal zone, results in a reduced volume of the white pulp in which noradrenergic fibers reside. By 17 months of age, further loss of both T-lymphocytes and macrophages in the white pulp is apparent; however, at this time, a decline in the density of noradrenergic innervation is also observed. Progressive loss of noradrenergic innervation and the loss of T-lymphocytes and macrophages in the spleen continues throughout the lifespan of the aged. With the normal aging process, a decline in the T cell-mediated immune functions is well documented. Whether this phenomenon is causally linked to the decline in noradrenergic innervation of secondary lymphoid organs remains to be tested (Bellinger et al., 1992).

The early presence of noradrenergic innervation in specific compartments of the thymus and the spleen during critical periods of development points toward a role of NE in the maturation/development of the immune system and "shaping" of the immune responsiveness. This is substantiated by recent observations showing that neonatally sympathectomized rats express alterations in T and B cell proliferation and NK cell activity, the extent of which is influenced by gender and age (Ackerman et al., 1991; Madden et al., 1993). Neonatal sympathectomy also results in reduced immunoglobulin (Ig) M production throughout development (Ackerman et al., 1991).

The bone marrow compartment contains the principal hematopoietic tissue and is the site of proliferation and maturation of multipotent stem cells into mature blood cells of different lineages. Murine bone marrow contains a substantial amount of CAs. NE and DA exhibit a daily rhythmicity, with peak values observed during the night. In addition, high and low values of NE and DA are associated with high and low values of their metabolites, whereas NE, but not DA or epinephrine, is positively associated with the proportion of cells in the G₂/M and S phases of the cell cycle (Maestroni et al., 1998). Thus, the SNS neural input of the bone marrow may be implicated in the regulation of hematopoiesis. Recently, Tang et al. (1999) demonstrated that cold exposure increased NE turnover rate in the murine bone marrow by 36%, whereas peritoneal Pseudomonas aeruginosa infection increased bone marrow NE turnover rate by 131%. Thus, environmental conditions and infectious agents known to elevate central sympathetic outflow induce local release of NE within the bone marrow. These findings also indicate that the noradrenergic innervation of the bone marrow is functionally dynamic and is responsive to generalized or immunological stress.

Administration of various doses of the ₁-AR antagonist prazosin were shown to increase the concentration of blood granulocytes and bone marrow granulocyte-macrophage colony forming units (GM-CFU) while decreasing the number of spleen B and T-lymphocytes (Maestroni et al., 1992; Maestroni and Conti, 1994b). These observations prompted Maestroni and Conti (1994a,b) to suggest that in vivo, myelopoiesis, and in particular the production of granulocytes and macrophages, are under a sympathetic inhibitory tone, whereas lymphocyte formation appears to need adrenergic stimulation. This is substantiated by the observations that ₁-ARs are present on bone marrow cells and NE inhibits the growth and differentiation of GM-CFU in vitro, a process mediated by ₁-ARs, and, to a lesser extend, by ₂-ARs (Maestroni and Conti, 1994a).

Stimulation of -ARs might also participate in the modulatory effects of endogenous CAs in hematopoiesis (see also above, signal transduction). Mouse bone marrow contains a primitive pluripotent cell (stem cell), which following transplantation produces macroscopic colonies in the spleen of heavily irradiated mice; each colony arises from a single cell. These cells are called colony forming units or CFU. In 1971, Byron (1971) demonstrated that upon exposure in vitro to low concentrations of isoproterenol, a -AR agonist, resting mouse spleen CFUs begin to synthesize DNA as shown by increased sensitivity to the cytotoxic effects of both high specific activity [³H]thymidine and hydroxyurea. These effects were blocked by propranolol, a -AR antagonist. Hence, it was suggested that early progenitors in mice may be very sensitive to small amounts of CAs and that -AR stimulation might serve to trigger hemopoietic stem cells into their cell cycle or to shorten their cell cycle (Byron, 1971). In animals, adrenergic agonists also stimulate erythropoiesis both in vivo (Fink and Fisher, 1977) and in vitro (Brown and Adamson, 1977). This appears to be also relevant to human erythropoiesis. Isoproterenol, when added to the culture over a wide range of concentrations, significantly enhanced human erythroid colony growth; this effect was mediated through ₂-ARs and required the presence of erythropoietin (Mladenovic and Adamson, 1984).

Although the above discussed evidence that the thymic cortex (a site where immature thymocytes develop and differentiate to mature thymocytes or T lymphocytes) receives rich sympathetic innervation, our knowledge about the functional significance of these neural inputs is currently limited (see Section IX.). The cell surface glycoprotein Thy-1 expressed on immune cells and neurons of rodents and humans is hypothesized to function in cell adhesion and signal transduction in T cell differentiation and proliferation. Singh and Owen (1976) demonstrated that in culture, higher proportions of 14-day fetal thymus cells expressed Thy-1 and TL antigens following treatment with isoproterenol, an -AR agonist; in contrast, a recent study demonstrated that isoproterenol caused concentration-dependent decreases in levels of Thy-1 mRNA in murine thymocytes, an effect prevented by the -AR antagonist propranolol (Wajeman-Chao et al., 1998). Futhermore, organ cultures of 14-day fetal thymic explants treated with phenylephrine, an ₁-AR agonist, showed increased proliferative activity when assayed in terms of ¹²⁵I-UdR incorporation and cell yields, in comparison with untreated cultures (Singh, 1979).

Thymocytes mature and differentiate into T cells in the thymus under the influence of a microenvironment that is created by a number of supporting cells. TECs, secreting several cytokines as key factors of the thymic microenvironment, are supposed to be the major cell type involved. Recently von Patay et al. (1998, 1999) demonstrated that NE stimulated IL-6 production by TEC cultures about 14-fold over basal values after 24 h. IL-6 in combination with interferon- and IL-2 induces the differentiation of cytotoxic T cells from immature thymocytes (Takai et al., 1988), whereas IL-6 together with IL-2 or IL-4 enhances the proliferation of CD4⁺CD8 or CD4⁺CD8⁺ single positive mouse thymocytes (Chen et al., 1989). Thus, NE may affect thymic development indirectly, through modulation of the secretion by TEC of key factors of the thymic microenvironment.

	VIII. Sympathetic Control of Lymphocyte Traffic and Circulation

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Lymphocyte migration, circulation, and traffic are under the influence of the CNS (for detailed reviews see Ottaway and Husband, 1992, 1994; Benschop et al., 1996) and the SNS plays a significant role in this process. Two phases are recognized after CA administration in humans: a quick (<30 min) mobilization of lymphocytes, followed by an increase of granulocytes with relative lymphopenia (maximal response at 2-4 h) (Benschop et al., 1996). CAs predominantly affect NK cells and granulocyte circulation, whereas T and B cell numbers remain relatively unaffected. Thus, infusion of both epinephrine and NE in humans induces a transient increase of total lymphocytes and numbers of CD3⁺ and CD8⁺ cells, and marked increases (between 400 and 600%) of NK cell numbers (CD16⁺, CD56⁺); two-color fluorescence analysis revealed that the changes in CD8⁺ cells are mainly due to alterations in NK cells coexpressing CD8 rather than CD8+ T cells (Benschop et al., 1996; Schedlowski et al., 1996). Similarly, acute psychological stress or physical exercise induce a transient increase in lymphocyte numbers, in particular the NK cell number. The role of increased CA levels in this phenomenon has also been documented (cf. Benschop et al., 1996; Schedlowski et al., 1996). By contrast, a reduction of Tc and NK cells, without significant alteration of Th cells, were observed after 7 days of treatment with terbutaline, a ₂-AR selective agonist, changes identical to that seen in congestive heart failure patients (Maisel et al., 1990b). Thus, a short term, acute increase of sympathetic activity or a single infusion of adrenergic agents might have the opposite effect on NK cell numbers, than does prolonged sympathetic activity: in the short term, CAs acutely mobilize NK cells from depots, whereas in the long term, chronically, CAs decrease the number of lymphocytes, and particularly of NK cells in the peripheral blood. This is substantiated by the observation that in humans, the percentage of NK cells in peripheral blood in normal subjects is negatively correlated to plasma epinephrine levels (Knudsen et al., 1996).

CAs appear to modulate NK cell circulation via spleen-dependent and spleen-independent ₂-AR mechanisms: these changes are demonstrated to also occur in splenectomized subjects, suggesting that reservoirs other than the spleen, probably the marginating pool in blood vessels (the so-called marginal position of white blood cells in the venular system, see below) are also utilized for this fast mobilization of lymphocytes, particularly NK cells (Schedlowski et al., 1996). Granulocyte increases, however, involve -AR stimulation, and these cells are predominantly released from the marginal pool, the lung and the bone marrow (Benschop et al., 1996).

The mechanisms by which CAs modulate lymphocyte distribution are not well established. One possible mechanism is that the SNS, which directly innervates the vascular smooth muscle, regulates the regional blood flow, and thereby changes the delivery of lymphocytes to postcapillary venules of tissues, and the opportunity for lymphocytes to enter tissue. Perhaps the lymph flow is also under sympathetic control. Thus, electrical stimulation of regional sympathetic nerves results in increased lymphatic pumping, which in turn has marked effects on lymphocyte output, whereas application of ₁-adrenoreceptor agonists into the afferent lymphatics of sheep popliteal lymph nodes increases the output of lymphocytes in efferent lymph (cf. Ottaway and Husband, 1992, 1994).

As evident, different lymphocyte subpopulations have different sensitivity to CA effect on lymphocyte distribution; however, it remains unclear whether this is a direct effect, or indirectly mediated, through hemodynamic alterations. A direct effect might simply reflect, as discussed above, different expression and activity of -ARs on lymphocyte subpopulations: although B cells have many -ARs, they generate little cAMP in response to CAs, and CAs do not affect their circulating numbers; Th cells, which are only modestly affected by CAs, have substantially less -ARs and generate only little cAMP in response to CAs compared with NK cells and Tc, which possess many more -ARs and are mainly affected by CAs. In this respect, alterations in the adhesive interaction of NK lymphocytes are believed to be responsible for the release of NK cells from the marginating pool of blood vessels into the circulating population. In vitro evidence supports this view, since both epinephrine and NE through ₂-AR stimulation, cause detachment of NK cells from cultured endothelium (Benschop et al., 1993).

	IX. Modulation of Lymphocyte Proliferation and K+ Channel Conductance

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Since selective K⁺ channel blockers such as quinine and 4-aminopyridine inhibit IL-2 production and T cell proliferation, it was suggested that the modulation of K⁺ channel conductance might be involved in control of the magnitude and extent of cellular activation and proliferation (DeCoursey et al., 1984; Premack and Gardner, 1991; Jensen et al., 1999). Thus, NE might be involved in the regulation of lymphocyte proliferation through modulation of K⁺ channel gating.

Several lines of evidence suggest that CAs or -AR agonists inhibit the T cell proliferation induced by mitogens (Hadden et al., 1970; Chambers et al., 1993). This is usually accompanied by an increase of cAMP in lymphocytes (Carlson et al., 1989a) and the amount of cAMP produced by T cells stimulated with isoproterenol, a -AR agonist, is proportional to the degree of inhibition of the proliferation (Bartik et al., 1993). Binding of lymphocyte CD3/T cell receptor (TCR) complex to an antigen on the surface of an antigen-presenting cell (APC) triggers the process of T cell activation. This TCR-antigen interaction leads to the generation of intracellular signals that in turn orchestrate a complex series of events that lead to T cell proliferation and cytokine secretion. Importantly, as in the case of mitogens, a similar inhibitory effect of isoproterenol was also observed for the proliferative response of highly purified human T cells stimulated with immobilized anti-CD3 monoclonal antibody through the CD3/TCR complex (Elliott et al., 1992; Bartik et al., 1993). The proliferative response of CD8⁺ T cells is inhibited to a greater extent than CD4⁺ T cells, presumably because CD8⁺ T cells have a higher number of -ARs (Bartik et al., 1993). The elevation of cAMP also inhibits IL-2 secretion by T cells (Bartik et al., 1993), thus suggesting that the inhibition of T cell proliferation might be due, at least in part, to the inhibition of the production of IL-2, a cytokine that is an important costimulatory molecule in T cell proliferation. However, the antigen-stimulated T cell proliferation seems to be more sensitive to a rise in cAMP than the IL-2-induced T cell proliferation (Bartik et al., 1993).

Activation of human T lymphocytes via the CD3/TCR complex or by mitogens produces an enhanced turnover of phosphatidylinositol (PI) cycle-related phospholipids accompanied by the increased production of DAG and phosphorylated derivatives of inositol (IP). Stimulation through the CD2 molecule, which is considered as a CD3/TCR alternative or costimulatory antigen-independent pathway of T cell activation, triggers identical PI cycle-related metabolic events. Recently Bismuth et al. (1988) demonstrated that increased intracellular cAMP inhibited the enhanced turnover of PI cycle-related phospholipids induced by anti-CD2 monoclonal antibodies, and, in parallel, both the DAG production and the IP release were strongly reduced. These results indicate that the increase of intracellular cAMP might interfere at one or more levels of the PI cycle metabolic process triggered through antigen-dependent CD3/TCR complex or antigen-independent CD2 pathway.

Using patch-clamp techniques, the presence of voltage-dependent potassium (K⁺) channels (i.e., the opening rate of the channel increases with membrane depolarization) in murine and human lymphocytes has been shown (DeCoursey et al., 1984; Lewis and Cahalan, 1988; Jensen et al., 1999). Potassium channels play a critical role in the modulation of T cell calcium signaling and subsequent regulation of proliferation. They represent the predominant ion channels in T cells (Premack and Gardner, 1991).

We have demonstrated that NE is able to inhibit in a concentration-dependent manner the outward voltage-dependent K⁺ currents recorded from rat thymocytes (Vizi et al., 1995). This effect is more pronounced at the end of the registered current, suggesting that NE may act predominantly through increasing the rate of inactivation of the voltage-dependent K⁺ current. Similarly, in CD8⁺ human peripheral lymphocytes, isoproterenol, a -AR agonist, decreases the peak current amplitude and increases the rate of inactivation of the K⁺ current (Soliven and Nelson, 1990). These effects were antagonized by the -AR-blocker propranolol, whereas dibutyryl cAMP and cholera toxin mimicked the effect of isoproterenol. Thus, the modulatory effects of NE and -AR agonists on voltage-dependent K⁺ channel conductance appear to be receptor-mediated and could involve cAMP-dependent pathways as well as G-proteins.

Several lines of evidence indicate that K⁺ channels are involved in the processes of lymphocyte activation, proliferation and differentiation. Thus, K⁺ channels are expressed very early in T cell differentiation, possibly before thymic processing (Schlichter et al., 1986) and the expression of K⁺ channels varies during mouse T cell development (McKinnon and Ceredig, 1986; Lewis and Cahalan, 1988; Ishida and Chused, 1993), while activation of T cells by mitogens and IL-2 up-regulates the expression of K⁺ channels and amplifies the potassium conductance (cf. Premack and Gardner, 1991). It is interesting to note that progesterone at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K⁺ channels (Ehring et al., 1998). This effect contributes to progesterone-induced immunosuppression. Thus, NE might be involved in the regulation of lymphocyte proliferation through modulation of K⁺ channel gating. Through the same mechanism, CAs might participate in the regulation of thymocyte or lymphocyte responsiveness to regulatory signals from blood-borne or locally released hormones and cytokines (Vizi et al., 1995).

	X. Modulation of cellular and humoral immunity by catecholamines

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Immune responses are regulated by APCs, such as monocytes/macrophages, dendritic cells, and other phagocytic cells, that are components of innate immunity, and by T helper (Th) lymphocyte subclasses Th1 and Th2, that are components of acquired (adaptive) immunity. Th1 cells primarily secrete IFN-, IL-2, and TNF-, which promote cellular immunity, whereas Th2 cells secrete a different set of cytokines, primarily IL-4, IL-10, IL-13 (Abbas et al., 1996; Fearon and Locksley, 1996; Mosmann and Sad, 1996), and IL-9 (Grohmann et al., 2000), which promote humoral immunity (Fig. 2).

View larger version (47K): [in this window] [in a new window]   Fig. 2.   Role of Th1 and Th2 cells, and type 1 (pro-inflammatory) and type 2 (anti-inflammatory) cytokines in the regulation of cellular and humoral immunity. Cellular immunity is stimulated by type 1 cytokines, secreted by APCs and Th1 cells, whereas humoral immunity is stimulated by type 2 cytokines secreted by APCs and Th2 cells. The cellular source of IL-4 that stimulates the Th0 to Th2 differentiation is not well defined. Cellular immunity provides protection against intracellular bacteria, protozoa, fungi, and several viruses, whereas humoral immunity provides protection against multicellular parasites, extracellular bacteria, some viruses, soluble toxins, and allergens. Systemic, Th2-driving effects of NE released from the postganglionic sympathetic nerve terminals in blood vessels and lymphoid organs, and of epinephrine, secreted from the adrenal medulla on the production of key regulatory type 1 and type 2 cytokines, Th1 and Th2 functions and, respectively, components of cellular and humoral immunity. Solid lines, stimulation; dashed lines, inhibition. Ag, antigen; T, T cell; B, B cell; Pl, plasma cell; Mø, macrophage.

Naive CD4⁺ (antigen-inexperienced) Th0 cells are clearly bipotential and serve as precursors of Th1 and Th2 cells. Among the factors currently known to influence the differentiation of these cells toward Th1 or Th2, cytokines produced by cells of the innate immune system are the most important (Fearon and Locksley, 1996). Thus, IL-12, produced by activated monocytes/macrophages or other APCs, is a major inducer of Th1 differentiation and hence cellular immunity; this cytokine acts in concert with NK-derived IFN- to further promote Th1 responses (Trinchieri, 1995). APC-derived IL-12 and TNF- in concert with natural killer (NK) cell- and Th1 cell-derived IFN- stimulate the functional activity of Tc, NK cells, and activated macrophages, i.e., the major components of cellular immunity. All three cytokines, IL-12, TNF- and IFN-, also stimulate the synthesis of NO and other inflammatory mediators that drive chronic delayed type inflammatory responses. Because of these crucial and synergistic roles in inflammation IL-12, TNF-, and IFN- are considered the major pro-inflammatory cytokines (Trinchieri, 1995; Fearon and Locksley, 1996; Mosmann and Sad, 1996).

Th1 and Th2 responses are mutually inhibitory (Fig. 2). Thus, IL-12 and IFN- inhibit Th2, and vice versa, IL-4 and IL-10 inhibit Th1 responses. IL-4 and IL-10 promote humoral immunity by stimulating the growth and activation of mast cells and eosinophils, the differentiation of B cells into antibody-secreting B cells, and B cell immunoglobulin switching to IgE. Of importance, these cytokines inhibit macrophage activation, T cell proliferation and the production of pro-inflammatory cytokines (Abbas et al., 1996; Fearon and Locksley, 1996; Mosmann and Sad, 1996). Thus, IL-4 and IL-10 are the major anti-inflammatory cytokines.

An increasing body of evidence suggests that CAs inhibit selectively Th1 functions, and favor Th2 responses, rather than causing generalized immunosuppression, as previously believed. CAs appear to suppress Th1 activities and cellular immunity and to boost Th2 and humoral responses at the level of APCs and Th1 cells, or act directly on the cellular components of both cellular and humoral immunity (Fig. 2). The effect of CAs on the production of different cytokines is summarized in Table 4.

View this table: [in this window] [in a new window]   TABLE 4 -ARs-mediated effects of CAs on cytokine and chemokine production

1. Effect on Antigen-Presenting Cells. We recently demonstrated that both NE and epinephrine potently inhibited the production of the main inducer of Th1 responses, IL-12, in human whole blood cultures stimulated with bacterial LPS ex vivo (Elenkov et al., 1996). These effects were mediated by stimulation of -ARs on monocytes, and epinephrine appears to be a strong inhibitor of IL-12 production, exhibiting an EC₅₀ of 10⁹ M. More recently, nonselective - and selective ₂-AR agonists were shown to inhibit the production of IL-12 by monocytes and dendritic cells in both in vitro and in vivo conditions (Panina-Bordignon et al., 1997; Hasko et al., 1998a). Since IL-12 is extremely potent in enhancing IFN- and inhibiting IL-4 synthesis by T cells, the inhibition of IL-12 production may represent one of the major mechanisms by which CAs affect the Th1/Th2 balance. Thus, in conjunction with their ability to suppress IL-12 production, ₂-AR agonists inhibited the development of Th1-type cells, while promoting Th2 cell differentiation (Panina-Bordignon et al., 1997). TNF- is another major pro-inflammatory cytokine that in concert with IL-12 plays a central pathogenic role in several type 1-driven autoimmune reactions. Several lines of evidence suggest that NE, epinephrine, and -AR agonists inhibit the production of TNF- by LPS-treated monocytes, microglial cells, and astrocytes (Hetier et al., 1991; Severn et al., 1992; van der Poll et al., 1994; Nakamura et al., 1998) and by human mast cells stimulated with IgE (Bissonnette and Befus, 1997). Increased extraneuronal concentration of CAs resulting from inhibition of their reuptake by desipramine was also recently associated with decreased neuronal-associated TNF- immunoreactivity in rat hippocampus and locus ceruleus (Ignatowski et al., 1997). CAs also suppress the production of IL-1 (Koff et al., 1986), another pro-inflammatory cytokine, an effect that is mostly indirect via inhibition of TNF- and potentiation of IL-10 production (van der Poll and Lowry, 1997a).

2. Effect on T Helper 1 Cells. Although Mohede et al. (1996) have shown that -AR agonists inhibit IL-4 production by human PBMC, more recent studies suggest that CAs do not affect directly Th2 cells cytokine production and functions. The finding that ₂-ARs are expressed on Th1 cells, but not on Th2 cells (Sanders et al., 1997) provides a mechanistic basis for the differential effect of CAs on Th1/Th2 functions. Of importance, in both murine and human systems, ₂-AR agonists inhibit IFN- production by Th1 cells, but do not affect IL-4 production by Th2 cells (Sanders et al., 1997; Borger et al., 1998). Furthermore, cAMP levels increase in Th1 cells following terbutaline exposure, but not in Th2 cells, even though AC is present and functional in both Th subsets (Sanders et al., 1997). IFN--producing Th1 cells induce B cells to produce IgG2a (in humans, IgG1), whereas IL-4-producing Th2 cells induce B cells to produce IgE and IgG1 (in humans, IgG4) (Fearon and Locksley, 1996). Thus, in the study of Sanders et al. (1997) the inhibition of IFN- production by Th1 cells induced by the ₂-AR agonist terbutaline was associated with subsequent suppressed IgG2a production by mouse B cells, whereas the lack of effect on IL-4 was associated with no changes in IgG1 production by B cells. In addition, isoproterenol, or other agents that activate the cAMP-PKA pathway, were reported to inhibit IL-2 production by T cells and to down-regulate their IL-2 and transferrin receptor expression (Chouaib et al., 1985; Feldman et al., 1987; Mary et al., 1987; Anastassiou et al., 1992).

3. In Vivo Effects. It is important to note that the differential effect of CAs on type1/type2 cytokine production also operates in in vivo conditions. Thus, increasing sympathetic outflow and endogenous production of CAs in mice by selective ₂-AR antagonists, or application of exogenous CAs or -AR agonists results in inhibition of LPS-induced TNF- (Elenkov et al., 1995; Szelényi et al., 2000a,b) and IL-12 production (Elenkov et al., 1995; Hasko et al., 1998d). Catecholamines also appear to exert tonic inhibition on the production of pro-inflammatory cytokines in vivo. Thus, application of propranolol, a -AR antagonist that blocks their inhibitory effect on cytokine-producing cells, results in substantial increases of LPS-induced secretion of TNF- (Elenkov et al., 1995) and IL-12 in mice (Elenkov et al., 1995; Hasko et al., 1998d,e), whereas, in IL-10 deficient C57BL/6 IL-10 (/) mice, plasma levels of IL-12 were about 70-fold higher than in their counterparts, suggesting tonic inhibitory effect of IL-10 on IL-12 production. Injection of isoproterenol, although augmenting the IL-10 response in C57BL/6 IL-10 (+/+) mice, inhibited IL-12 production in both C57BL/6 IL-10 (+/+) and C57BL/6 IL-10 (/) mice (Hasko et al., 1998d). Thus, the inhibition of IL-12 production appears to be independent of the increased release of IL-10. In humans, the administration of the ₂-AR agonist salbutamol results in inhibition of IL-12 production ex vivo (Panina-Bordignon et al., 1997), whereas acute brain trauma that is followed by massive release of CAs triggers secretion of substantial amounts of systemic IL-10 (Woiciechowsky et al., 1998). Phosphodiesterase type IV inhibitors that increase intracellular cAMP in lymphoid cells suppress, both in vitro and in vivo, the production of type 1 cytokines such as IL-12, TNF-, and IFN-, whereas they up-regulate the production of IL-10, a type 2 cytokine (see Section XIV.). Furthermore, in animals or humans, exogenous CAs increased sympathetic outflow and endogenous production of CAs by selective ₂-AR antagonists, excessive exercise, immobilization stress, or elective craniotomy via a -AR-mediated pathway that potentiates the production of IL-6 in in vivo conditions (DeRijk et al., 1994; Takaki et al., 1994; Hasko et al., 1995a; Heesen et al., 1996; Papanicolaou et al., 1996).

4. Local Responses. The above-mentioned evidence, obtained from both human and animal studies, indicates that systemically, CAs inhibit type 1 but stimulate type 2 cytokine secretion, respectively. In local responses, in specific compartments, the effect of CAs may be different. Thus, NE, via stimulation of ₂-ARs, can augment LPS-stimulated production of TNF- from mouse peritoneal macrophages (Spengler et al., 1990), whereas experimental hemorrhage in mice, a condition associated with elevations of systemic CA concentration, increases the expression of TNF- and IL-1 by lung mononuclear cells through stimulation of -AR (Le Tulzo et al., 1997). Since the response to -AR agonist stimulation wanes during maturation of the human monocyte to a macrophage (Baker and Fuller, 1995), it is possible that in certain compartments of the body, the -AR-mediated effect of CA becomes transiently dominant. Through this mechanism, CAs may actually boost local cellular immune responses in a transitory fashion. This is further substantiated by the finding that CAs, through stimulation of ₂-ARs potentiate the production of IL-8 from human monocytes and epithelial cells of the lung (Linden, 1996; van der Poll and Lowry, 1997b), thus probably promoting recruitment of polymorphonuclear leukocytes to this organ (see Table 4). Thus, in summary, whereas CAs suppress Th1 responses and pro-inflammatory cytokine secretion and boost Th2 responses systemically, they may differ in how they affect certain local responses. The possible mechanisms that determine the difference between systemic and certain local effects of CAs are discussed in details below (see Section X.D.2.).

Interleukin-8, produced by monocytes, macrophages, and endothelial cells, is a major factor in neutrophil-mediated inflammation, by activating and recruiting neutrophils into inflamed tissue compartments. IL-8 also induces expression of surface adhesion molecules, production of reactive oxygen metabolites, and degranulation. As mentioned above, CAs, through -AR-mediated mechanisms, potentiate the production of IL-8 from LPS-stimulated monocytes and epithelial cells of the lung (Linden, 1996; van der Poll and Lowry, 1997b). Recent evidence suggests that epinephrine might promote IL-8 production in human leukocytes via an effect on platelets. Thus, IL-8 levels in samples containing platelets and stimulated with LPS and epinephrine were significantly higher than control samples containing no platelets (Engstad et al., 1999). In fact, as shown by Kaplanski et al. (1993) activated platelets are able to induce endothelial secretion of IL-8. These observations may provide a novel relationship between coagulation, Cas, and inflammation. Interestingly, IL-8 was also suggested to be a mediator of sympathetic pain; IL-8 evoked hyperalgesia in a rat paw pressure test by a prostaglandin-independent mechanism (Cunha et al., 1991). Thus, IL-8 might be the first endogenous mediator to be identified as evoking hyperalgesia involving the SNS.

The CC-chemokine MIP-1 is produced by a number of cells including neutrophils, activated lymphocytes, and fibroblasts. MIP-1 is chemotactic for leukocytes, monocytes/macrophages, and T lymphocytes, particularly CD8⁺ T cells. In addition, it potently activates macrophages to secrete TNF-, IL-1, and IL-6. MIP-1 appears to contribute to lung leukocyte recruitment and capillary leak age, the early mortality in endotoxemia, and the pathogenesis of rheumatoid arthritis. Recently Hasko et al. (1998b) demonstrated that exogenous and endogenous CAs inhibit the production of MIP-1 via a -AR-mediated mechanism. Thus, CAs are probably important endogenous regulators of MIP-1 expression in inflammation. The overall effect of SNS on the chemokine system, however, as well as the effect of CAs on the production of the other members of chemokine - and -subfamily, is currently not understood.

D. Effects of Catecholamines and Drugs on the Cellular Components of Immunity

1. Natural Killer Cell Activity. CAs appear to have a dual effect on NK cells. On one hand, CAs (mostly epinephrine) mediate an acute, short lasting, and transient increase of NK cells numbers due to their mobilization from depots (see text above); on the other hand, however, CAs appear to mediate, both acutely and chronically, an inhibition of NK cell activity. This is substantiated by several in vitro and in vivo studies. Thus, application of epinephrine and isoproterenol in vitro elevates cAMP about 2.5-fold and induces an inhibition of NK cell activity (Hellstrand and Hermodsson, 1989; Whalen and Bankhurst, 1990). This effect is blocked by propranolol and mimicked by terbutaline, a 2-AR agonist, indicating the involvement of 2-ARs in this process (Hellstrand and Hermodsson, 1989; Whalen and Bankhurst, 1990).

2. Macrophage Activity. Peripheral, circulating monocytes after activation and/or migration to a particular organ differentiate into macrophages. Although peripheral monocytes appear to express only the -ARs and the effect of CAs on their function and cytokine production appear to be more clear-cut (see above), the effect of CAs on macrophage functions appears to be more complex (see Fig. 3) and the subject of some controversies.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Simplified scheme of the effect of locally released mediators on the secretion of TNF- and IL-12, and macrophage activity. TNF- and IL-12 stimulate the activity of activated macrophages, NK cells, and Tc, the major component of cellular immunity. Excessive production of TNF- and IL-12 play a key role in the inflammatory activity and the tissue damage observed in organ-specific autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Apart from the modulatory effects of NE through 2- and 2-adrenoreceptors (the 2-adrenoreceptor-mediated inhibitory effect is most likely the dominant one; see text), macrophage activity might me affected by locally secreted SP by peptidergic/sensory nerve fibers or histamine, secreted by mast cells. Note that the release of SP and CRH may trigger the secretion of histamine, which, in turn, mediates vasodilatation, acute inflammation, or neurogenic inflammation through stimulation of H1 histamine receptors. Thus, the "anti-inflammatory effect", in general, of catecholamines exerted through stimulation of 2-adrenoreceptors depends on the presence and expression of -adrenoreceptors on macrophages and mast cells, and other local mediators or factors (see text for details).

3. T Cytotoxic Lytic Activity. Relatively few data are available about the effect of CAs on Tc (CD8⁺) cell cytotoxicity. Epinephrine, NE, and isoproterenol suppress the in vitro generation of anti-MOPC-315 tumor cytotoxicity by mouse splenic Tc lymphocytes (Cook-Mills et al., 1995). Increasing cAMP with either a cAMP analog or the -ARs agonist metaproterenol significantly inhibits the in vitro development of memory Tc activity in mice using an anti-influenza cytotoxic Tc assay (Bender et al., 1993). In contrast, CAs or -AR agonists, when added at the initiation of a 5-day sensitization phase, increased the generation of Tc-mediated cytotoxicity using the mixed lymphocyte culture method in BALB/c mice (Hatfield et al., 1986). Thus, CAs may exert enhancing effects on the initiation of Tc responses, in contrast to inhibition of effector cell function.

4. Neutrophil Functions. CAs inhibit both neutrophil phagocytosis and the release of lysosomal enzymes from neutrophils (Zurier et al., 1974). Low doses of isoproterenol also inhibit the respiratory burst of neutrophils associated with degranulation (Nielson, 1987). Furthermore the superoxide generation and formation of oxygen radicals that play an important microbicidal role are both suppressed at nanomolar concentrations of epinephrine, an effect prevented by ₂-AR blockade (Weiss et al., 1996; Barnett et al., 1997). It was revealed that -AR stimulation decreased the maximal rate of superoxide production and increased the rate of termination of superoxide production (Gibson-Berry et al., 1993). In the presence of two powerful chemoattractants, leukotriene B4 and formyl-methionyl-leucyl-phenylalanine, -AR stimulation also inhibits the chemotaxis of human neutrophils (Harvath et al., 1991).

Binding of antigen to B cells induces an activation and subsequent proliferation and differentiation of these cells into antibody-secreting cells plasma cells. However, to proceed through these steps, the B cells require "help", and CD4⁺ Th are the cells that provide this help through cell contact- and cytokine-mediated signals (Fig. 2). When B cells and Th cells are exposed to Th cell-dependent antigens, NE, through stimulation of ₂ receptors, exerts an enhancing effect on B cell antibody (Ab) production (Sanders, 1995; Sanders et al., 1997). One mechanism for this enhancement may involve a ₂-AR-induced increase in the frequency of B cells differentiating into Ab-secreting cells. Thus, when the number of cells secreting anti-trinitrophenyl (TNP) IgM Ab was determined by a TNP-specific ELISPOT assay, it was found that there was about 2-fold increase in the precursor frequency of anti-TNP IgM-secreting cells in the cultures treated with terbutaline, a ₂-AR agonist (Sanders, 1998). The ₂-AR-mediated increase of intracellular cAMP may be particularly important for B cell activation and Ab production for two other reasons. First, the expression of the B7 molecule on B cells that determines the effectiveness of a T cell-B cell interaction for both T cell and B cell activation, is up-regulated by either MHC-restricted T cell interaction or by agents that elevate cAMP (Watts et al., 1993). Second, it appears that a critical threshold level of intracellular cAMP must be obtained before the conjugated B cell can be activated (Pollok et al., 1991). Thus, ₂-AR stimulation during the critical Th/B lymphocyte interaction may augment the cAMP in those conjugated B cells that did not reach a critical threshold level of cAMP (Sanders, 1998).

Moreover, Th cells not only activate B cells during cell-to-cell interaction, but they (Th2 cells) also provide the cytokines necessary for B cell growth. Here again CAs may play an important modulatory role through their differential effect on type 1 and type 2 cytokine production (see text above and Fig. 2). Recent studies are in support of this hypothesis. Thus, the -AR agonists salbutamol and fenoterol potentiate IL-4 induced IgE production by human PBMC, although they inhibit IFN- production by these cells (Coqueret et al., 1995). Furthermore, salbutamol induces an increase of the ex vivo release of IL-4, IL-6, and IL-10 (Coqueret et al., 1994a). This might have resulted from the disinhibition of the restraining inputs of type 1 cytokines on Th2 cells and by a direct potentiation of the production of IL-6 and IL-10 by APC. Taken together, the enhancement of Ab production (and specifically of IgE by -AR agonists) further supports the hypothesis that CAs, via -AR stimulation selectively inhibit Th1 functions and cellular immunity and mediate a Th2 shift that potentiate humoral immunity (see also text above).

	XI. Role of Growth Factors in Sympathetic Nervous System Development and Modulation of the Immune Response

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Glucocorticoids, thyroid hormones, and a family of neurotrophic proteins such as nerve growth factor (NGF), neurotrophin 3 (NT3), NT4, and brain-derived neurotrophic factor (BDNF) play a major role in SNS development. Sympathoadrenal progenitors are bipotential when cultured in vitro. In the presence of NGF, they differentiate to form sympathetic neurons, and in the presence of glucocorticoids, they form chromaffin-like cells (Anderson and Axel, 1986). In fact, targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development; in glucocorticoid receptor / mice, not only is the capacity of chromaffin cells to synthesize epinephrine abolished, but the number of chromaffin cells are reduced substantially. This is accompanied by a normal survival of noradrenergic chromaffin cells (Cole et al., 1995). Similarly, in mice lacking a functional corticotropin-releasing hormone receptor 1, the medulla of the adrenal gland is atrophied (Timpl et al., 1998).

Thyroid hormones such as thyroxine (T4) and triiodothyronine (T3), apart from regulating energy metabolism, influence growth and development. In chromaffin cells, T3 induces the enzyme TH, which is involved in catecholamine synthesis. This action is similar but apparently independent from NGF (Timiras and Nzekwe, 1989). The production of NGF, however, is thyroid hormone-responsive, and it seems likely that some of the effects of thyroid hormones on autonomic and CNS development are mediated through induction of NGF and other growth factors (Fisher et al., 1982).

NGF was the first discovered, and so far, the best characterized neurotrophic factor for the development and differentiation of sympathetic neurons in both developing and adult animals. The essential role of NGF was first demonstrated by injecting NGF antibodies in rodents. This resulted in the death of peripheral sympathetic neurons (cf. Aloe, 1998). NT3 plays both a complimentary and overlapping role with NGF in the development and maturation of sympathetic neurons (Zhou and Rush, 1996). NGF and NT3 are neurotrophic factors that are synthesized by effector tissues and are retrogradely transported by postganglionic neurons to prevent cell death. NGF has been found in various lymphoid organs such as spleen, lymph nodes, and thymus (Aloe et al., 1997). The spleen also contains high levels of mRNA for BDNF and NT3 (Yamamoto et al., 1996). In these organs NGF apparently regulates the sympathetic neurite outgrowth toward these organs (Kannan et al., 1996). The induction of NGF production in lymphoid organs appears to be mediated partly by IL-1 (Kannan et al., 1996).

Apart from its effects on SNS development and differentiation, NGF is now known to affect cells of the immune and endocrine systems in a variety of ways (cf. Simone et al., 1999). NGF influences B- and T-lymphocyte proliferation, is an autocrine survival factor for memory B lymphocytes, stimulates immunoglobulin production, and promotes human hemopoietic colony growth factor secretion and differentiation (cf. Aloe, 1998). In addition, hypothalamic NGF concentration increases following stressful events, and it stimulates the secretion of pituitary ACTH and adrenal glucocorticoids. The circulating levels of NGF increase in inflammatory responses, in various autoimmune diseases, in parasitic infections, and in allergic diseases (Aloe et al., 1997; Aloe, 1998). Thus, this neurotrophic factor is in fact a pleiotropic cytokine that plays an important role at the interface between the nervous and the immune systems.

Recent studies indicate that NGF is synthesized by cells of immune system lineage, its level increases during inflammatory responses, and that cytokines such as IL-1 and TNF- are potent inducers of NGF secretion (Aloe et al., 1997; Aloe, 1998). NGF-specific mRNA is present in mouse T lymphocytes of both the CD4⁺ and CD8⁺ phenotypes, and in splenic B cells. In CD4⁺ cells, NGF is present in both Th1 and Th2 antigen-specific clones, but an increase of NGF-specific message is detected after antigenic challenge only in Th2 cells (Santambrogio et al., 1994). Human Th2 clones also produce and release NGF (Bonini et al., 1999). Splenic mononuclear cells from allergen-sensitized mice produced NGF in response to allergen (Braun et al., 1998). They respond to exogenously added NGF with an increase in IL-4 and IL-5 production and augmented IgE and IgG1 synthesis (Braun et al., 1998). In rat peritoneal mast cells NGF causes an increase in the mRNAs for IL-3, IL-4, IL-10, and TNF- (Bullock and Johnson, 1996). Thus, an allergic inflammation might be accompanied by enhanced local secretion of NGF that acts as an amplifier for Th2 effector functions. This is substantiated by the observations that NGF also enhances histamine release (Bischoff and Dahinden, 1992), although NGF and IL-4 act as cofactors for IL-3-induced histamine production by basophils cells (Richard et al., 1992).

In mouse astrocytes IL-4, IL-5, and IL-10 induce an increase in NGF secretion, whereas IFN-, IL-2, IL-3, and IL-6 do not (Awatsuji et al., 1993; Brodie, 1996). However, IFN- appears to antagonize the increase in NGF secretion, induced by IL-10 (Brodie, 1996). This indicates that type 2 cytokines such as IL-4, IL-5, and IL-10 may provide neurotrophic support to injured neurons via induction of NGF synthesis. Of interest, treatment with ₂-AR agonists up-regulates NGF secretion in astrocyte cultures, and NGF appears to mediate the neuroprotective effect of the ₂-AR agonist clenbuterol in vitro and in vivo (Culmsee et al., 1999). In addition evidence was obtained (Charon et al., 1998; Cruz-Aguado et al., 1999; Culmsee et al., 1999; Matsuoka et al., 1999; Semkova et al., 1999; Silva et al., 2000) that ₂-adrenoceptor activation increases cAMP level, and it is in correlation with neuroprotection actions and NGF production (Cruz-Aguado et al., 1999; Culmsee et al., 1999; Silva et al., 2000).

	XII. Physiologic Control of the Sympathetic-Immune Interface: -Adrenergic Receptor Expression, Coupling, and Desensitization

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The physiologic regulation of the sympathetic-immune interface appears to be a rather complex phenomenon, exerted at different levels. Centrally, the SNS output appears to be affected by different neurotransmitter pathways, stress, diurnal rhythms, and immune responses (see text above). Several cytokines appear to control neurotransmitter plasticity, its release, and receptor excitability at the level of the sympathetic ganglia (Jonakait, 1993). Thus, TNF- and IFN- modulate nicotinic responses, Ca²⁺ currents and NE secretion and inactivation, whereas transforming growth factor (TGF)- modulates the development of neuronal excitability by regulating the expression of voltage-gated K⁺ channels in sympathetic ganglia (Soliven and Albert, 1992a,b; Soliven and Wang, 1995; Phelan et al., 1997). Furthermore, IL-1, and to a lesser extent TNF-, induce substance P in sympathetic ganglia through the induction of leukemia inhibitory factor (Shadiack et al., 1993; Ding et al., 1995), whereas IL-6 induces about a 6-fold increase in choline acetyltransferase mRNA in cultured rat sympathetic neurons (Oh and O'Malley, 1994).

At the level of sympathetic nerve terminals, presynaptic receptors regulate the output of NE released in lymphoid organs and blood vessels (see text above and Fig. 1). Although some cytokines (such as TNF- and IL-1) have been shown to inhibit presynaptically the release of NE in the median eminence, hippocampus, myenteric plexus, and human atria (Elenkov et al., 1992a; Hurst and Collins, 1994; Ignatowski and Spengler, 1994; Abadie et al., 1997), currently there is not enough convincing evidence that cytokines are able to modulate "presynaptically" the release of NE from the sympathetic nerve terminals in lymphoid organs. Glucocorticoids and estrogens, however, are potent inhibitors of the extraneuronal uptake of NE; thus, through this mechanism these hormones may increase local levels of CAs (Salt, 1972).

The input of the SNS on lymphoid cells can also be regulated postsynaptically at different levels: by regulation of the expression of - and/or -ARs and the subtype of G-protein coupled to these receptors (see text above for the differential expression of -ARs on different subpopulations of lymphocytes and their coupling to adenylate cyclase); by regulation of both PDE and -adrenergic receptor kinase (ARK) activity, and through the process of homologous and heterologous desensitization of G-protein-coupled receptors.

Human lymphocyte -ARs demonstrate characteristic down-regulation after chronic treatment with ₂-AR agonists. This process appears to differ among different mononuclear cell subsets: after treatment of healthy volunteers for 7 days with terbutaline, a ₂-AR agonist, the ₂-AR number decreased by more than 50% in Tc, but the reduction was much smaller in NK and Th cells and absent in B cells. The -AR down-regulation in these cells was related to a decreased cAMP response to isoproterenol; the cAMP generation in response to prostaglandin E1 was also reduced, suggesting heterologous type of desensitization (Maisel et al., 1989). The -AR down-regulation appears to be relevant to in vivo conditions since Pende et al. (1991) found an inverse correlation between the ₂-AR density on mononuclear leukocytes and the basal plasma NE in healthy human volunteers.

However, both an increase and decrease of the number of -ARs and the cAMP response to isoproterenol are reported after activation of human and murine lymphocytes with mitogens (Radojcic et al., 1991; Carlson et al., 1994; Cazaux et al., 1995). After activation of quiescent Tc clones in IL-2-containing media, the cAMP response to ₂-AR agonists, histamine, and prostaglandins increases, peaking 4 to 5 days after stimulation. Carlson et al. (1994) suggested that mitogens prevent both the sequestration of the ₂-AR and its dissociation from the G_s protein in response to isoproterenol stimulation, whereas Radojcic et al. (1991) also reported some increase in the number of ₂-ARs. In apparent contrast, a decrease in ₂-AR numbers on murine T lymphocytes and diminished response to isoproterenol at the peak of the proliferative response to a mitogen was recently reported (Cazaux et al., 1995). In vivo, immunization with bovine serum albumin (BSA) induces a reduction in the density of -ARs 3 days after antigenic challenge, followed by a significant increase in receptor number 7 and 15 days after immunization (Morale et al., 1992). Cytokines such as IL-1, IL-1, and TNF- increase the density of human -ARs, whereas glucocorticoids also increase -AR density and markedly potentiate the effect of these cytokines (Stern and Kunos, 1988; Nakane et al., 1990). Glucocorticoids are well known to exert a permissive effect on cAMP-elevating agents in various cell types (Malbon et al., 1988), and the gene for the human ₂-AR contains a glucocorticoid response element (Hadcock et al., 1989). Furthermore, glucocorticoids appear to sensitize cAMP formation in resting human lymphocytes by altering the AC activity (Michel et al., 1994). Other hormones, such as insulin, also up-regulate the expression of ₂-ARs and their coupling to AC in mononuclear leukocytes (Sager et al., 1990).

Recent evidence indicates that G-proteins, and the signals they regulate, such as ion channels and AC (G_s/i) and phospholipase C (G and G_11/15-16) are differentially regulated in lymphoid cells in a maturation- and lineage-dependent manner. As already discussed the expression of the G-proteins G₁₅ (murine) and G₁₆ (humans) has been shown to be hemopoietic lineage-restricted with particularly high expression in pre-B cell lines (Wu et al., 1995). Of interest, there is a progressive and marked down-regulation of the expression of G₁₆ correlating with differentiation through mature human resting B cells to "follicular" B cells (Grant et al., 1997). Murine thymocytes, splenic T cells, and human tonsillar T cells show little or no protein expression of any of the G isoform tested, except G_s, whereas G_i1 seem to be expressed in the late stages of human B cell development (Grant et al., 1997). Although it is a matter of speculation, since G_s stimulate AC, whereas G_i inhibit its activity, this may explain the differences in cAMP responses between T and B cells, and particularly the poor or absent cAMP response in B cells (see text above).

On the other hand, since cAMP is considered anti-proliferative and although there does not appear to be an absolute correlation between proliferation and decreased G_s expression, the process of cell activation and proliferation appears to involve down-regulation of G_s and increased expression of G_I in activated or proliferating B and T cells (Grant et al., 1997). These findings may suggest that altered G-protein expression may provide a mechanism for "escaping" the anti-proliferative signal, cAMP; i.e., presumably the CAs--ARs-cAMP pathway in activated or transformed lymphoid cells.

Agonist-induced increases in cAMP-PDE activity represent a potentially important mechanism of functional desensitization. Prolonged -AR stimulation has been reported to up-regulate cAMP-PDE activity in human monocytes, accompanied by a mirror-image decrease in cAMP responsiveness (Chan et al., 1982). Although resting monocytes expressed both mRNA and protein for PDE4A, PDE4B, and PDE4D, prolonged -AR stimulation up-regulated the message for PDE4A and PDE4B, whereas mRNA for PDE4D was not detected in treated cells (Manning et al., 1996).

Cellular responses to many hormones and neurotransmitters wane rapidly despite continuous exposure of cells to these stimuli. This phenomenon is termed desensitization. In the case of -AR desensitization this process does not appear to require internalization of the receptors, but rather an alteration in the functioning of -ARs themselves that uncouples the receptors from the stimulatory G-protein G_s. Two patterns of rapid desensitization have been characterized for G-protein-coupled receptors: homologous desensitization, which mainly involves G-protein-coupled receptor kinases (GRK) and arrestins, and heterologous desensitization, which mainly involves PKA and PKC (Chuang et al., 1996). ARK is a serine-threonine kinase involved in the process of homologous desensitization of G-protein-coupled receptors. ARK is a member of a multigene family, consisting of six known subtypes, also named GRKs (De Blasi et al., 1995). Among the six known GRKs, four (GRK-2, GRK-3, GRK-5, and GRK-6) are highly expressed in peripheral blood leukocytes. Agonist occupancy triggers translocation of ARK from cytosol to plasma membranes, where it phosphorylates agonist-occupied receptors. A substantial increase of ARK is observed after stimulation of lymphocytes with mitogens, a process that appears to be PKC-mediated. A significant increase in ARK1 and ARK2, but not GRK-5 and GRK-6, was observed 48 h after mitogen stimulation (De Blasi et al., 1995). Of interest, a recent study by Daaka et al. (1997) performed in HEK293 cells, demonstrates that a mechanism previously shown to mediate uncoupling of the ₂-AR from G_s and thus heterologous desensitization (PKA-mediated receptor phosphorylation), also serves to "switch" coupling of this receptor from G_s to G_i, and, thus, initiate a new set of signaling events.

In summary, the expression of -ARs on lymphoid cells and their coupling to different G-proteins and intracellular effectors is a dynamic process that depends of the state of activation and differentiation of the cell and appear to be subject to regulation by several endogenous ligands, such as CAs, different hormones, and cytokines. Since the level of these endogenous ligands varies during an immune response, the regulation of - or -ARs might have physiological and pathophysiological importance and it may participate in regulatory mechanisms.

	XIII. Clinical Implications

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A major factor governing the outcome of infectious diseases is the selection of Th1 versus Th2 predominant adaptive responses during and after the initial invasion of the host (Abbas et al., 1996; Fearon and Locksley, 1996; Mosmann and Sad, 1996). Thus, hyperactive SNS or stress-related increases of CA levels through induction of a Th2 shift may have a profound effect on the susceptibility of the organism to and/or may influence the course of an infection, the defense against which is primarily through Th1-driven cellular immunity mechanisms (Table 5).

View this table: [in this window] [in a new window]   TABLE 5 Putative pathophysiologic roles of CA-induced alterations of Th1/Th2 balance in certain infections, infectious complications after major injury, autoimmune/inflammatory, allergic or neoplastic diseases (modified from Elenkov and Chrousos, 1999)

Cellular immunity, and particularly IL-12 and IL-12-dependent IFN- secretion in humans, seems essential in the control of mycobacterial infections (Altare et al., 1998). In the 1950s, Thomas Holmes (cf. Lerner, 1996) reported that individuals who had experienced stressful life events were more likely to develop tuberculosis and less likely to recover from it. Although it is still a matter of some speculation, stress hormone-induced (CAs and glucocorticoids) inhibition of IL-12 and IFN- production and the consequent suppression of cellular immunity may amply explain the pathophysiologic mechanisms of these observations (Elenkov et al., 1996; Elenkov and Chrousos 1999).

Helicobacter pylori infection is the most common cause of chronic gastritis, which in some cases progresses to peptic ulcer disease. The role of stress in promoting peptic ulcers has been recognized for many years (see Levenstein, 1998; Levenstein et al., 1999) Thus, increased systemic CAs and glucocorticoid levels, in concert with an increased local concentration of histamine, induced by inflammatory or stress-related mediators, may skew the local responses toward Th2 and, thus, allow the onset or progression of a H. pylori infection (Elenkov et al., 1998).

HIV+ patients have IL-12 deficiency versus elevated levels of IL-10, whereas disease progression has been correlated with a Th2 shift (cf. Haraguchi et al., 1995a). The innervation (primarily sympathetic/noradrenergic) of lymphoid tissue may be particularly relevant to HIV infection, since lymphoid organs represent the primary site of HIV pathogenesis. In fact, NE, the major sympathetic neurotransmitter released locally in lymphoid organs (see text above) was recently reported to directly accelerate HIV-1 replication by up to 11-fold in acutely infected human PBMCs (Cole et al., 1998). The effect of NE on viral replication is transduced via the -AR-AC-cAMP-PKA signaling cascade (Cole et al., 1998). In another recent study Haraguchi et al. (1995a,b,c) found that the induction of intracellular cAMP by a synthetic, immunosuppressive, retroviral envelope peptide caused a shift in the cytokine balance and led to suppression of cell-mediated immunity by inhibiting IL-12 and stimulating IL-10 production. Thus, on one hand CAs may suppress cellular immunity and directly accelerate HIV replication, whereas, in contrast, retroviruses may suppress cell-mediated immunity using the same pathways by which CAs alter the Th1/Th2 balance.

In a recent study, an association was demonstrated between stress and the susceptibility to the common cold among 394 persons who had been intentionally exposed to five different upper respiratory viruses. Psychological stress was found to be associated in a dose-dependent manner with an increased risk of acute infectious respiratory illness, and this risk was attributed to increased rates of infection rather than to an increased frequency of symptoms after infection (Cohen et al., 1991). Thus, stress hormones (CAs and glucocorticoids) through their selective inhibition of cellular immunity (Elenkov et al., 1996) may play substantial roles in the increased risk of an individual to acute respiratory infections caused by common cold viruses.

Major injury (serious traumatic injury and major burns) or major surgical procedures often lead to severe immunosuppression that contributes to infectious complications and, in some cases to sepsis, the most common cause of late death after trauma. The neuroendocrine response is an essential component of the adaptive process to trauma, brain injury, and major surgery. Generally, after brain or extracerebral injury there is a biphasic pattern of response, with a sympathoadrenal storm associated with variable and altered stimulation of the HPA axis during the ebb phase; the first phase is followed by a decrease in both responses (Chiolero and Berger, 1994). It is important that the intensity of these changes (particularly with CAs and cortisol plasma levels) correlates with the severity of both cerebral and extracerebral injury and an unfavorable prognosis (Jarek et al., 1993; Chiolero and Berger, 1994; Rothwell and Lawler, 1995; Rothwell et al., 1996). In patients with traumatic major injury and in animal models of burn injury, the suppressed cellular immunity is associated with diminished production of IFN- and IL-12 and increased production of IL-10, i.e., a Th2 shift (O'Sullivan et al., 1995). This is further substantiated by the observation that TNF- production is reduced in LPS-stimulated whole blood after trauma (Fabian et al., 1995), whereas the production of type 1 cytokines, such as IFN-, TNF-, and IL-2, is down-regulated on postoperative day 1 after conventional but not laparoscopic surgery (Brune et al., 1999). A recent study by Woiciechowsky et al. (1998) indicated that systemic release of IL-10 triggered by SNS activation might be an important mechanism of immunosuppression after injury. Thus, high levels of systemic IL-10 documented in patients experiencing a "sympathetic storm" due to acute accidental or iatrogenic brain trauma were associated with high incidence of infection. Furthermore, this study demonstrates that the -AR blockade by propranolol dose dependently inhibited CA-induced release of IL-10 and prevented the increase of circulating IL-10 in the rat model (Woiciechowsky et al., 1998).

Therefore, all the above-mentioned data suggest that CA secretion triggered by major injury, via an induction of a Th2 shift, may contribute to the severe immunosuppression observed in these conditions. As recently stated by Plata-Salaman (1998), these observations may have two important implications: 1) they provide direct evidence in humans of a neuroendocrine pathway, the sympathoadrenal system coupled with a systemic immunosuppressive response that triggers high incidence of infections and complications; 2) they emphasize the importance of neurotransmitter/hormone-associated modulation of immunity.

As mentioned above, the suppression of host defense mechanisms associated with major surgery or trauma was proposed to determine susceptibility to infectious complications and to the development of sepsis. Apart from suppression of type 1 cytokines and potentiation of type 2 cytokine production, major surgery and trauma was also associated with impaired lymphocyte proliferation, delayed-type hypersensitivity skin test response, cell surface MHC class II antigen expression, and suppressed neutrophil functions, including chemotaxis, phagocytosis and oxygen radical production. During established sepsis, however, hyporeactivity of the immune system may be followed by a state of hyperactivity that is characterized by the excessive production of multiple inflammatory cytokines and is associated with high mortality. The molecular mechanisms responsible for this functional conversion of the immune system have not been identified as yet (cf. Hensler et al., 1998).

In the case of Gram-negative bacterial sepsis, the pro-inflammatory cytokines TNF- and IL-1, produced in response to endotoxin, appear to be critical components in the dysregulated host response during sepsis. It is believed that these cytokines trigger a cascade of events, including synthesis of prostaglandins, leukotrienes, reactive oxygen metabolites, and platelet-activating factor (PAF) that results in septic shock and multiple organ dysfunction syndrome. An important point is that TNF- appears to be a key mediator of enhanced NO production by the macrophage-type inducible nitric oxide synthase that contributes to the development of hypotension, peripheral vasodilatation, and vascular hyporeactivity to vasoconstrictor agents in endotoxin shock. However, thus far, the results of anti-TNF- strategies, the administration of IL-1 receptor antagonist, as well as inhibitors of PAF and NO have been largely disappointing and failed to demonstrate a survival benefit (Pastores et al., 1996; Eigler et al., 1997). This may be related, in part, to the incomplete understanding of the complex timing of mediator release and balance during sepsis.

The differential effects of ₂-AR antagonists and ₂-AR agonists on type 1 and type 2 cytokine production, such as inhibition of TNF-, IL-12 and augmentation of IL-10 production (see Section X.B.), clearly offers a potential for pharmacologic manipulation of cytokine production during septic shock. The first study to examine the effect of epinephrine pretreatment on cytokine production in healthy human volunteers challenged with endotoxin in vivo confirmed the inhibition of TNF- appearance and increase in IL-10 release (van der Poll et al., 1996). Furthermore, isoproterenol, not a subtype selective -AR agonist, appears to suppress NO production by macrophages and prevents the LPS-induced suppression of vascular contractility to NE in the thoracic aorta ex vivo (Szabó et al. 1997; Hasko et al. 1998a).

Pharmacological cardiovascular support of human septic shock is currently not well standardized (Pastores et al., 1996). Such support includes the administration of -adrenergic agonists to maintain perfusion pressure, -adrenergic agonists (including dobutamine, which acts mostly as a ₁-AR agonist) to improve cardiac output and oxygen delivery; and dopamine-receptor agonists to augment renal and mesenteric perfusion (for more details see Pastores et al., 1996). Despite early claims of improved outcome, more recent studies add a word of caution regarding the use of dobutamine and suggest worsening survival and increased end-organ injury (Hayes et al., 1994). Moreover, in humans, ₂- but not ₁-ARs appear to control monocyte/macrophage cytokine profiles. Therefore, currently, a clear recommendation for a specific catecholamine regimen in septic shock is impossible. Systemic examination will be necessary to determine the combination and the right choice of adrenergic agents that would satisfy an optimal balance between cardiovascular support and immunomodulation during septic shock.

Further complexity is added by the recent observations of Hensler et al. (1998) showing that low preoperative IL-12 secretion by monocytes precedes the onset of sepsis. This is consistent with the concept discussed above that suppression of both innate and T cell-dependent mechanisms predisposes patients with major surgery or trauma for the development of septic complications. Furthermore, recently obtained in vitro evidence indicates that CAs can dramatically increase the growth of Gram-negative bacteria such as Escherichia coli and Yersinia enterocolitica (cf. Lyte, 1992). Thus, an early intervention and suppression of IL-12 production and possibly an increase in bacterial growth by application of adrenergic agents might even have a harmful effect.

Recent studies applying anti-TNF- strategies demonstrated that patients with high systemic levels of IL-6 might benefit most from anti-cytokine therapy (cf. Eigler et al., 1997). Therefore, patient stratification based on the individual inflammatory response condition should greatly improve the benefits of immune therapy. Thus, it was proposed that immune stimulatory protocols should prove beneficial for patients showing hyporeactive immune response conditions (a Th2 shift?), whereas patients with a hyperactive immune system (a Th1 shift?) may selectively benefit from anti-inflammatory therapy (Hensler et al., 1998). In conclusion, patient stratification, the right choice of timing, and type of intervention, including adrenergic agents, might be an important factor in determining the success of therapeutic strategies in sepsis and its complications (see Section XIII.B. and the beginning of this section).

Several autoimmune diseases are characterized by common alterations of Th1 versus Th2 and IL-12/TNF- versus IL-10 balance (Table 5). In rheumatoid arthritis (RA), multiple sclerosis (MS), type 1 diabetes mellitus, autoimmune thyroid disease (ATD) and Crohn's disease the balance is skewed toward Th1 and an excess of IL-12 and TNF- production, whereas Th2 activity and the production of IL-10 are deficient (Wilder, 1995; Mosmann and Sad, 1996; Elenkov et al., 1997). This appears to be a critical factor that determines the proliferation and differentiation of Th1-related autoreactive cellular immune responses in these disorders (Segal et al., 1998). On the other hand, systemic lupus erythematosus (SLE) is associated with a Th2 shift and an excessive production of IL-10, whereas IL-12 and TNF- production appear to be deficient (Maini et al., 1994; Horwitz et al., 1998).

The autonomic/sympathetic nervous system and HPA axis, both involved in stress responses, influence autoimmunity in a complex way (Arnason et al., 1988; Chrousos, 1995; Wilder, 1995; Rogers and Fozdar, 1996). In consideration of Th2-driving effects of CAs systemically, one could postulate that a hypoactive SNS may facilitate/sustain the Th1 shift in MS or RA, and vice versa, SNS hyperactivity may intensify the Th2 shift and induce/facilitate flares of SLE. Animal studies and certain clinical observations support this hypothesis. Thus, Fischer (F344) rats, which have a hyperactive stress system, are extremely resistant to experimental induction of Th1-mediated autoimmune states, including arthritis, uveitis, and experimental allergic encephalomyelitis (EAE) (Wilder, 1995). Similarly, women in the third trimester of pregnancy, who have increased levels of cortisol and probably CAs (Cohen et al., 1988; Magiakou et al., 1996), experience remission of Th1 type-mediated autoimmune diseases, such as RA, MS, type 1 diabetes mellitus, and ATD, possibly via suppression of pro-inflammatory and potentiation of anti-inflammatory cytokine production. Through a reciprocal mechanism, Th2 type-mediated autoimmune disorders mainly driven by IL-10, such as SLE, may flare in high cortisol and CAs output states, i.e., during stress or pregnancy (Wilder, 1995; Elenkov et al., 1997). There are different opinions (Lin et al., 1993; Sacks et al., 1998, 1999) suggesting that there is dysregulation between the maternal nonspecific and specific immune responses.

Conversely, Lewis (LEW) rats, which exhibit a hypoactive stress system are extremely prone to develop experimentally induced Th1-mediated states, such as arthritis, uveitis, or EAE (Wilder, 1995). Similarly, clinical situations associated with blunted stress system activity are associated with heightened susceptibility to or activity of Th1 type-mediated autoimune diseases such as RA, MS, and ATD. This might include the postpartum period and the period that follows cessation of chronic stress or a rebound effect upon relief from stressors (Chrousos, 1995; Wilder, 1995; Elenkov et al., 1996).

Several lines of evidence suggest that the sympathetic-immune interface is defective in MS and its experimental model, EAE. Thus, sympathetic skin responses are decreased and lymphocyte -ARs are increased in progressive MS (Karaszewski et al., 1990). The density of -ARs on CD8⁺ T cells are increased between 2- and 3-fold, compared with age-matched controls; no changes of the density of these receptors on monocytes, B cells, and CD4⁺ cells were observed (Arnason et al., 1988; Karaszewski et al., 1993). Similarly, in the preclinical stage of EAE the NE content in spleen is reduced, accompanied by an increase of splenocyte -ARs density (Mackenzie et al., 1989). A defective or hypoactive SNS is most likely to be a "causative" factor for the up-regulation of -ARs observed in MS (Arnason et al., 1988); however, the "up-regulating" effects of cortisol or IL-1 on -ARs receptor expression cannot be ruled out (Zoukos et al., 1992).

The "protective" role of the SNS is further substantiated by the observations that chemical sympathectomy augments the severity of EAE (Chelmicka-Schorr et al., 1988), whereas a uniformly increased splenic NE content was observed at peak disease (Leonard et al., 1991). Neurochemically it means that the release of NE was reduced, giving further support that SNS is protective in EAE. Moreover, chemical sympathectomy with 6-OHDA produced a significant depletion of splenic NE alone, which resulted in increased disease severity, despite the fact that circulating glucocorticoids were elevated. Furthermore, isoproterenol and terbutaline, -AR and ₂-AR agonists, respectively, were reported to suppress chronic/relapsing EAE in LEW rats (Chelmicka-Schorr et al., 1989; Wiegmann et al., 1995). The latter observation might have resulted from the previously discussed effects of CAs and -AR agonists on the production of type 1 cytokines. This is further substantiated by recent evidence that the drug rolipram, by selective inhibition of phosphodiesterase type IV and subsequently the production of type 1/pro-inflammatory cytokines, such as TNF-, IFN-, and IL-12, prevents and ameliorates the course of EAE (see text below).

The role of SNS in RA is less clear and more controversial. In a recent study, Baerwald et al. (1997) demonstrated that in RA patients the number of -ARs on CD8⁺ lymphocytes, but not on CD4⁺ lymphocytes, was significantly decreased compared with cells from healthy donors. An even more pronounced decrease of -ARs on synovial fluid lymphocytes compared with peripheral lymphocytes was also observed. Furthermore, these changes were accompanied by a diminished effect of CAs on OKT3-induced PBMC activation, suggesting a functional significance of ₂-AR alterations. Thus, it was suggested that impaired control of the immune response by the ANS may contribute to the pathogenesis of RA (Baerwald et al., 1997). In contrast, Lombardi et al. (1999) failed to observe differences of -AR number on PBMC between RA patients and healthy donors. Interestingly, they found a decrease in GRK activity in RA subjects that was mirrored by a decrease in GRK-2 and GRK-6 protein expression. The decrease in GRK-2 activity appears to be mediated by cytokines such as IL-6 and IFN-. Thus, local pro-inflammatory cytokines or altered activity of the SNS may mediate changes in coupling of -ARs to G-proteins observed in RA patients.

In the arthritis-prone LEW rats, sympathectomy with 6-OHDA enhanced the severity of adjuvant-induced arthritis (Felten et al., 1992). In this animal model of arthritis, selective sympathetic denervation of the reactive secondary lymphoid organs (the popliteal and inguinal lymph nodes) was achieved with local injection into the fat pads surrounding these lymph nodes (Felten et al., 1992). This denervation resulted in earlier onset and enhanced severity of inflammation and bone erosions compared with nondenervated rats. In contrast, Levine et al. (1988) provided evidence in adjuvant-induced arthritis in Sprague-Dawley rats that chronic administration of the -AR blocker propranolol induced a delayed onset and attenuation of the severity of joint injury.

These discrepancies might have resulted from different experimental conditions, rat strain differences in sensitivity and susceptibility to experimentally induced arthritis, a nonspecific effect of rather high doses of propranolol used in the study of Levine et al. (1988), or more likely, they may reflect the complex regulation of macrophage functions by the SNS (see text above). As already mentioned, the SNS alone, or in conjunction with secretory mediators from sensory neurons might up-regulate, at some point, local macrophage functions and/or type 1 cytokine production that may trigger an exacerbation of disease activity. Thus, the systemic effect of the SNS might be different from certain local responses, and particularly the effect of sympathetic innervation in the joints. The local effect of the SNS in the joints might even be different from the local effect of CAs, centrally, in microglia or astrocytes (see above). In addition, the role of the SNS at the time of the induction of experimental arthritis, including its effect on type 1 cytokine production might be distinct from its role much later in the effector phase. Thus, during an established arthritis local inflammatory mediators may directly affect sympathetic nerve terminals releasing pro-inflammatory mediators.

In fact, recent evidence indicates that continuous perfusion of bradykinin induces significant plasma extravasation in the knee joint of the rat (Green et al., 1994). Employing this experimental model of inflammation, it has been suggested that the potent inflammatory mediator bradykinin acts on postganglionic sympathetic nerve terminals to cause the release of mediators (specifically PGE2), which then promote inflammation (Green et al., 1998). Interestingly, the action of bradykinin to produce plasma extravasation via a sympathetic mechanism was found not to require electrical activity in sympathetic neurons and was not dependent on vesicular release of the neurotransmitter NE (Miao et al., 1996). Thus, peripheral sympathetic terminals may have two different functions: the classical transmission of impulse activity with concomitant release of NE and mediation of inflammatory processes, independent of activity and neurotransmitter vesicular release (Green et al., 1998). Another important local factor might be the presence of mast cells in joints (Malone et al., 1987). Thus, locally, in the RA synovium stress- or inflammation-induced release of certain mediators released from both sympathetic and sensory nerve terminals, such as SP and CRH (Malone et al., 1987) might exert potent pro-inflammatory effects via release of histamine from mast cells (Theoharides et al., 1995, 1998; Elenkov et al., 1999). Certainly, the above-mentioned discrepancies about the role of SNS in RA demand further studies.

However, a recent study suggests that the protective role of the SNS in RA might prevail over the "permissive" one. Thus, Malfait et al. (1999) demonstrated that the ₂-AR agonist salbutamol is a potent suppressor of established collagen-induced arthritis in mice. This drug had a profound protective effect as assessed by clinical score, paw thickness, and joint histology. Additionally, in in vitro experiments salbutamol reduced IL-12 and TNF- release by peritoneal macrophages and blocked mast cell degranulation in joint tissues.

Patients with unexplained chronic pain and/or fatigue have been described for centuries in the medical literature, although the terms used to describe these symptom complexes have changed frequently. The currently preferred terms for these syndromes are fibromyalgia and chronic fatigue syndrome (CFS) (for details see Clauw and Chrousos, 1997).

Fibromyalgia is the second most common rheumatologic disorder, behind osteoarthritis. To fulfill the criteria for fibromyalgia, an individual must have both chronic widespread pain and the presence of "tender points" on examination. The current definition of CFS requires that the affected individual display severe chronic fatigue without a defined cause, as well as the presence of four of the eight following symptoms: myalgia, arthralgia, sore throat, tender nodes, cognitive difficulty, headache, postexertional malaise, or sleep disturbance (cf. Clauw and Chrousos, 1997). There has been little study of underlying pathophysiologic mechanisms of fibromyalgia and CFS. Clauw and Chrousos (1997) recently suggested that a blunting of human stress response predisposes and/or mediates these syndromes. This may be manifested as: blunting of one or more hypothalamic-pituitary axes, globally increased peripheral and/or visceral nociception, or instability of the autonomic nervous system. In fact, several lines of evidence indicate that a dysregulation of the autonomic nervous system might play a role in fibromyalgia. Thus, muscle sympathetic activity appears to be reduced in fibromyalgia (Elam et al., 1992). Qiao et al. (1991) demonstrated decreased microcirculatory vasoconstrictor responses to both cold and auditory stimulation and a high baseline skin conductance. More recently, a blunted sympathetic response to stressors was reported when heart rate variability or tilt table testing has been used to analyze autonomic responses (Clauw et al., 1996a,b). Perhaps the most consistent finding regarding autonomic function is that fibromyalgia patients have an impaired sympathetic ability to respond to stressors such as exercise, muscle contraction, and noise (Qiao et al., 1991; Elam et al., 1992; van Denderen et al., 1992). The autonomic nervous system has not been as extensively studied in CFS, although these patients have been found to experience a high prevalance of neurally mediated hypotension on tilt table testing, which is related to autonomic dysfunction (Rowe et al., 1995), since ₂-AR antagonists increase sympathetic outflow (see Section XIV.) and inhibit TNF- production (Haskó et al., 1995a; Elenkov et al., 1996). Taking these interactions into account, ₂-AR antagonists are recommended for the treatment of fibromyalgia and chronic fatigue syndrome.

The amount of IL-12 available at the tumor site appears to be critical for tumor regression (Colombo et al., 1996). Thus, low levels of IL-12 have been associated with tumor growth, as opposed to tumor regression observed with administration of IL-12 delivered in situ or systemically. On the other hand, local overproduction of IL-10 and TGF-, by inhibiting the production of IL-12 and TNF- and the cytotoxicity of Tc, NK cells, and macrophages, seems to play an inappropriate immunosuppressive role, allowing increased malignant tumor growth (Chouaib et al., 1997). These and others studies suggest that local and/or systemic Th1 functions and cellular immunity are down-regulated during tumor growth.

Few recent studies suggest that CA-mediated effects might be involved in an increased susceptibility to tumors, tumor growth, and metastases. In animals, -AR stimulation suppresses NK cell activity and compromises resistance to tumor metastases (Shakhar and Ben-Eliyahu, 1998), whereas stress has been reported to decrease the potential of spleen cells to turn into antitumor Tc against syngeneic B16 melanoma, and it significantly suppresses the ability of tumor-specific CD4⁺ cells to produce IFN- and IL-2 (Li et al., 1997). Mice subjected to unilateral superior cervical ganglionectomy showed slowed growth of two breast cancer lines after implantation in the sympathetically denervated skin (Romeo et al., 1991). In humans, the augmentation of the rate of tumor progression and cancer-related death has been associated with stress (cf. Li et al., 1997). In nonmedicated advanced cancer, patients showing long symptomless periods had all normal values, whereas those who remained free of symptoms for only a short time had raised NE, epinephrine, and cortisol levels (Lechin et al., 1990). These data suggest that CA-mediated suppression of cellular immunity may play a role in increased growth of certain tumors.

	XIV. Pharmacological Manipulation of the Sympathetic-Immune Interface

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In the CNS, the adrenergic inhibition of sympathetic discharges is assigned to ₂-ARs. The release of NE from varicosities of sympathetic nerve terminals in response to axonal activity is subjected to negative feedback modulation via ₂-ARs (Starke, 1981): NE released from the varicosity reduces its own release. It has been shown that the presynaptic ₂-ARs, responsible for negative feedback modulation of NE release (Elenkov and Vizi, 1991; Hasko et al., 1995b), are sensitive to prazosin, an _2B/C antagonist (cf. Docherty, 1998), and sympathetic varicose terminals do not make synaptic contact with the immune cells. A very similar interaction exists between noradrenergic varicosities and other neurons (cf. Vizi and Kiss, 1998; Vizi, 2000).

Thus, application of selective ₂-AR antagonists brings about a type of disinhibition that results in increased release of NE and increased sympathetic output (cf. Vizi, 1979, 2000; Vizi and Labos, 1991). We have shown that mice treated with the highly selective ₂-AR antagonist CH-38083 (Vizi et al., 1986) showed a blunted LPS-induced TNF- response (Hasko et al., 1995a; Elenkov et al., 1996). This effect was blocked by propranolol, indicating that the excessive stimulation of -ARs is responsible for the action of the ₂-AR antagonist. In addition, in in vivo condition, it was also shown that ₂-AR activation by clonidine (Szelényi et al., 2000a,b) increased TNF- production that had been reduced by isoproterenol in mice pretreated with reserpine. Under this condition, the sympathetic innervation is impaired by reserpine, and the effect of clonidine can be as direct effect on cells producing TNF-. These findings together with those obtained with ₂-AR antagonists (Haskó et al., 1995a; Elenkov et al., 1996) suggest that the plasma levels of TNF-, i.e., the in vivo production of this cytokine mainly directed by NE(E) via ₂-ARs overcoming ₂-ARs. The central stimulant and sympathomimetic amphetamine evokes the release of NE by displacing cytoplasmic NE, which is released by a carrier-mediated process from the noradrenergic nerve terminal. It has been shown that in vivo administration of this drug inhibits the proliferative response of mouse lymph node cells and IL-2 production from spleen cells (Heilig et al., 1993).

Morphine administration activates the sympathoadrenal system and increases plasma CA concentration via stimulation of opioid µ-receptors at discrete hypothalamic and brainstem sites (cf. Bencsics et al., 1997a). Thus, morphine, apart from this direct effect, may exert immunomodulatory properties via stimulation of SNS. In fact, recent studies in mice indicate that the suppressive effect of morphine, in vivo, on the proliferative response of splenic cells to mitogens or LPS-induced TNF- production was prevented by chlorisondamine, a ganglionic blocker or -AR antagonists, suggesting that these effects require intact sympathetic outflow (Fecho et al., 1993, 1996; Bencsics et al., 1997a). In addition, it appears that morphine potentiates the -AR responsiveness of human mononuclear cells through stimulation of µ-receptors (Pende et al., 1995).

Peripherally, the pharmacological modulation of the sympathetic-immune interface can be achieved at different levels. As already discussed, the release of NE in lymphoid organs can be modulated via several presynaptic receptors. For example, the application of the highly selective ₂-AR antagonist CH-38083, by blocking the presynaptic negative feedback exerted by NE, increases the release of endogenous NE in lymphoid organs (Elenkov and Vizi, 1991; Vizi et al., 1995). This presynaptic effect may contribute, at least in part, to the above-mentioned inhibitory effect of this drug on TNF- production, in vivo. Prazosin, an ₁ (but also an _2B/C-AR antagonist) had a similar effect on TNF- production in mice (Elenkov et al., 1996). Neomycin, gentamycin, and streptomycin, aminoglycoside antibiotics induce high affinity agonist binding of G_s-protein-coupled -AR receptors (Herrmann et al., 1989). Thus, these antibiotics may amplify the effect of CAs transmitted via G-protein-coupled -ARs.

Moreover, isoproterenol, a -AR agonist, inhibits LPS-induced TNF- production while potentiating IL-6 and IL-10 production in mice (Elenkov et al., 1995; Hasko et al., 1995a, 1998a). Treatment with the ₂-AR agonist salbutamol inhibits IL-12 production in humans while potentiating the ex vivo release of Th2-type cytokines in mice (Panina-Bordignon et al., 1997). These effects of -AR agonists might be related to the already mentioned beneficial effect of isoproterenol in EAE (see text above). The -AR antagonist propranolol removes the inhibitory effect of endogenously released NE on cytokine-producing cells, and is able to significantly increase the TNF- and IL-12 production induced by LPS in mice in a dose-dependent manner (Elenkov et al., 1995; Hasko et al., 1998a).

Rolipram, an antidepressant extensively studied in humans, also increases the intracellular availability of cAMP in lymphoid cells by selective inhibition of phosphodiesterase type IV. Several recent studies indicate that this drug inhibits, both in vitro and in vivo, the production of type 1/pro-inflammatory cytokines, such as TNF-, IFN-, and IL-12 (Ross et al., 1997; Hasko et al., 1998b; Liang et al., 1998). These effects, however, are independent from the simultaneous increase of IL-10 production (Ross et al., 1997; Hasko et al., 1998b). Moreover, the suppressive effects of this drug on type 1 cytokines, in parallel with the increase of IL-10 have been recently linked to rolipram-induced prevention and amelioration of the course of experimental collagen-induced arthritis and EAE in rodents and nonhuman primates, and diabetes in NOD mice (Genain et al., 1995; Ross et al., 1997; Liang et al., 1998). Recent evidence indicates that all these experimental models of autoimmune diseases are driven by overproduction of type 1 cytokines, particularly IL-12 and TNF- (Segal et al., 1998).

	XV. Conclusions

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The presence of sympathetic/noradrenergic nerve fibers in lymphoid organs, the release of NE from the sympathetic nerve terminals in these organs, and the expression of adrenoreceptors on lymphoid cells, which are able to respond functionally to stimulation, suggests that NE may meet the criteria for neurotransmitter/neuromodulator in lymphoid organs. The varicose axon terminals of the sympathetic nerve do not make synaptic contact with immune cells. Similar to many organs in the periphery, the release of NE is subject to presynaptic modulation via _2A/C-ARs. NE released from sympathic axon terminals diffuses far away from the release site; therefore NE transmits its signals nonsynaptically. Thus, the SNS may provide major integrative and regulatory pathway between the CNS and the immune system.

Sympathetic-immune interactions are undoubtedly complex. A few recent studies suggest that endogenous CAs modulate the function of primary lymphoid organs, such as the bone marrow and the thymus. However, the role of sympathetic innervation and endogenous CAs in regulation of hematopoiesis and thymocyte development remains poorly understood. In addition, there is almost complete lack of knowledge about how CAs might affect mucosal immunity.

Evidence accumulated in the last decades indicates that, peripherally, both NE released from the nonsynaptic sympathetic nerve terminals in lymphoid organs and blood vessels and epinephrine released from the adrenal medulla are involved in fine tuning of immune responses. Very similarly, steroid synthesis and secretion in the adrenal cortex is also under direct local tuning by NE and/or DA released from nonsynaptic noradrenergic varicosities in the zona glomerulosa. Morphological and neurochemical evidence indicates that a substantial proportion of the noradrenergic nerve endings lie in close proximity to zona glomerulosa cells without making synaptic contact, thus providing evidence for a direct local modulatory role of catecholamines in steroid secretion (Vizi et al., 1992, 1993; Bornstein and Vaudry, 1998; Szalay et al., 1998; Vizi, 1998). These noradrenergic neurons, like other noradrenergic neurons in the central nervous system, are able to take up DA, convert it partly into NE, and to release both NE and DA together with the cotransmitter ATP when neuronal activity drives them to do so (Vizi et al., 1993).

The effects of CAs are quick, within minutes. This modulation might be ideally designed for quick adjustment of immune responsiveness. Another important role of CAs in the periphery might be a tonic inhibition of certain immune functions, and particularly, the production of type 1/pro-inflammatory cytokines. Importantly, CAs appear to exert systemically differential, opposite effects on cellular and humoral immunity. By inhibiting type 1 and potentiating type 2 cytokine production and by acting directly on effector cells, CAs suppress cellular and boost humoral immunity. The Th2-driving effects of CAs may have, however, under certain conditions, both beneficial and detrimental consequences.

Although interest in the Th2 response was initially directed at its protective role in helminthic infections and its pathogenic role in allergy, this response may have important regulatory functions in countering the tissue-damaging effects of macrophages and Th1 cells (Fearon and Locksley, 1996). Thus, the above-mentioned Th2-driving force of CAs, in concert with the effect of glucocorticoids, might be a part of an important feedback mechanism (Fig. 1). Thus, an excessive immune response, through activation of the stress system, and hence, through glucocorticoids and Cas, suppresses the Th1 response and causes a Th2 shift. These beneficial effects may protect the organism from "overshooting" by type 1/pro-inflammatory cytokines and other products of activated macrophages with tissue damaging potential.

On the other hand, the substantial Th2-driving force of endogenous CAs can be amplified to a great extent during certain conditions such as severe acute, subacute, or chronic stress. For example, in major injury, a condition followed by a sympathetic storm, these effects of CAs may contribute to serious infectious complications. Therefore, a defect in the sympathetic-immune interface, or an abnormal activity of the SNS in either direction, might contribute at a certain point to the pathophysiology of common human diseases, where a selection of Th1 (type 1) versus Th2 (type 2) responses plays a significant role. These include several infections, major injury and its complications, allergic (atopic) reactions, autoimmune/inflammatory diseases, and tumor growth.

Locally, as stated above, the SNS may exert pro- or anti-inflammatory effects. This may be influenced by several factors, such as the presence or absence of antigen, the nature of antigen, and/or the presence and relative expression of particular receptor subtypes on the surface of immune cells (e.g., ₂- versus ₂-adrenergic receptors), the type of G-protein coupled to the ₂-AR (e.g., G_s versus G_i), the stage of activation/differentiation of the cell, or the presence or absence of a particular receptor (e.g., ₂-ARs on Th1 but not on Th2 cells). Thus, the precise mapping and type of adrenoreceptor expression on different lymphoid cells and their coupling to intracellular pathways, according to their stage of maturation, differentiation, and tissue localization, clearly need further studies. This may provide new tools for pharmacologic tailoring of inflammatory conditions.

In summary, the immune system is not autonomous; the SNS and HPA axis may represent the major communication channels through which the CNS superimposes its control on the immune system. Better knowledge and understanding of the physiology and pathophysiology of the sympathetic-immune interface may help the development of new therapeutic strategies for common human diseases. Thus, blocking the effects of SNS or stress by ₂-AR antagonists or administration of ₂-AR agonists (used as antihypertensive drugs) may result in the boosting of Th1 responses that may be useful in the management of certain intracellular infections or tumors. The administration of ₂-AR agonists, ₂-AR antagonists, and/or selective inhibitors of PDEIV may help in the management of Th1-mediated autoimmune diseases, such as RA^. and MS. In addition, ₂-AR-antagonists able to increase noradrenergic activity might have therapeutic benefits in the treatment of fibromyalgia and chronic fatigue syndrome.