I. Background

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The T cell-dependent antibody response is a critical component of adaptive immunity and serves as both a sentinel and a defender against bacterial and viral infections. In addition, the potential exists for the T cell-dependent antibody response to contribute to the development of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus (reviewed in Boitard, 1992; Goodnow, 1997). In light of this potential for antibody production to both protect and damage the body, the immune system has developed a number of autoregulatory mechanisms to augment the antigen-specific response directed against a foreign antigen and, at the same time, to prevent responses directed against autoantigens. These regulatory mechanisms govern both B cell and T cell activation, as well as effector function during the T cell-dependent antibody response.

The two-signal hypothesis of B cell activation, as first described by Bretscher and Cohn (1970), represents one of these autoregulatory mechanisms (Fig. 1A). They proposed that B cell activation requires two signals, with signal 1 originating from stimulation of the antigen-specific B cell receptor (BCR) by a foreign antigen. Upon stimulation of the BCR, the B cell begins to prepare itself to produce antibody. However, without receiving another signal originating from the CD4⁺ T-helper (Th) cell, the B cell will not differentiate into either an antibody-secreting plasma cell or a memory B cell. This second signal from the Th cell was originally proposed to be in the form of cytokines. Thus, during a T cell-dependent antibody response, the B cell will differentiate into either an antibody-secreting plasma cell or a memory cell following both BCR stimulation and CD4⁺ T cell cytokine production.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Two-signal hypotheses of B and T cell activation. A, two-signal hypothesis of B cell activation (Bretscher and Cohn, 1970). Signal 1 to the B cell occurs upon stimulation of the B cell receptor (BCR) by antigen (Ag). Following signal 1, the B cell prepares to produce antibody (Ab) and awaits signal 2, which is delivered in the form of cytokine receptor stimulation. Only upon delivery of signal 2 will the B cell differentiate into either a plasma cell or a memory B cell. Without this costimulatory signal, the B cell will undergo apoptosis or anergy. B, two-signal hypothesis of CD4+ T cell activation (Lafferty and Cunningham, 1975). A resting CD4+ T cell receives the first activation signal following stimulation of the T cell receptor (TCR) by the MHC class II-antigen (Ag) peptide complex expressed by a professional antigen-presenting cell (APC). If the T cell does not receive signal 2, a costimulatory signal, the T cell is either anergized or induced to undergo apoptosis. However, if the T cell does receive a costimulatory signal, the T cell is activated to produce cytokines.

Similar to the process of B cell activation, an antigen-specific Th cell also requires two distinct signals to become activated to provide "help" to a B cell (Fig. 1B). As first proposed by Lafferty and Cunningham (1975), the first of these signals is generated by the recognition of the peptide antigen by the antigen-specific TCR expressed on the Th cell surface, which is now known to be presented by the B cell or another antigen-presenting cell in the context of MHC class II (the peptide-MHCII complex). In addition, if the Th cell receives costimulatory signals from the B cell, then the Th cell becomes fully activated to produce cytokines that provide the second signal, or "help", required by the B cell to differentiate into either an antibody-secreting plasma cell or a memory B cell. However, if the Th cell does not receive the additional costimulatory signals required for cell activation, such as a B7:CD28 interaction, the cell is either anergized or induced to undergo apoptosis (Schwartz, 1990). Thus, the antigen-specific physical interaction between the CD4⁺ Th cell and the B cell represents a potent regulator of the Th cell-dependent antibody response (Sanders et al., 1986, 1988), which includes antibody secretion from plasma cells, affinity maturation of the BCR, antibody isotype switching, and memory B cell formation (Liu and Banchereau, 1997).

In addition to regulatory mechanisms that are provided by immune cells, it is now known that complex bidirectional interactions (Fig. 2) between the cells of the immune system and the nervous system contribute to additional regulatory mechanisms that influence the function of cellular activities associated with both systems (reviewed in Sanders and Munson, 1985a; Ader et al., 1990; Madden and Felten, 1995; Straub et al., 1998; Kohm and Sanders, 2000).

View larger version (53K): [in this window] [in a new window]   Fig. 2.   Pathways of communication between the central nervous and immune systems. The presence of sympathetic nerve fibers in lymphoid organs and the release of norepinephrine from nerve terminals located in the direct vicinity of immune cells provide a mechanism by which norepinephrine might influence immune cell function. Upon release, norepinephrine binds to the 2-adrenergic receptor expressed on the surface of a variety of immune cells to influence their activity. The activity of sympathetic nerves originating in the central nervous system (CNS) may be influenced by products of activated immune cells because circulating cytokines and cells are actively transported into the CNS to influence their activity centrally and, also, by stimulation of cytokine receptors expressed on peripheral nerves to influence their activity peripherally. More importantly, a small number of lymphocytes actively patrol the normal CNS, but upon activation, increased numbers of lymphocytes enter the CNS and produce both cytokines and antibodies that can either protect against or contribute to a number of CNS pathologies. Finally, hormone production resulting from activation of the hypothalamic-pituitary-adrenal (HPA) axis may also influence a variety of systemic immune cell activities.

One mechanism by which signals from the immune system may regulate nervous system activity is via the stimulation of cytokine receptors expressed both on cells within the CNS (reviewed in Rothwell et al., 1996) and on peripheral sympathetic nerves and ganglia (Hart et al., 1993; Gadient and Otten, 1996; Marz et al., 1996; Cunningham et al., 1997). Cytokine receptor stimulation on peripheral nerves and ganglia alters the level of CNS activity possibly by afferent signal transduction. But more importantly, alterations in the level of CNS activity may alter the level of efferent nerve activity and neurotransmitter release in the periphery. Thus, peripheral cytokine production may influence efferent nerve activity and neurotransmitter release by binding to cytokine receptors expressed on peripheral nerves. In addition, cytokines produced in the periphery may cross the blood-brain barrier (BBB) via a number of specific cytokine transporter systems within the BBB to directly affect targets within the CNS (Banks and Kastin, 1987, 1991; Banks et al., 1991; Gutierrez et al., 1993; Plotkin et al., 1996). Additionally, activated immune cells may pass through the BBB to release cytokines directly into the CNS (reviewed in Weller et al., 1996), thus bypassing the need for afferent signaling pathways from the periphery. The integrity of the BBB can be disrupted under certain pathological conditions, such as viral infection of the CNS, allowing immune cells and other blood-borne mediators to enter the CNS (reviewed in Persidsky, 1999). Therefore, several mechanisms exist for the immune system to communicate with the CNS. However, in order for the CNS to influence an immune response, reciprocal pathways of communication from the CNS to the immune system must also exist.

The sympathetic nervous system is typically associated with the physiological "fight or flight" response, such that it is involved in the regulation of cardiovascular and respiratory function, especially during times of critical need. In addition, the sympathetic nervous system regulates gastrointestinal tract smooth muscle contraction/relaxation, gastric secretions, and other autonomic functions. Sympathetic neurotransmission from the CNS to the periphery occurs via projections extending from the paraventricular nucleus of the hypothalamus, rostral ventrolateral medulla, ventromedial medulla, and caudal raphe nucleus to preganglionic neurons of the spinal cord (Sawchenko and Swanson, 1982). The preganglionic cell bodies of sympathetic nerves reside in the intermediolateral cell column of the lateral horn of the spinal cord at T1-L2. These cell bodies send myelinated projections that exit the spinal cord via the ventral roots to synapse primarily on the superior mesenteric ganglia. From these ganglia, a second projection follows the vasculature to innervate target organs. Within the target organ, sympathetic nerves form terminals from which the sympathetic neurotransmitter norepinephrine (NE) is released to bind to adrenergic receptors expressed by various cell populations.

Most studies of sympathetic innervation of lymphoid organs incorporated immunohistological techniques in which the rate-limiting enzyme of norepinephrine synthesis, tyrosine hydroxylase, was detected. These studies demonstrated a rich sympathetic innervation of all primary (thymus and bone marrow) and secondary (spleen and lymph nodes) lymphoid organs (Calvo, 1968; Reilly et al., 1979; Williams and Felten, 1981; van Oosterhout and Nijkamp, 1984; Felten et al., 1988a,b). Additionally, these studies reported the presence of sympathetic innervation in both the splenic capsule and trabeculae, but more importantly, in the immune cell compartment of the spleen (the white pulp), especially the T cell-rich periarteriolar lymphoid sheath, the B cell-rich marginal zone, and marginal sinus areas (Felten et al., 1985, 1987a,b; Livnat et al., 1985; Ackerman et al., 1987; Felten and Olschowka, 1987). Whereas innervation is prominent in the white pulp, little innervation is present in the red pulp and represents less than 1% of the total splenic innervation. Electron microscopic studies of the white pulp reveal that sympathetic nerve terminals are in direct apposition to T cells and adjacent to both interdigitating dendritic cells and B cells (Felten et al., 1987a,b), with the neuro-immune junction being approximately 6 nm wide (Felten and Olschowka, 1987), in contrast to a typical CNS synapse that is approximately 20 nm wide. The close proximity of sympathetic nerve terminals to immune cells provides a mechanism not only for specific targeting of norepinephrine release to immune cells, but also for the containment of neurotransmitter release, possibly to permit differential modulation of only resident immune cells, depending on the specific immune response being evoked.

Finally, GAP-43 (a marker for an activated neuron)-positive sympathetic fibers enter the outer periarteriolar lymphoid sheath, marginal zone, and marginal sinus within the spleen following immunization, suggesting that not only can the immune response influence sympathetic outflow, but also that immune cell-derived neurotrophic factors may direct innervating fibers to the site of the response (Yang et al., 1998; Besser and Wank, 1999) to release norepinephrine to bind to -adrenergic receptors (ARs) expressed on immune cell populations. Thus, a complete "circuit" appears to exist between the immune system and the CNS, such that the initiation of an immune response in the periphery signals the CNS, resulting in subsequent regulation of the immune response via activation of the sympathetic nervous system.

In summary, whereas behavioral conditioning studies provided the initial suggestion that an interaction between the CNS and immune system existed (Ader and Cohen, 1975; Rogers et al., 1976; Wayner et al., 1978; Cohen et al., 1979; Exton et al., 1998), research findings over the past 20 to 30 years have documented a number of complex bidirectional interactions between the nervous system and the immune system that appear to be necessary for the maintenance of homeostasis in both systems, as well as for the regulation of immune responses during the development and progression of immune-related disease states.

The catecholamine norepinephrine is released from both postganglionic sympathetic nerve terminals found innervating all internal organs and from chromaffin cells residing in the adrenal medulla. Norepinephrine is the principal neurotransmitter of the sympathetic nervous system and is released into the periphery upon activation of the sympathetic nervous system. Norepinephrine is produced via multiple enzymatic alterations of tyrosine, of which the hydroxylation of tyrosine by tyrosine hydroxylase is the rate-limiting step (Zigmond et al., 1989). This enzymatic cascade is initiated upon the activation of sympathetic postganglionic nerve fibers. The final step in norepinephrine synthesis occurs within the nerve terminal storage vesicles and is mediated by the membrane-bound dopamine -hydroxylase. Various fates await norepinephrine upon release from the nerve terminal, such as metabolization into normetanephrine by catechol-O-methyltransferase, reuptake back into the nerve terminal, diffusion, or receptor binding to influence target cell function.

The -adrenergic family of receptors (ARs) binds norepinephrine and contains three subtypes: the 1AR, the 2AR, and the 3AR (reviewed in Bylund et al., 1994). The AR is a seven-transmembrane receptor that classically leads to heterotrimeric guanine nucleotide-binding protein (G-protein) activation upon stimulation. Historically, the signaling capacity of the AR has been attributed to the association of the cytoplasmic tail of the receptor with stimulatory G-proteins, in which stimulation of the receptor results in adenylyl cyclase activation, increased intracellular accumulation of adenosine 3',5'-cyclic monophosphate (cAMP), and increased protein kinase A (PKA) activity (reviewed in Kobilka, 1992; Meinkoth et al., 1993). Upon activation, PKA regulates the activity of multiple targets via phosphorylation, including various transcription factors, such as NF-B. Although stimulation of each of the three AR-subtypes results in adenylyl cyclase activation, the 2AR appears to be more efficiently coupled to adenylate cyclase than is the 1AR or 3AR (reviewed in Strosberg, 1997).

However, over the last 5 to 10 years, a number of other signaling pathways have been reported to be activated following 2AR stimulation. One such pathway that is also relevant to lymphocyte function is the activation of protein kinase C (PKC). 2AR stimulation induces PKC activity (Kelleher et al., 1984), which may then mediate a number of intracellular events, including down-regulation of 2AR surface expression (Kelleher et al., 1984), positive or negative effects on adenylyl cyclase activity (reviewed in Houslay, 1991), and activation of Bruton's tyrosine kinase (reviewed in Mohamed et al., 1999) which ultimately activates the mitogen-activated protein kinase (MAPK) pathway. These same 2AR-induced signaling pathways are also involved in BCR signaling and, therefore, represent a mechanism by which 2AR stimulation may influence intracellular events in B cells following antigen recognition. Similarly, 2AR and BCR stimulation both result in Src kinase activation, which may induce down-regulation of 2AR expression (Daaka et al., 1997; Cornall et al., 1998; Lankar et al., 1998) and Ras activation (Daaka et al., 1997), as well as a number of additional intracellular events associated with BCR stimulation. Thus, as will be discussed later, due to the existence of overlapping intracellular signaling pathways associated with stimulation of the 2AR and the BCR, it is not surprising that stimulation of the 2AR by either an agonist or norepinephrine may influence B cell function.

	II. Evidence and Mechanisms for the Release of Norepinephrine in Lymphoid Organs

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As previously discussed, lymphoid organs are heavily innervated by sympathetic nerve fibers. However, in order for norepinephrine to influence immune cell function, it must be released at the immediate site of action, since it is either rapidly degraded by catechol-O-methyltransferase and monoamine oxidase, diffused into the circulation, or taken back up into the nerve terminal following release (reviewed in Glowinski and Baldessarini, 1966). Therefore, if norepinephrine is to influence immune cell function in response to antigen, it may be critical that mechanisms exist for enhancing the normally low basal level of norepinephrine released within the microenvironment in which immune cells reside (findings summarized in Table 1).

Splenic norepinephrine is derived from local sympathetic nerve terminals, as opposed to circulating catecholamine (Williams et al., 1981; Shimizu et al., 1994), and the electrical stimulation of the postganglionic splenic nerve results in norepinephrine release within the spleen (Lundberg et al., 1989). The rate of norepinephrine release from sympathetic nerve terminals is regulated by both positive and negative feedback mechanisms. For example, the release of norepinephrine from sympathetic nerve terminals is inhibited via stimulation of the ₂-adrenergic receptors (2AR) expressed on the presynaptic nerve terminal itself but is enhanced by stimulation of the presynaptic 2AR (Elenkov and Vizi, 1991; Hasko et al., 1995; Vizi et al., 1995). In addition, although there are conflicting reports concerning the level of sympathetic nerve activity and the release of norepinephrine within the spleen during an immune response, there is increasing evidence that immune-derived factors may also influence the rate of norepinephrine release within lymphoid organs.

A. Lipopolysaccharide- and Antigen-Induced Norepinephrine Release

1. Infection/Endotoxin. For many years, it has been known that systemic infection induces sympathetic nervous system activity via the endotoxin released from bacterial cell walls. Early studies reported alterations in the level of sympathetic nerve activity both in times of infection and shock by measuring circulating levels of norepinephrine and epinephrine as an indirect indicator of systemic sympathetic nerve activity (Heiffer et al., 1960; Rosenberg et al., 1961; Spink et al., 1966; Devereux et al., 1977; Feuerstein et al., 1981). In all of these studies, endotoxin exposure increased the levels of circulating norepinephrine, suggesting enhanced sympathetic nerve activity and norepinephrine release. In other studies, the total tissue content of norepinephrine was determined as a measure of sympathetic nerve activity following infection with Escherichia coli or injection of E. coli-derived endotoxin. Immunization of animals with endotoxin resulted in a significant decrease in the total tissue content of norepinephrine in the spleen (Zetterstrom et al., 1964; Pohorecky et al., 1972), possibly via altering norepinephrine reuptake mechanisms (Pardini et al., 1982). Such observations were interpreted in several ways, i.e., the decreased splenic norepinephrine levels may have been due to decreased norepinephrine production, increased norepinephrine release, increased norepinephrine diffusion/metabolism, and/or decreased reuptake of norepinephrine back into the nerve terminal.

2. Particulate Antigens/Sheep Red Blood Cells. In addition to infectious challenge, other types of immune stimuli may also influence the rate of norepinephrine release within lymphoid organs. One of the earliest studies reporting a correlation between the splenic content of norepinephrine and an ongoing immune response to a particulate antigen was performed by Besedovsky et al. (1979). Immunization of animals with the particulate T cell-dependent antigen sheep red blood cells (sRBC) decreased the total norepinephrine content of the spleen in comparison with control animals. In later studies, this same group extended their findings to note that 3 days after immunization of rats with sRBC, the total norepinephrine content was lower in the spleen, lymph nodes, and thymus of immunized animals in comparison with nonimmunized control animals (Del Rey et al., 1981). Importantly, the effect of sRBC-induced immune cell activation on sympathetic nerve activity may be influenced by central regulatory mechanisms. For example, since central norepinephrine inhibits hypothalamic neuronal activity and efferent sympathetic nerve activity, the observation by Besedovsky and colleagues (1983) that sRBC-induced immune cell activation decreased the rate of norepinephrine release in the hypothalamus suggested less norepinephrine-mediated inhibition of CNS activity and increased sympathetic nerve activity in the periphery. Taken together, these findings support the hypothesis that sRBC-induced immune cell activation decreases the total lymphoid tissue content of norepinephrine by increasing the level of sympathetic nerve activity and norepinephrine release.

3. Soluble Protein Antigen. In light of the previously discussed studies concerning the role of infectious challenge and particulate antigens on the level of sympathetic nerve activity, one study has used an antigen-specific model system to investigate the effects of a cognate soluble protein antigen on the rate of norepinephrine release in lymphoid organs by norepinephrine turnover analysis (Kohm et al., 2000). Severe combined immunodeficient (scid) mice were reconstituted with keyhole limpet hemocyanin (KLH)-specific Th2 cell clones and freshly isolated trinitrophenyl (TNP)-specific B cells prior to immunization with the cognate antigen TNP-KLH. Activation of Th2 cells and B cells increased the rate of norepinephrine release in the spleen and bone marrow 18 to 25 h, but not 1 to 8 h, following immunization. Since the rate of norepinephrine release was not measured between 8 and 18 h following immunization in these studies, it is possible that immune cell activation increased the rate of norepinephrine release at a time earlier than 18 h after immunization. Importantly, it was shown that immunization of scid mice reconstituted with antigen-specific cell populations with a noncognate antigen (fluorescein ovalbumin) that would not activate either the Th2 cells or B cells, but would activate resident macrophages, did not alter the rate of norepinephrine release in the spleen and bone marrow. Thus, these findings suggested that macrophage activation and inflammatory cytokine production are not responsible for the soluble protein antigen-induced increase in sympathetic nerve activity in this model system and that a cognate interaction between Th2 cells and B cells is necessary for soluble protein antigen-induced enhancement in norepinephrine release by a currently undetermined mechanism.

The hallmark experiments of Besedovsky et al. (1983) suggested that activated immune cells secrete "soluble factors" into the circulation that ultimately enter the CNS to stimulate neuronal activity in both the hypothalamus and brainstem. These studies were some of the first to show that soluble factors produced by cells of the immune system could alter noradrenergic nerve activity in the brain, as measured by changes in hypothalamic and brainstem norepinephrine content following -methyl-p-tyrosine inhibition of norepinephrine synthesis. It is now known that these soluble factors were cytokines and that their transport into the CNS represents one possible mechanism of immune-to-brain communication. However, in order for cytokines to leave the blood and enter the CNS, a major obstacle must be overcome. The BBB, which is characterized by the astrocyte-mediated formation of tight junctions between endothelial cells composing the CNS vasculature, limits the entry of blood-borne proteins and cells into the CNS. The passage of molecules across the BBB is regulated on a variety of levels, including size, charge, lipophilicity, and adhesion molecule expression (Banks and Kastin, 1985a,b, 1987). Thus, the BBB serves as a biological filter for entry into the CNS. However, although several mechanisms exist for either the passage of, or signaling by, blood-borne cytokines into the CNS, this communication pathway is not considered a primary line of communication from the immune system to the CNS for a number of reasons, including: 1) the low concentration of cytokines present at the BBB, 2) the lack of specificity of cytokine signaling directly to the CNS, and 3) the observation that certain cytokine-related illnesses occur in the absence of detectable serum cytokine elevation (Kluger, 1991). Thus, although mechanisms exist for the transport of cytokines into the CNS, one alternative mechanism for immune cell-derived cytokines to signal the CNS is through the stimulation of cytokine receptors expressed on peripheral sensory nerves. By this mechanism, immune responses occurring near sites of sensory innervation could easily communicate signals to the CNS.

The interleukin-1 receptor (IL-1R) was the first cytokine receptor reported to be expressed on peripheral sensory nerves and, thus, became the focus of early studies concerning cytokine communication from the periphery to the CNS. IL-1 is a primary product of activated macrophages (Dinarello, 1998) and was a leading candidate for a mediator of the LPS-induced increase in sympathetic nerve activity and norepinephrine release. In addition, early studies suggested the presence of the IL-1R on peripheral nerves, because the peripheral administration of IL-1 increased CNS activity (Saphier and Ovadia, 1990; Dunn, 1992). However, these studies did not directly measure the expression of IL-1 receptors on peripheral nerves. A number of other studies have reported that peripheral administration of IL-1 resulted in increased vagus nerve activity, suggesting not only that IL-1 receptors are expressed on peripheral nerves, but that stimulation of these receptors by their specific cytokines may induce afferent nerve activity to the CNS (reviewed in Maier et al., 1998). In addition, some of these studies reported a CNS-localized effect of peripheral IL-1 administration as measured by cytokine-induced hyperalgesia, which can be blocked via administration of an IL-1R antagonist. Thus, not only did peripheral administration of IL-1 stimulate IL-1 receptors expressed in the periphery to induce vagal nerve activity, but in addition, it altered the CNS response to pain.

In later studies, the role of the vagus nerve in transmitting IL-1-induced signals to the CNS was further explored. For example, the injection of either LPS, a bacterial protein product that activates macrophages to secrete cytokines, including IL-1, or the injection of IL-1 itself into the peritoneal cavity of mice and rats resulted in fever, hypothalamic norepinephrine depletion, and increased c-fos and acetylcholine expression in the brain (Fleshner et al., 1995; Gaykema et al., 1995; Sehic and Blatteis, 1996). Importantly, the effect of peripheral IL-1 on hypothalamic levels of norepinephrine were blocked by subdiaphragmatic vagotomy, suggesting a role for vagal afferents in mediating the effect of IL-1 on norepinephrine levels within the CNS (Fleshner et al., 1995). These findings were later supported by the observation that vagal paraganglia express IL-1 receptors, providing a direct mechanism by which IL-1 can directly activate vagal nerve afferent fibers (Goehler et al., 1997). Finally, others have shown that cultured sympathetic neurons express a functional IL-1R and that stimulation of this receptor results in the activation of NF-B (Bai and Hart, 1998). Thus, it appears that the expression of functional IL-1 receptors on sympathetic nerves, such as the vagus nerve, provides one mechanism by which immune-derived cytokines can signal the CNS.

In addition to IL-1 receptors, the expression of other cytokine receptors on sympathetic nerves has been studied to a lessor extent. For example, sympathetic neurons appear to express too low a level of IL-6R to allow a functional effect of endogenous IL-6 on the neuron, but the exposure of sympathetic neurons to soluble IL-6R in vitro results in IL-6-induced neuron survival (Marz et al., 1998). This may be explained by the fact that the IL-6R ligand binding subunit does not possess tyrosine kinase activity, and therefore, IL-6-stimulated signaling relies on the dimerization of the ligand binding subunit of the IL-6R with the signaling subunit gp130 (reviewed in Dinarello, 1998). These studies suggest that although sympathetic neurons may express low levels of the ligand binding subunit of the IL-6R, they do express adequate levels of the signaling gp130 subunit. Thus, although sympathetic neurons may not constitutively express adequate levels of IL-6 binding subunits to respond to endogenous IL-6, either soluble IL-6R production or nerve injury may enhance the level of functional IL-6R expression on sympathetic nerves.

Finally, one study has detected the expression of IL-2 receptors on sympathetic neurons (Haugen and Letourneau, 1990). Using immunofluorescence staining, sympathetic neurons were shown to express detectable levels of IL-2R on their surface. In addition, treatment of cultured sympathetic neurons to IL-2 enhanced neurite outgrowth, suggesting that the IL-2R expressed by these cells is functional. Therefore, the presence of cytokine receptors on peripheral nerves provides a potential mechanism by which local immune cell-derived cytokines produced in the periphery may transmit signals to the CNS or to the peripheral nerve directly.

As previously discussed, the effect of antigen-specific Th2 cell and B cell activation on the rate of norepinephrine release in lymphoid organs was significantly decreased by ganglionic blockade (Kohm et al., 2000). These studies suggested that the activation of antigen-specific cell populations induced the local release of norepinephrine via a mechanism that relied partially on ganglionic transmission. In light of these findings, it is reasonable to hypothesize that an immune cell-derived signal stimulated a neuronal reflex mechanism of norepinephrine release dependent upon structures at, or above, the sympathetic ganglia. Because the diffusion of locally produced immune-derived factors into the circulation would produce extremely low concentrations of circulating cytokine and, thus, would unlikely be able to induce CNS-regulated norepinephrine release in the spleen, it was more plausible to hypothesize that some local mechanism existed that was capable of responding to immune-derived signals to induce norepinephrine release from local sympathetic nerve terminals.

An early study noted the presence of afferent unmyelinated type C nerve fibers in the spleen (Herman et al., 1982), although others have observed that a small percentage of afferent fibers of the splenic nerve are myelinated (Utterback, 1944; Calaresu et al., 1984). Later studies suggested that approximately 5% of the splenic nerve is composed of afferent nerve fibers as determined by horseradish peroxidase retrograde tracing (Baron and Janig, 1988). These afferent splenic nerve fibers arose from the spinal cord at levels ranging from T4 to L2. However, the most significant origin of sympathetic afferent fibers (approximately 60%) appeared to be from levels T10 to T13. Importantly, the stimulation of these splenic afferent nerve fibers activated a reflex response via the splenic nerve increasing the level of cardiopulmonary sympathetic efferent nerve activity (Herman et al., 1982). Because activation of splenic afferents influenced cardiac efferent sympathetic nerve activity in these studies, such a mechanism may also play a role in the low level of cardiac norepinephrine release following activation of antigen-specific cell populations (Kohm et al., 2000). Interestingly, afferent signals from the spleen did not seem to originate from the capsule innervation but, instead, from vasculature-associated interior innervation, which is the location of cytokine-producing cells in the spleen (Herman et al., 1982). Finally, other studies reported that afferent fibers supplying the spleen may be activated by immune-derived stimuli (Niijima et al., 1991; Fleshner et al., 1995). Taken together, these findings support the participation of afferent innervation in transmitting the signals induced by locally produced immune-derived products to increase the rate of local norepinephrine release in lymphoid organs.

Interestingly, other studies in rats reported the absence of afferent innervation of the spleen using techniques similar to those previously discussed (Nance and Burns, 1989). The origin of these conflicting data is currently unclear. However, because these conflicting studies were performed in different animal species, the presence of afferent fibers in the splenic nerve may be a species-dependent observation. The existence of splenic afferent innervation is further supported by the report of afferent nerve fibers in another species, the guinea pig (Elfvin et al., 1992). Thus, the presence of afferent innervation in the spleen provides a specific mechanism by which locally produced cytokines or other immune cell-derived products may stimulate sympathetic nerve activity and norepinephrine release.

Cytokines, which were once thought to only influence immune cell function, have now been shown to affect glial cell proliferation, neuron survival, neuronal proliferation and differentiation, and neurotransmitter expression (Giulian and Lachman, 1985; Yamamori et al., 1989; Jonakait and Schotland, 1990; Barbany et al., 1991; Freidin and Kessler, 1991; Hart et al., 1991; Schwartz et al., 1991; Brenneman et al., 1992). In addition, a number of studies have reported that a variety of cytokines may influence peripheral sympathetic nerve activity and the rate of norepinephrine release.

As previously discussed, numerous studies have suggested that exposure of animals to infectious challenge or bacterial products, such as endotoxin, increases the rate of norepinephrine release in lymphoid organs. In light of the critical role of macrophage activation and IL-1 production in clearing infections and the role of norepinephrine in regulating macrophage activity (Miles et al., 1996), it is not surprising that a significant number of studies have investigated the role of IL-1 in regulating the level of norepinephrine release in vivo.

One indication that IL-1 may influence the level of sympathetic nerve activity is demonstrated by its ability to influence CNS activity. Because the hypothalamus is an area within the CNS that controls efferent sympathetic nerve activity, an IL-1-induced increase in hypothalamic activity may enhance the level of efferent sympathetic nerve activity and the rate of norepinephrine release in the periphery. For example, peripheral injection of IL-1 enhanced both hypothalamic nerve activity and the level of CRF secretion from the hypothalamus (Sapolsky et al., 1987; Akiyoshi et al., 1990; Dunn, 1992; Fleshner et al., 1995). Also, peripheral IL-1 administration induced c-fos expression in CRF-producing cells in the paraventricular nucleus of the hypothalamus, suggesting that IL-1 increased hypothalamic neuronal activity (Ericsson et al., 1994). Because a number of studies have reported that peripheral IL-1 increases neuronal activity in the hypothalamus, it is reasonable to hypothesize that these IL-1-induced alterations in hypothalamic activity may translate into alterations in efferent sympathetic nerve activity.

Using a nonisotopic technique employing either -methyl-p-tyrosine to measure norepinephrine turnover in the spleen (Akiyoshi et al., 1990) or direct measurements of sympathetic nerve electrical activity (Niijima et al., 1991), it was shown that peripheral administration of IL-1 increased the rate of norepinephrine turnover in the spleen 1 to 6 h following exposure in a dose-dependent manner. Other studies measuring the level of sympathetic nerve electrical activity reported that peripheral IL-1 exposure increased the level of sympathetic nerve activity within 10 to 15 min of exposure in a dose-dependent manner (Takahashi et al., 1992) and that the rate of norepinephrine release in the spleen peaks within 40 min after peripheral IL-1 exposure (Ichijo et al., 1992; Shimizu et al., 1994). Finally, the effect of IL-1 on sympathetic nerve activity was specific for certain nerves, because it increased the rate of norepinephrine release in the spleen, but not in the heart (Akiyoshi et al., 1990). Because these studies administered IL-1 directly, it is not surprising that the rate of norepinephrine turnover increased much quicker than that in a study in which immune cells were activated via antigen exposure (Kohm et al., 2000). In contrast, others have reported an IL-1-induced inhibition of splenic sympathetic nerve activity as measured by microdialysis or inhibition of [³H]norepinephrine release from atria (Bognar et al., 1994; Abadie et al., 1997). Although the reason for these conflicting findings is currently unknown, the concentration of IL-1 used in these studies does not seem to be the source of these conflicting findings, inasmuch as studies reporting an IL-1-mediated enhancement of splenic norepinephrine release have used varying concentrations of IL-1.

Although the exact mechanism by which peripheral IL-1 increases the level of sympathetic nerve activity and the rate of norepinephrine release is currently unknown, prostaglandin synthesis may be a critical mediator of IL-1's effect on sympathetic nerve activity. For example, peripheral administration of cyclo-oxygenase inhibitors blocked the effect of IL-1 on sympathetic nerve activity in the spleen, suggesting a role for IL-1-induced prostaglandin synthesis in regulating norepinephrine release (Niijima et al., 1991). In addition, the production of CRF within the CNS appears to be another critical mediator of IL-1's effect on norepinephrine release, because central administration of a neutralizing antibody directed against CRF blocked the ability of peripherally administered IL-1 to increase the level of splenic norepinephrine release (Ichijo et al., 1992; Shimizu et al., 1994).

In summary, although there are conflicting reports concerning the level of splenic sympathetic nerve activity and norepinephrine release during an immune response, it appears that IL-1 may play an important role in mediating the level of sympathetic outflow in the spleen. However, IL-1-induced regulation of norepinephrine release may only occur during immune responses involving macrophage activation, because these cells are the principal source of the cytokine. Therefore, a few studies have determined the role of other cytokine receptors in modulating norepinephrine release in lymphoid organs.

For example, whereas IL-6 does not affect the uptake of [³H]norepinephrine into sympathetic nerve terminals, IL-6 does exert dose-dependent effects on sympathetic nerve activity. For example, 1 ng/ml IL-6 stimulated, 10 ng/ml IL-6 had no effect, and 100 ng/ml IL-6 inhibited [³H]norepinephrine release from sympathetic nerve terminals in vitro within 2 h of cytokine exposure (Ruhl et al., 1994). Importantly, the combination of subthreshold concentrations of IL-6 (10 ng/ml) and IL-1 (0.1 ng/ml) significantly suppressed the level of sympathetic nerve activity and was blocked by an antagonist of either the IL-6 or the IL-1 receptor. Finally, others have shown that low concentrations of both IL-2 (Bognar et al., 1994) and TNF- (Foucart and Abadie, 1996; Abadie et al., 1997) inhibited the rate of norepinephrine release in the spleen. Thus, IL-6, IL-2, and TNF- may either enhance, inhibit, or have no effect on the rate of norepinephrine release, depending on both the cytokine concentration and the presence of other cytokines in the microenvironment of the nerve terminal.

Taken together, these studies suggest that a physical mechanism for immune cell-derived cytokines to influence local sympathetic nerve activity is in place. Several studies have reported the presence of afferent innervation in the spleen, the presence of cytokine receptors on peripheral nerves, the ability of cytokine receptor stimulation to initiate afferent signals to the CNS resulting in alterations in hypothalamic neuronal activity, and finally, cytokine-induced alterations in sympathetic nerve activity and the rate of norepinephrine release in lymphoid organs. In light of these findings, immune cell-derived cytokine production may represent one mechanism by which an ongoing immune response may influence the rate of local norepinephrine release. Importantly, different types of antigen may lead to the activation of different populations of immune cells and affect which cytokines are produced during an immune response. The specific cytokines produced may, in turn, determine the mechanism that regulates the level of norepinephrine release within immune organs. Finally, it is possible that greater levels of infection involve the CNS-mediated regulatory mechanisms, whereas lower levels of infection may involve only local regulatory mechanisms of sympathetic nerve activity. However, in order for local norepinephrine release to influence immune cell function, lymphocytes must express receptors for the neurotransmitter.

	III. -Adrenergic Receptor Expression on CD4+ T and B Lymphocytes

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A. CD4⁺ T Lymphocytes

1. Receptor Expression. Although few studies have reported the presence of ARs on T cells, early studies suggested the presence of a functional AR on their surface. An important premise that made these studies possible was that stimulation of the AR was found to increase the level of adenylyl cyclase activity and intracellular cAMP accumulation in other nonlymphoid cell types (reviewed in Wolfe et al., 1977). Therefore, using AR agonists, early reports demonstrated that the exposure of lymphocytes to AR agonists resulted in adenylyl cyclase activation and increased cAMP production (Bourne and Melmon, 1971; Makman, 1971; Bach, 1975). Thus, although AR expression would not be measured directly on lymphocytes for another 6 years, early pharmacological and biochemical data suggested their functional presence. A recent review provides comprehensive discussion concerning the expression of adrenergic receptors on immune cells (Sanders et al., 2001). Figure 3 summarizes 2AR expression on both CD4⁺ T cells and B cells.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   2-Adrenergic receptor expression on CD4+ cells and B cells. The predominant adrenergic receptor expressed on resting and activated B cells is the 2AR. Similarly, naive CD4+ T cells also predominantly express the 2AR. However, whereas 2AR expression is retained on clones and newly generated Th1 cells, 2AR expression is repressed on clones and newly generated Th2 cells.