Valentin A Pavlov,1 Hong Wang,1 Christopher J Czura,1 Steven G Friedman,1,2 and Kevin J Tracey1
Abstract
This review outlines the mechanisms underlying the interaction between the nervous and immune systems of the host in response to an immune challenge. The main focus is the cholinergic anti-inflammatory pathway, which we recently described as a novel function of the efferent vagus nerve. This pathway plays a critical role in controlling the inflammatory response through interaction with peripheral α7 subunit–containing nicotinic acetylcholine receptors expressed on macrophages. We describe the modulation of systemic and local inflammation by the cholinergic anti-inflammatory pathway and its function as an interface between the brain and the immune system. The clinical implications of this novel mechanism also are discussed.
INTRODUCTION
Inflammation is a normal response to disturbed homeostasis caused by infection, injury, and trauma. The host responds with a complex series of immune reactions to neutralize invading pathogens, repair injured tissues, and promote wound healing (1,2). The onset of inflammation is characterized by release of pro-inflammatory mediators including tumor necrosis factor (TNF), interleukin (IL)-1, adhesion molecules, vasoactive mediators, and reactive oxygen species (1–3). The early release of pro-inflammatory cytokines by activated macrophages has a pivotal role in triggering the local inflammatory response (2). Excessive production of cytokines, such as TNF, IL-1β, and high mobility group B1 (HMGB1), however, can be more injurious than the inciting event, initiating diffuse coagulation, tissue injury, hypotension, and death (2,4–6). The inflammatory response is balanced by anti-inflammatory factors including the cytokines IL-10 and IL-4, soluble TNF receptors, IL-1 receptor antagonists, and transforming growth factor (TGF)β. Although simplistic (7,8), the pro-/anti- terminology is widely used in the discussion of the complex cytokine network. Apart from their involvement in local inflammation, TNF and IL-1β are signal molecules for activation of brain-derived neuroendocrine immunomodulatory responses. Neuroendocrine pathways, such as the hypothalamo-pituitary-adrenal (HPA) axis and the sympathetic division of the autonomic nervous system (SNS) (9–15), control inflammation as an anti-inflammatory balancing mechanism. The host thereby mobilizes the immunomodulatory resources of the nervous and endocrine systems to regulate inflammation.
Restoration of homeostasis as a logical resolution of inflammation does not always occur. Insufficient inflammatory responses may result in increased susceptibility to infections and cancer. On the other hand, excessive responses are associated with autoimmune diseases, diabetes, sepsis, and other debilitating conditions. When control of local inflammatory responses is lost, pro-inflammatory mediators can spill into the circulation, resulting in systemic inflammation that may progress to shock, multiple organ failure, and death. Effective therapies for diseases of excessive inflammation are needed.
We recently discovered the anti-inflammatory role of the vagus nerve (16,17) in an animal model of endotoxemia and shock. This previously unrecognized immunomodulatory circuit termed the “cholinergic anti-inflammatory pathway” is a mechanism for neural inhibition of inflammation (18,19), and interfaces the brain with the immune system. Can it be a “missing link” in neuroimmunomodulation that will validate the notion of a mind-body connection?
This review outlines brain-derived control mechanisms of immune function and specifically the role of cholinergic anti-inflammatory pathway in the regulation of inflammation.
INTERACTION BETWEEN THE IMMUNE SYSTEM AND BRAIN IN RESPONSE TO IMMUNE CHALLENGE
Communication between the immune, nervous, and endocrine systems is essential for host defense and involves a variety of mediators including cytokines, neurotransmitters, hormones, and humoral factors. The influence of the brain on immune function and the mechanisms involved in these interactions have been elucidated over the past 3 decades (17–19). Two important questions arise when describing the brain-derived immunomodulation: 1) How is the brain initially signaled by cytokines to trigger corresponding neural and neuroendocrine responses; and 2) how is immunomodulation achieved through these mechanisms?
Immune-To-Brain Communication
The brain can monitor immune status and sense peripheral inflammation through 2 main pathways: neural and humoral (Figure 1; for a review, see 20, 21).
Neural pathway
The neural mechanism relies upon activation of vagus nerve afferent sensory fibers that signal the brain that inflammation is occurring. Immunogenic stimuli activate vagal afferents either directly by cytokines released from dendritic cells, macrophages, and other vagal-associated immune cells, or indirectly through the chemoreceptive cells located in vagal paraganglia (22). For instance, intraperitoneal administration of endotoxin can induce IL-1β immunoreactivity in dendritic cells and macrophages within connective tissues associated with the abdominal vagus nerve and subsequently in vagal paraganglia and afferent fibers (23). Visceral vagus afferent fibers, residing in the nodose ganglion, terminate primarily within the dorsal vagal complex (DVC) of the medulla oblongata. The DVC consists of the nucleus tractus solitarius (NTS), the dorsal motor nucleus of the vagus (DMN), and the area postrema (AP) (24). The DMN is the major site of origin of preganglionic vagus efferent fibers; cardiovascular vagal efferents also originate within the medullar nucleus ambiguous. The AP, which lacks a blood-brain barrier, is an important circumventricular organ and site for humoral immune-to-brain communication, as described below. The main portion of vagal sensory input is received by neurons in the NTS that coordinate autonomic function and interaction with the endocrine system (25). Ascending projections from the NTS reach forebrain sites including hypothalamic nuclei, amygdala, and insular cortex. One of the hypothalamic nuclei receiving input from the NTS is the paraventricular nucleus (PVN). The PVN is associated with the synthesis and release of corticotropin releasing hormone (CRH), an important substance in the HPA axis.
This ascending link between the NTS and PVN provides a pathway that can modulate neurohormonal anti-inflammatory responses. Synaptic contacts also exist between the neurons in the NTS and C1 neurons in the rostral ventrolateral medulla (RVM), which occupies an important role in control of cardiovascular homeostasis. The RVM neurons in turn project to the locus coeruleus (LC), which is the main source of noradrenergic innervations of higher brain sites, including the hypothalamus and PVN. Projections emanate from the RVM and LC to sympathetic preganglionic neurons in the spinal cord. There are also descending pathways from the PVN to the RVM and NTS. These ascending and descending connections provide a neuronal substrate for interaction between HPA axis and SNS as an immunomodulatory mechanism.
The transmission of cytokine signals to the brain through the vagal sensory neurons depends upon the magnitude of the immune challenge. Subdiaphragmatic vagotomy inhibits the stimulation of the HPA axis (26) and norepinephrine (NE) release in hypothalamic nuclei (27) in response to intraperitoneal administration of endotoxin or IL-1β. Intravenous endotoxin administration induces expression of the neural activation marker c-Fos in the brainstem medulla, regardless of the integrity of the vagus nerve (28). Vagotomy fails to suppress high dose endotoxin-induced IL-1β immunoreactivity in the brain (29) and increases blood corticosterone levels (30). It is likely that the vagal afferent neural pathway plays a dominant role in mild to moderate peripheral inflammatory responses, whereas acute, robust inflammatory responses signal the brain primarily via humoral mechanisms.
Humoral pathway
A large body of evidence supports the involvement of humoral mechanisms in the immune-to-brain communication, especially in cases of systemic immune challenge (21, 31–33). The question remains, however, of how the circulating cytokines interact with brain structures involved in the anti-inflammatory response, and how circulating cytokines induce central cytokine production associated with fever and sickness. Blood-borne IL-1β and TNF can cross the blood-brain barrier and enter cerebrospinal fluid and the interstitial fluid spaces of the brain and spinal cord by a saturable carrier-mediated mechanism (34) that may function only at very high plasma cytokine concentrations. Cytokines also can bind to receptors at the surface of the endothelium of the brain capillaries and can enhance the synthesis and release of soluble mediators such as prostaglandins and nitric oxide, which diffuse into the brain parenchyma and modulate the activity of specific groups of neurons (21,35,36). It has been suggested that prostaglandins mediate fever and HPA axis activation (13).
Cytokine-to-brain communication also may occur via circum-ventricular organs that lack normal blood-brain barrier function. Among the circumventricular organs, the AP appears to represent the best candidate for such a transduction site (for a review, see 37). The AP is located in the floor of the caudal 4th ventricle (38) and dendrites of neurons in the NTS and DMN penetrate both the AP and floor of the 4th ventricle (39,40). The close proximity of AP to NTS and RVM and the existing neural connections provide a way of signaling the SNS and HPA axis. Cytokine-induced production of prostaglandins within the AP, NTS, and RVM may activate the catecholamine projections to the PVN, resulting in subsequent HPA axis activation (37). This is one possible interaction between the neural and humoral mechanisms of immune-to-brain communication through which the brain mediates anti-inflammatory responses.
Apart from their function in signaling the brain for immunomodulatory responses, cytokines play a multifunctional role in brain injury and neurodegenerative diseases (for review, see 41–43).
Brain-To-Immune Communication
The brain exerts strong modulatory effects on immune function by activation of the HPA axis and the SNS, which results in increased synthesis and release of glucocorticoids and catecholamines (see Figure 1). The immunomodulating properties of α-melanocyte stimulating hormone (α-MSH) and estrogens also are known. The HPA axis is a neurohormonal pathway; its role in the regulation of the immune function has been widely studied (for reviews, see 14,44–46). The components of the HPA axis are the hypothalamic PVN, the anterior pituitary gland, and the adrenal cortex. Specialized neurons in PVN synthesize CRH, which is released into the pituitary portal blood system and stimulates the synthesis of adrenocorticotropin hormone (ACTH) from the anterior pituitary. ACTH is the main inducer of the synthesis and release of immunosuppressive glucocorticoids (cortisol in humans and corticosterone in rats) from the adrenal cortex. Pro-inflammatory cytokines trigger the HPA axis via the neural or humoral mechanisms described above. At both the hypothalamic and pituitary level, the HPA axis is subject to a classical negative feedback loop by the final product: glucocorticoids. ACTH also inhibits the synthesis of CRH from the PVN. The hypothalamo-pituitary circuit of the HPA axis is regulated by neural mechanisms including acetylcholine (ACh)-, catecholamine-, GABA-, serotonin-, and histamine-mediated modulation.
Glucocorticoids exert their effects by binding to intracellular receptors and subsequently triggering up-regulation or down-regulation of gene expression (47). Apart from triggering the activation of the HPA axis, cytokines such as IL-1 and IL-6 also can alter peripheral glucocorticoid effects by directly influencing the function of corresponding glucocorticoid receptors (45). Immunosuppressive glucocorticoid influence is mainly linked to suppression of nuclear factor-κB activity (14,48,49), which plays an important role in regulating cytokine synthesis (50). As summarized by Webster and others (14), glucocorticoids inhibit the synthesis of pro-inflammatory cytokines, such as TNF, IL-1, IL-8, IL-11, IL-12, and interferon-γ; and they activate the synthesis of the anti-inflammatory cytokines IL-4 and IL-10. Inhibition of neutrophil, eosinophil, monocyte, and macrophage infiltration, and adhesion molecule expression are attributed to glucocorticoid suppression of local inflammation (14,45). Glucocorticoids are also potent clinical anti-inflammatory agents (45).
The SNS plays a dual role in the regulation of inflammation, because it mediates both pro- and anti-inflammatory activities; it is thus an integral component of the host defense system against injury and infection (15,51). The locus coeruleus (LC) and RVM, brain functional components of the SNS, project to sympathetic preganglionic cholinergic neurons in the spinal cord. Sympathetic innervation of primary (thymus and bone marrow) and secondary (spleen, lymph nodes, and tissues) lymphoid organs is the anatomic basis for modulation of immune function by the SNS (15,51,52). Sympathetic postganglionic norepinephrine (NE)-ergic and neuropeptide Y-ergic neurons also innervate blood vessels, the heart, the liver, and the gastrointestinal tract (15,52). NE, released from sympathetic postganglionic nerve endings, exerts anti-inflammatory effects by interacting with adrenoceptors expressed on lymphocytes and macrophages.
Adrenoceptors belong to the G-protein coupled receptor superfamily and are divided into α and β subtypes, which can be further subdivided. Sympathetic control on cytokine production is achieved by 2 mechanisms: (1) synaptic-like junctions with corresponding adrenoceptors (as in the spleen) and (2) nonsynaptic, distant mechanisms, such as NE diffusion through the parenchyma before interaction with the receptor. The nonsynaptic mechanism plays a dominant physiological role (15). The release of NE is subject to complex presynaptic regulation, involving the effects of neuropeptide Y, acetylcholine, dopamine, prostaglandins, and other micro-environmental factors. Sympathetic immunomodulation also is mediated via epinephrine, and to a lesser extent, by NE released from the chromaffin cells of the adrenal medulla. The chromaffin cells represent homologs of the sympathetic ganglia. Activation of the preganglionic sympathetic neurons innervating these cells leads to an increase in the release of catecholamines in the bloodstream, which can act systematically as hormones. Thus, sympathetic neural regulation is converted into “hormonal regulation” within the adrenal glands. The adrenals are therefore an important peripheral component of the CNS-controlled immunoregulation responsible for the synthesis of glucocorticoids (from the cortical cells) and catecholamines (from the medullar chromaffin cells).
SNS activation protects the organism from the detrimental effects of pro-inflammatory cytokines. Activation of β-adrenoceptors leads to marked inhibition of endotoxin induced serum TNF, IL-1, IL-12, interferon-γ, and nitric oxide production, and elevation of IL-6 and IL-10. Stimulation of β-adrenoceptors also is accompanied by suppression of the TNF and IL-1 expression caused by hemorrhagic shock. NE and epinephrine inhibit production of pro-inflammatory cytokines through stimulation of β2-adrenoceptor-cAMP-protein kinase A pathway, and they stimulate the synthesis of anti-inflammatory cytokines. Healthy volunteers receiving epinephrine and subsequent endotoxin treatment developed significantly decreased TNF levels and elevated IL-10 levels, as compared with controls exposed to endotoxin alone (53), raising the possibility that pharmacological control of cytokine production in septic patients could be achieved by selective adrenoceptor agonists and antagonists. A catecholamine-based strategy for treating sepsis and its complications is difficult, however, because of the broad effects of these agents and the need to balance the cardiovascular and immunomodulatory drug effects (15).
During the early stages of some cases of inflammation, stimulation of the SNS can be associated with activation of the local inflammatory responses and neutrophil accumulation (1,54). NE stimulation of the α2–subtype adrenoceptor has been linked to an increase in endotoxin-induced production of TNF and other cytokines (15,55). This dual role of the SNS in immunomodulation agrees with the general mode of SNS function, depending on the peripheral receptors involved. A classic example of dual effects is the “fight-or-flight” response, leading to increased perfusion of the heart, skeletal muscles, and brain, and release of glucose from the liver. Gastrointestinal motility and the blood supply to the skin are simultaneously depressed.
Anti-inflammatory properties of α-MSH have been shown in animal models of inflammation including sepsis and rheumatoid arthritis (for a review, see 56, 57). In rodents α-MSH is mainly produced from the intermediate lobe of the pituitary gland. The cells of the pituitary pars distalis and various extrapituitary cells, such as monocytes, astrocytes, and keratinocytes, are the source of this hormone production in humans. The activity of α-MSH is mediated through G-protein coupled melanocortin receptors widely distributed in peripheral tissues and in the brain. Immunomodulatory effects of α-MSH are associated with stimulation of its receptors on peripheral immune target cells and on glial cells in brain; α-MSH may also exert indirect effects via a brain-spinal cord pathway and sympathetic neurons (58). Although the exact molecular mechanisms of α-MSH immunosuppression and pro-inflammatory cytokine downregulation are not well understood, hormonal inhibition of nuclear factor κB may play an important role. The administration of endotoxin to normal human subjects is accompanied by an increase in α-MSH blood concentrations (in addition to ACTH) and decreased TNF plasma levels, demonstrating the role of α-MSH in the inflammatory response (59). It is not clear how pro-inflammatory cytokines cause stimulation of pituitary α-MSH secretion, but the ascending pathways from NTS to the hypothalamus underlying the HPA axis activation may play a role.
Female sex hormones and estrogens, in particular, also exert immunomodulatory and anti-inflammatory effects. Because their synthesis and blood concentrations are under control of hypothalamo-pituitary hormones, estrogen-affected immune functions can be imputed to the brain. Female sex hormones play immunoregulatory roles during pregnancy and in diseases like rheumatoid arthritis and osteoporosis (60). The classic mechanism of steroid hormone action (as described above for glucocorticoids) may contribute to estrogen immunomodulating activity. Estrogens are neuroprotectors (61); they prevent cartilage degradation during inflammation associated with increased production of IL-1 (62); and they inhibit the production of pro-inflammatory cytokines at different stages of their synthesis (63,64).
CHOLINERGIC ANTI-INFLAMMATORY PATHWAY
The sympathetic and parasympathetic parts of the autonomic nervous system rarely operate alone; autonomic responses represent the interplay of both parts. A link between the parasympathetic part of the autonomic nervous system and immunoregulatory processes was suggested 30 years ago, when alleviation of T-lymphocyte cytotoxicity by muscarinic cholinergic stimulation was noted (65). Despite this observation, the role of the parasympathetic/vagal efferents in immunomodulation is not completely understood (66–68).
We recently demonstrated the existence of a parasympathetic pathway of modulation of systemic and local inflammatory responses (16,69), which focuses attention on neural immunomodulatory mechanisms via the vagus nerve.
Evidence for Parasympathetic (Vagus Nerve) Control of Systemic And Local Inflammation
Acetylcholine is an important neurotransmitter and neuromodulator in the brain. It mediates neural transmission in the ganglion synapses of both sympathetic and parasympathetic neurons, and is the principle neurotransmitter in the postganglionic parasympathetic/vagal efferent neurons. Acetylcholine acts through 2 types of receptors: muscarinic (metabotropic) (70) and nicotinic (ionotropic) (71). In addition to the brain and “wire-innervated” peripheral structures, the RNA for these receptor subtypes (muscarinic) and subunits (nicotinic) has been detected on mixed populations of lymphocytes and other immune and non-immune cytokine-producing cells (72–77). Most of these cells can also produce acetylcholine (78).
We recently discovered that the α7 subunit of the nicotinic acetylcholine receptor is expressed on macrophages (16). Acetylcholine significantly and concentration-dependently decreases TNF production by endotoxin-stimulated human macrophage cultures via a post-transcriptional mechanism. Using specific muscarinic and nicotinic agonists and antagonists, we demonstrated the importance of an α-bungarotoxin-sensitive nicotinic receptor in the inhibition of TNF synthesis in vitro by acetylcholine. Acetylcholine also is effective in suppressing other endotoxin-inducible pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-18, by a post-transcriptional mechanism; release of the anti-inflammatory cytokine IL-10 from endotoxin-stimulated macrophages is not affected by acetylcholine (16).
Because of the immunosuppressive effects of acetylcholine in vitro, we studied the possible immunonodulatory role of the parasympathetic division of the autonomic nervous system in vivo. In a rat model, vagotomy without electrical stimulation significantly increases serum and liver TNF levels in response to intravenously administered endotoxin (Figure 2A and 2B), suggesting a direct role of efferent vagus neurons in the regulation of TNF production in vivo. Augmentation of efferent vagus nerve by direct electrical stimulation significantly attenuates endotoxin-induced serum and hepatic TNF (see Figure 2A and 2B). TNF amplifies inflammation by activating the release of pro-inflammatory mediators such as IL-1, HMGB1, nitric oxide, and reactive oxygen species (3,6).
TNF also plays an essential role in endotoxin-induced shock by inhibiting cardiac output, activating microvascular thrombosis, and modulating capillary leakage syndrome (4,79). These activities of TNF are consistent with the finding that attenuation of serum TNF via cervical vagus nerve stimulation prevents hypotension and shock in animals exposed to lethal doses of endotoxin (see Figure 2C) (16). Animals subjected to vagotomy without vagus nerve stimulation develop profound shock more quickly than sham-operated animals (see Figure 2C), demonstrating a role for vagus nerve efferent signaling in maintaining immunological homeostasis. Importantly, the immunomodulatory effects of the efferent vagus nerve also play a role in localized peripheral inflammation, because electrical stimulation of the distal vagus nerve also inhibits the local inflammatory response in a standard rodent model of carrageenan-induced paw edema (69). Pretreatment with acetylcholine, muscarine, or nicotine localized within the site of inflammation also prevents the development of hind paw swelling (69). Vagal efferents are distributed throughout the reticuloendothelial system and other peripheral organs, and the brain-derived motor output through vagus efferent neurons is rapid. The cholinergic anti-inflammatory pathway is therefore uniquely positioned to modulate inflammation in real time.
The most abundant dietary sources of choline—a precursor to acetylcholine—are animal fats such as egg yolks, cream, fatty cheeses, fatty fish, fatty meats, and liver. Non-animal sources include avocadoes and almonds.
Aerobic exercises influence acetycholine production.