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PHYSIOLOGICAL REVIEWS Vol. 79 No. 1 January 1999, pp. 1-71
Copyright ©1999 by the American Physiological Society
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California; and North Western Injury Research Centre, University of Manchester, Manchester, United Kingdom
I. INTRODUCTION
A. Hormones and Cytokines: Definitions
B. Concept of Bidirectional Communication Between Immune and Neuroendocrine Systems: a Historical Perspective
C. Cytokines
D. Hypothalamic-Pituitary-Adrenal Axis
II. CYTOKINE INFLUENCE ON HYPOTHALAMIC-PITUITARY-ADRENAL AXIS SECRETORY ACTIVITY IN VIVO
A. Animal Studies
B. Human Studies
III. PHYSIOLOGICAL/PATHOPHYSIOLOGICAL CIRCUMSTANCES IN WHICH ENDOGENOUS CYTOKINES PLAY A ROLE IN REGULATION OF HYPOTHALAMIC-PITUITARY-ADRENAL AXIS
A. Viral Disease
B. Endotoxin Treatment
C. Local Inflammation
D. Physical and Psychological Stress
E. Basal Hypothalamic-Pituitary-Adrenal Activity
IV. CYTOKINE ACTIONS ON THE CENTRAL NERVOUS SYSTEM, PITUITARY, AND ADRENAL
A. Evidence That Cytokines Activate the Hypothalamic-Pituitary-Adrenal Axis Primarily at the Level of the Central Nervous System
B. Evidence for Direct Effects of Cytokines on Pituitary Adrenocorticotropic Hormone Secretion
C. Evidence for Direct Actions of Cytokines on Adrenal Glucocorticoid Secretion
V. MECHANISMS OF HYPOTHALAMIC-PITUITARY-ADRENAL AXIS ACTIVATION BY INTERLEUKIN-1
A. Direct Actions on Pituitary and Adrenal
B. Penetration of Cytokines Into Brain
C. Role of Readily Diffusible Intermediates
D. Induction of Intermediates at Blood-Brain Barrier Interface
E. Actions at Circumventricular Organs
F. Potential Role of Catecholamines and Evidence of Activation of Medullary Cell Groups
G. Activation of Vagal Afferent Fibers
H. Cytokine Synthesis Within Brain
I. Local Interleukin-1 Induction of Circulating Interleukin-6
VI. CONCLUSIONS
REFERENCES
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 Top References
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Turnbull, Andrew V., and Catherine L. Rivier. Regulation of the Hypothalamic-Pituitary-Adrenal Axis by Cytokines: Actions and Mechanisms of Action. Physiol. Rev. 79: 1-71, 1999. 
Glucocorticoids are hormone products of the adrenal gland, which have long been recognized to have a profound impact on immunologic processes. The communication between immune and neuroendocrine systems is, however, bidirectional. The endocrine and immune systems share a common "chemical language," with both systems possessing ligands and receptors of "classical" hormones and immunoregulatory mediators. Studies in the early to mid 1980s demonstrated that monocyte-derived or recombinant interleukin-1 (IL-1) causes secretion of hormones of the hypothalamic-pituitary-adrenal (HPA) axis, establishing that immunoregulators, known as cytokines, play a pivotal role in this bidirectional communication between the immune and neuroendocrine systems. The subsequent 10-15 years have witnessed demonstrations that numerous members of several cytokine families increase the secretory activity of the HPA axis. Because this neuroendocrine action of cytokines is mediated primarily at the level of the central nervous system, studies investigating the mechanisms of HPA activation produced by cytokines take on a more broad significance, with findings relevant to the more fundamental question of how cytokines signal the brain. This article reviews published findings that have documented which cytokines have been shown to influence hormone secretion from the HPA axis, determined under what physiological/pathophysiological circumstances endogenous cytokines regulate HPA axis activity, established the possible sites of cytokine action on HPA axis hormone secretion, and identified the potential neuroanatomic and pharmacological mechanisms by which cytokines signal the neuroendocrine hypothalamus.
A. Hormones and Cytokines: Definitions
Defining what is meant by the term hormone, and what is meant by the term cytokine, is certainly no easy task. Indeed, the work reviewed in this article has changed many people's opinion as to what our definitions of these classes of cell-cell signaling molecules should be. However, we are faced at the outset with conveying to the reader what "we" mean when we use the terms cytokines and hormones throughout this review. A classical endocrinology textbook definition of a hormone is "a biomolecule, which is produced by a specialized cell type, is secreted from a ductless gland directly into the bloodstream, and acts on distant target cells/tissues, to regulate pre-existing cellular activities." Chemically, hormones are small to large polypeptides, proteins, glycoproteins, derivatives of aromatic amino acids, or steroids. In the case of the pituitary peptide hormone adrenocorticotropin (ACTH), it is produced by corticotropes (specialized cell type) within the anterior pituitary (a ductless gland), is secreted into the general circulation, and acts predominantly on the adrenal cortex (a distant target) to enhance glucocorticoid secretion. This is perhaps a narrow view of a hormone, because many "classical" hormones are also synthesized and act within the brain and are produced and act locally within the periphery. On the other hand, it is perhaps too broad, because under this definition CO2 produced by exercising muscle and stimulating respiration might have to be considered a hormone also. However, it is a fairly accurate description of what most people understand when thinking of a hormone acting in a "classical endocrine fashion." In contrast to hormones that have most commonly been associated with the "endocrine system," cytokines have been classically associated with the "immune system." Defining a cytokine is even more difficult than defining a hormone. For the purposes of the work described within this review, we found the definition used in The Cytokine Handbook (862) most useful. Here cytokines are defined as "regulatory proteins secreted by white blood cells and a variety of other cells in the body; the pleiotropic actions of cytokines include numerous effects on cells of the immune system and modulation of inflammatory responses." This definition is somewhat narrow in that it probably overemphasizes the importance of the immune system as a source and target, but probably reflects most accurately people's first thoughts when they think of cytokines. It certainly reflects best the definition that would have been applied at the time when the majority of the work described in this article was performed. Under any definition, the term cytokine encompasses the "monokines" (monocyte/macrophage-derived mediators) and "lymphokines" (lymphocyte-derived mediators), which were terms commonly used in the earlier studies described in this review. Table 1 was compiled of what is presently known of the features of hormones and cytokines. Although this is again not definitive, it is fair to say that if the majority of characteristics of the substance under consideration fit in the cytokine column, then the substance is a cytokine, and if the majority of characteristics fit in the hormone column, then it is a hormone. It should be pointed out that the same chemical substance could be classified differently depending on the "setting" under consideration. For example, prolactin produced by the pituitary and acting on the mammary gland is clearly acting in an "endocrine hormone" fashion. However, prolactin can also be produced by, and act on, lymphocytes, a situation in which it might be better classified as a cytokine. Perhaps the key difference between cytokines and hormones that we indicate in Table 1 is that cytokines are regulators of predominantly local tissue processes, whereas hormones function as regulators predominantly of "systemic" or "whole body" homeostasis.
B. Concept of Bidirectional Communication Between Immune and Neuroendocrine Systems: a Historical Perspective
Regulation of the immune system by the adrenal gland was observed as early as the middle of the 19th century when Thomas Addison (3) documented that a patient with adrenal insufficiency had an excess of circulating lymphocytes. In agreement with this observation, removal of the adrenal gland of the rat was found to produce hypertrophy of the thymus (an organ responsible for the manufacture of mature lymphocytes) (363). Perhaps the best known of early experimental studies were those of Hans Selye (764, 765), who found that enlargement of the adrenal gland and involution of the thymus were communal features of an animal's response to stress, regardless of the nature of the injurious insult. These early studies clearly suggested a close association between adrenal gland physiology and immune activity. The isolation of the active principal of the adrenal cortex, cortisone, by Kendall and Reichstein in the late 1940s, and the demonstration of its ability to suppress inflammation (335), gave support to the hypothesis that adrenal glucocorticoid (GC) secretion plays a significant role in regulating immunologic processes. However, even though marked elevations in the plasma blood concentration of GC are observed after all types of stressful stimuli, studies showing that GC produce immunosuppression were assumed (by the majority of workers) to be of pharmacological, rather than of physiological, significance. These findings advented the widespread use of GC-based therapies for autoimmune and inflammatory disease. It was not until the late 1970s and the pioneering work of Besedovsky, del Rey, Sorkin, and colleagues that a physiological role for GC in preventing overactivity, and preserving the specificity, of immune reactions became established (67, 191). In a significant review article of the early 1980s, Munck et al. (567) reinforced this concept. These authors proposed the now commonly held view that endogenous GC act to prevent "overshoot" of immune/inflammatory responses, thus limiting the host defense response to fighting the aggressor (e.g., invading pathogen) without the deleterious effects to the host of a hyperactive immune system (e.g., autoimmunity). More recent work has indicated that the influence of GC on immunologic processes is more complex than a generalized suppression of immune activity and depends on the type of immune activity and the subset of immunologic cells involved (see Ref. 531 for extensive review). However, it is clear that endogenous GC are key regulators of immune system function. Further work by Besedovsky et al. (73) suggested that not only do GC have a profound impact on immune activity, but that the converse is also true and immune activity influences GC secretion. This hypothesis grew out of experimental observations that during the development of an immune response to a foreign antigen (sheep red blood cells), rats mount a parallel endocrine response characterized by elevated plasma levels of GC (corticosterone) (73). Furthermore, mice injected with supernatants from concanavalin A-stimulated peripheral blood or spleen cells produce one or more GC-increasing factors (GIF) that increase the blood concentrations of corticosterone (68). These studies therefore suggested the existence of an immune-neuroendocrine regulatory feedback mechanism in which immune cells limited their own activity by secreting molecules that stimulate the secretion of adrenal GC. The concept of "bidirectional communication" between immune and endocrine systems became firmly established with the seminal works of Edwin Blalock and co-workers in the early 1980s. These workers began to describe molecular basis for such bidirectional communication (reviewed in Refs. 80-82, 952, 953). Their early studies showed a commonality in the pathways of action of immunoregulators (e.g., interferon) and hormones (e.g., norepinephrine) (84). This group went on to discover that a number of classical hormones are not only secreted by classical endocrine glands (e.g., pituitary) but are also made by cells of the immune system (e.g., lymphocytes). For example, they showed that lymphocytes synthesize ACTH, the pituitary hormone which is the major physiological regulator of adrenal GC secretion (792). In addition, they demonstrated that not only do lymphocytes produce hormones such as ACTH, endorphins, thyrotropin, and growth hormone (792, 794, 951) but that these hormones were able to influence immunologic processes (79, 83, 370, 371). Subsequent studies have demonstrated that a number of hormones (e.g., prolactin, insulin-like growth factor) are produced by lymphocytes (372, 556, 557, 621). The finding that lymphocytes are able to synthesize an ACTH-like molecule, combined with the demonstration that mice whose pituitary gland had been removed (hypophysectomy) still produced a corticosterone response to infection with the Newcastle disease virus (NDV), led Blalock and co-workers (793) to propose the concept of lymphoid-adrenal axis. According to this hypothesis, ACTH produced by virus-stimulated lymphocytes acts on the adrenal to increase corticosterone secretion. However, subsequent studies have failed to replicate the persistance of an NDV-induced corticosterone response in hypophysectomized mice (64, 218, 221, 604), and the hypothesis of a lymphoid-adrenal axis involving lymphoid production of ACTH molecule has fallen out of favor. Furthermore, Besedovsky et al. (71) showed that stimulated lymphocytes secrete GIF that increase plasma ACTH and corticosterone levels in rats and that this corticosterone response was prevented by hypophysectomy. Given that the electrical and neurochemical activities of the hypothalamus are also altered during the course of an immune response (69, 72), Besedovsky et al. (71) proposed that the effects of such GIF on adrenal GC secretion were probably mediated at the hypothalamic component of the hypothalamic-pituitary-adrenal (HPA) axis, rather than on the pituitary or adrenal glands directly. The chemical identity of putative "GIF" became apparent with the recognition that classical endocrine hormones are not the only class of mediators involved in immune-endocrine communication. Indeed, in the mid 1980s, it became readily apparent that immunoregulatory cytokines also form a key link between immune and neuroendocrine systems (70, 331, 532, 966). Blalock and co-workers (966) showed that the monokines interleukin (IL)-1 and IL-6 (or hepatocyte-stimulating factor) stimulate ACTH secretion from the corticotropic tumor cell line AtT20.1 A year later, Besedovsky et al. (70) demonstrated that systemic administration of monocyte-derived or recombinant IL-1 increases plasma ACTH and GC concentrations in normal mice. Furthermore, Besedovsky et al. (70) demonstrated that neutralization of endogenous IL-1 inhibits the GC response to experimental viral infection (NDV) in rats. This latter experiment clearly indicated that the observations of stimulatory effects of cytokines on neuroendocrine secretion were not merely pharmacological phenomena and suggested that IL-1 plays an important endogenous role in regulating the HPA axis during viral disease. Indeed, these landmark studies by Besedovsky and Blalock indicated that cytokines could be the extrahypothalamic corticotropin-releasing factors (CRF) released by injured tissue which had previously been reported by Broddish and co-workers during the 1970s (112, 113, 497). Subsequent work by three independent laboratories resulted in articles being published back to back in a 1987 issue of Science (55, 62, 730). One of these studies (62) demonstrated a direct action of IL-1 on ACTH secretion from primary cell cultures of rat anterior pituitary cells, thus supporting the earlier studies by Blalock and co-workers (966) suggesting a direct action of IL-1 on the pituitary gland to secrete ACTH. Conversely, the other two Science papers (55, 730) found that IL-1 does not stimulate ACTH secretion from anterior pituitary cells in primary culture, despite the fact that IL-1 in vivo elevates plasma ACTH and GC concentrations. These latter two groups (55, 730) showed that IL-1-induced ACTH secretion in vivo is dependent on the secretion and action of the hypothalamic 41-amino acid peptide CRF, which is the major hypothalamic ACTH secretagogue. These findings clearly implicate the hypothalamus as the site at which the HPA axis response to IL-1 is mediated and gave great support to the idea that immunoregulators could influence the activity of the central nervous system (CNS). Controversy over the likely primary site of IL-1 action (CNS, pituitary gland, or possibly adrenal glands) in stimulating pituitary-adrenal secretion has continued for many years and is considered in detail in sections IV and V. However, a large body of evidence has now accumulated that indicates that IL-1 and other cytokines can signal the brain. In parallel with studies investigating the relationship between the immune system and HPA axis, a large number of studies indicated that fever caused by invading pathogens occurs as a result of the elaboration from immune cells of an "endogenous pyrogen" capable of signaling the CNS (reviewed in Refs. 23, 420). This endogenous pyrogen was putatively identified as IL-1 (reviewed in Ref. 420). Furthermore, administration of IL-1 produces many CNS-mediated changes including changes in behavior (reduced exploration, reproductive activity, food-motivated behavior, and increased sleep), changes in autonomic outflow, metabolic rate, and the activity of a number of neuroendocrine axes (see Refs. 54, 65, 408, 409, 439, 440, 529, 683, 708 for relevant reviews). Collectively, these studies have provided strong evidence for the regulation by IL-1 of CNS responses to peripheral changes in immune activity. Furthermore, the common mediator of the effects on various CNS responses provides a molecular basis for the observations of the stereotypical responses to immune challenges of diverse origins. This "acute phase response" to sickness is characterized by fever, appetite suppression, anorexia, alterations in plasma cation concentrations, synthesis of specific liver proteins (known as acute phase proteins), and changes in neuroendocrine secretions (441). It is now firmly established that acute phase responses are produced by the actions of, and complex interactions between, IL-1 and numerous other cytokines. Since the landmark studies by the groups of Besedovsky and Blalock, it has become apparent that IL-1 has potent effects on the secretion of the majority of hormones under neuroendocrine control (see Table 2). Furthermore, more recent studies have shown that alterations in neuroendocrine secretion are produced not only by IL-1, but also by many other immunoreglatory cytokines. The HPA axis has remained the most extensively studied neuroendocrine system with respect to the influence of cytokines, and the ability to increase the secretory activity of this axis is a biological property of several interleukins, tumor necrosis factors, chemokines, hematopoietins, interferons, growth factors, and neurotrophic factors.
This article reviews published findings that demonstrate 1) which cytokines influence hormone secretion from the HPA axis, 2) under what physiological/pathophysiological circumstances endogenous cytokines may influence HPA axis secretory activity, 3) at which level (hypothalamus, pituitary, or adrenal) cytokines primarily act, and 4) what anatomic and pharmacological pathways mediate the actions of cytokines on the neuroendocrine hypothalamus. To achieve this aim, we have divided this article into sections corresponding to these overall objectives. We begin by providing brief introductions to relevant aspects of cytokine biology (see sect. IC) and the functional anatomy of the HPA axis (see sect. ID). C. Cytokines
1. Cytokines and cytokine receptor families
I. INTRODUCTION
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References
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TABLE 1.
Features of cytokines and classical endocrine hormones
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TABLE 2.
Influence of interleukin-1 on neuroendocrine secretion
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TABLE 3.
Cytokine families
and -1
; interferon- and -; and tumor necrosis factor-, -) most cytokines share little sequence similarity. Consequently, classification of cytokines has been based on either functional attributes, target receptors, or cells of origin. Most commonly, cytokines have been classified into families of interleukins, tumor necrosis factors (TNF), interferons (IFN), chemokines, hematopoietins (or neuropoietins), and colony-stimulating factors (CSF). Because of their similar actions particularly within the CNS and peripheral nervous system, growth factors (GF) and neurotrophins (NT) have also been considered to fall under the umbrella term cytokine. Their overlapping actions lead to a number of cytokines belonging to more than one family (see Table 3). For example, IL-6 is not only an interleukin, but also a member of a family of either hematopoietic or (neuropoietic) factors that utilize an identical receptor subunit (gp130) for cell signaling (416). Furthermore, IL-1, IL-3, IL-5, and IL-6 are also CSF.
, have many common biological activities despite the utilization of distinct cell surface receptors (see Table 4).
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Although there is some evidence that cytokines may play a role in some "normal" physiological processes such as sleep (605, 606, 838, 839), exercise (138, 139, 815, 920), and ovulation (2, 137), the expression of most cytokines in most normal healthy tissues is very low. However, cytokine production increases markedly during "tissue stress" produced by diverse cellular challenges, including periods of rapid cellular growth, tissue remodeling, disease, infection, or trauma. The particular cytokines produced in response to a threat to tissue homeostasis depends on the nature of the threat (e.g., bacterial, viral, inflammatory), the cellular or tissue type being threatened, the hormone milieu, and to a large extent the profile of other cytokines that are being produced. A further striking feature of cytokines is the multiple interactions between different individual cytokines. Many types of interactions are apparent, including stimulatory or inhibitory actions at the level of cytokine synthesis. For example, the proinflammatory cytokines TNF-
and IL-1 potently stimulate the production of a number of other cytokines, including each other, as well as IL-6, IL-8, IL-9, macrophage inflammatory protein (MIP), and CSF (206). In contrast, anti-inflammatory cytokines such as IL-4, IL-10, and IL-13 abrogate the production of many proinflammatory cytokines (e.g., IL-1, IL-8, IL-12, TNF-, IFN-
, and CSF) (122, 563), whereas IL-6 inhibits the production of IL-1 and TNF- but stimulates the production of the endogenous IL-1 receptor antagonist IL-1ra (379, 743). Furthermore, there are numerous examples of a particular cytokine influencing the cellular responses to another cytokine. For example, nearly all biological responses to either TNF- or IL-1 can be enhanced when the two are administered together (206), whereas activin functionally antagonizes the actions of gp130-signaling cytokines (114). The great propensity for cytokine-cytokine interactions is illustrated by the large number of different cytokines that may be produced by a single threat to cellular/tissue homeostasis. For example, endotoxemia has been reported to cause the increased synthesis and/or secretion of IL-1, IL-1, IL-1ra, IL-6, IL-8, IL-10, IL-12, TNF-, MIP, macrophage migrating inhibitory factor (MIF), IFN-, leukemia inhibitory factor (LIF), and granulocyte-macrophage CSF. It is therefore apparent that during the course of a threat to tissue homeostasis, the physiological outcome is determined by the net effect of the interactions between a number of cytokines.
Although many different cytokines have been shown to influence HPA axis secretory activity, by far the majority of studies have focused on the cytokines IL-1, IL-6 and TNF-. These three cytokines share many biological activities (see Table 4). Section IC2 gives a brief outline of their structure, biosynthesis, and receptors.
2. IL-1, IL-6, and TNF-
and IL-1, share ~25% sequence homology, are distinct gene products, and exhibit the same activities in numerous biological test systems (205). Both are synthesized as 31-kDa precursor molecules. Pro-IL-1 is biologically inactive and requires proteolytic cleavage by the IL-1 converting enzyme (ICE, also known as caspase-1) (401). In addition, an endogenous antagonist at IL-1 receptors (IL-1ra) has been described, which shares significant homology with IL-1 and IL-1, binds IL-1 receptors, but lacks intrinsic biological activity (225, 318). Interleukin-1ra has been used extensively as a pharmacological tool to explore the role of IL-1-IL-1 receptor interactions in physiological responses (203). More interestingly, however, endogenous IL-1ra is secreted by similar cell types, and in response to similar stimuli, as those which produce the IL-1 agonists, and endogenous IL-1ra plays an important role in regulating the physiological responses to endogenous IL-1 (260, 485). Recently, a fourth member of the IL-1 family has been proposed. Structural alignment of mouse IL-18 (also known as IFN- inducing factor, IGIF) demonstrated a 12 and 19% structural homology of this newly cloned cytokine with IL-1 and IL-1, respectively (46). Interleukin-18 was thus tentatively termed IL-1 (46). Although there is, as yet, little information about this new cytokine (850), it is known that lipopolysaccharide (LPS)-stimulated production of mature IL-18/IL-1 requires ICE (249, 287, 307). Furthermore, IL-18/IL-1 and IL-1 itself share similar signaling pathways (428) and functional activities (351), again indicating a close relationship between this novel cytokine and the IL-1 family.
Two distinct mammalian, membrane-bound IL-1 receptors have been described and designated IL-1R1 and IL-1R2 (784). Both are glycoproteins belonging to the immunoglobulin supergene family and possess a single transmembrane domain. Each receptor binds IL-1, IL-1, and IL-1ra, but with differing affinities (784). It has been proposed that the biological actions of IL-1 are mediated exclusively through IL-1R1 (442, 785), with IL-1R2 functioning solely as a decoy receptor that limits the availability of IL-1 for interaction with IL-1R1 (163, 784, 786). In contrast, some studies have demonstrated that a monoclonal antibody (ALVA 42) raised against IL-1R2 inhibits some actions of IL-1 within the brain (493, 548), although the ability of this antibody to bind IL-1R2 has been questioned (282). In addition to the two receptor isoforms, an accessory protein (IL-1RAcP) has been identified that enhances binding of IL-1 to IL-1R1 (304, 482) and plays a critical role in cell signaling through this receptor (348, 433, 994). Several additional members of the IL-1 receptor family have been identified on the basis of sequence homology (53, 94, 278, 488, 489, 550, 627, 989). One of these proteins (IL-1 receptor-related protein, IL-1Rrp) has recently been identified as a functional receptor for IGIF/IL-18/IL-1 (869).
Tumor necrosis factor also occurs in - and -forms, which share ~50% homology. Tumor necrosis factor- (lymphotoxin-) is produced predominantly by activated lymphocytes. In contrast, TNF- (also known as cachectin) is expressed on a wide variety of hemopoietic and nonhemopoietic cells as a 26-kDa membrane-associated molecule. This can be processed to give a secreted 17-kDa soluble form that mediates a range of inflammatory and cellular immune responses. Tumor necrosis factor is one of 10 known members of a family of ligands that activate a family of structurally related receptors. These include receptors for TNF- and TNF-, lymphotoxin-, Fas ligand, nerve growth factor (NGF), and CD40 ligand (47). All the ligands for these receptors consist of three polypeptide chains, and the majority are transmembrane proteins that act mainly through cell-to-cell contact. However, TNF, as indicated, is also secreted.
Actions of TNF- are exerted through interactions with two distinct receptors: the 55-kDa (TNF-R1) and 75-kDa (TNF-R2) receptors (47). These two receptors are both transmembrane proteins with a single transmembrane span and are expressed at low levels on most cell types. Although the extracellular domains of these two receptors show a similar architecture, the intracellular domains of these two receptors bear no significant homology, suggesting that they utilize separate signaling pathways (467). Indeed, studies of the effects of receptor-specific agonistic antibodies (233, 241, 283, 851, 968) and of TNF-R-deficient mice (234, 642, 707) indicate that these two receptors mediate effects that are largely, but not exclusively, nonoverlapping. Recent molecular studies have shed a considerable light on the activities of the two receptors (reviewed in Ref. 182). Binding of TNF to either receptor activates the proinflammatory transcription factor NF
B. In the case of TNF-R2, signal transduction occurs via heteterodimerization of the receptor with two TNF-R2 associated factors, TRAF1 and TRAF2, and it is TRAF2 that appears to mediate TNF-R2-induced activation of NFB. In contrast, TNF-R1, upon ligand binding, recruits a protein called TRADD. Like TRAF2, TRADD causes NFB activation but, unlike TRAF2, also causes apoptosis via an ICE-like protease. This explains why TNF-R1, but not TNF-R2, causes apoptosis. However, the NH2-terminal domain of TRADD interacts directly with TRAF2, and overexpression of a dominant negative TRAF2 blocks not only TNF-R2 but also TNF-R1-induced NFB activation. Thus activation of the two TNF receptors elicits separate signaling pathways that can interact with one another, thus explaining the distinct and overlapping signals generated by the two TNF receptors.
Interleukin-6 is a single 21- to 28-kDa glycoprotein produced by both lymphoid and nonlymphoid cells and regulates immune responses, acute-phase protein synthesis, and hematopoiesis. Human IL-6 is synthesized as a precursor polypeptide of 212 amino acids that is processed by cleavage of a 28-amino acid NH2-terminal signal sequence into a mature form of 184 amino acids.
One IL-6 receptor has thus far been identified. This IL-6 specific receptor (IL-6R) is responsible only for binding of its ligand (IL-6). Interleukin-6 belongs to a family of cytokines that includes ciliary neurotropic factor (CNTF), oncostatin M (OM), LIF, IL-11, and cardiotropin-1 (CT-1), which share a common signal-transducing mechanism (reviewed in Refs. 415, 416). All these cytokines are bound by receptors that interact with the common cell-surface protein gp130. Ligand-receptor complexes that share gp130 trigger signaling by the formation of either homodimers of gp130 or heterodimers between gp130 and LIFR. In the case of IL-6, signaling is initiated by the homodimerization of gp130 induced by the interaction with the IL-6/IL-6R complex. Either homodimerization of gp130 or heterodimerization of gp130 with LIFR activates JAK kinases, followed by the tyrosine-specific phosphorylation and nuclear translocation of a member of the STAT family (STAT3) of transcription factors. In addition, there is another signaling pathway that involves the activation of the RAS-MAP kinase cascade followed by the activation of transcription factors such as nuclear factor-IL-6 (NF-IL-6). Such sharing of receptor complexes and subsequent activation of similar signaling pathways by members of the IL-6 family of cytokines is a clear example of how a number of different cytokines display similar biological activities (i.e., cytokine redundancy).
Receptors for IL-1, IL-6, and TNF- occur not only in membrane-bound forms, but also as truncated soluble products, which are capable of binding their ligand (257, 332, 703). These receptors are generated either by proteolytic cleavage at the cell surface or are synthesized as alternatively spliced mRNA species. The ability of IL-1 and TNF soluble receptors to bind their ligands limits the availability of either IL-1 or TNF- for interaction with their membrane-bound receptors and therefore confers antagonistic properties to these truncated receptors. In contrast, the soluble IL-6 receptor, upon binding its ligand, can interact with gp130 and elicit a cellular response. Indeed, coincubation of IL-6 with its soluble receptor has been demonstrated to confer IL-6 sensitivity to previously IL-6-insensitive cells and enhances the effectiveness of IL-6 in vivo (506, 641, 747). Thus the biological activity of a particular cytokine is determined not only by its own concentration and the concentration of cytokines which influence its activity but also by the presence of its soluble receptor.
D. Hypothalamic-Pituitary-Adrenal Axis
1. HPA axis organization
Over the last 10-15 years, there have been over 1,000 published articles concerning the activation of the HPA axis by cytokines. This relative abundance of work is due to the large number of cytokines discovered, the complexity of the organization of the HPA axis (see Fig. 1), and the functional importance of activation of the HPA axis during stressful situations. Basal secretion of GC is necessary for the normal function of most tissues, and even small deviations from normal circulating levels of these steroids produce changes in a wide variety of physiological and biochemical parameters. Interactions between the endocrine system and the CNS result in a diurnal rhythm of GC secretion with a peak occurring at the time of awakening and a nadir during the first few hours of sleep. Blood levels of circulating GC increase in response to virtually any type of stimulus that poses, or is perceived to pose, a threat to bodily homeostasis. Glucocorticoids act on multiple targets to enhance or inhibit various cellular activities, actions that are aimed at providing the altered metabolic, endocrine, nervous, cardiovascular, and immunologic needs that promote survival. Not surprisingly, therefore, the regulation of blood levels of GC is subject to diverse sensory inputs, and this information is integrated at the level of the hypothalamus.
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The primary CNS nucleus involved in the regulation of pituitary-adrenal axis is the paraventricular nucleus (PVN) of the hypothalamus. The PVN is the principal CNS source of the 41-amino acid peptide CRF, which is the major physiological regulator of pituitary ACTH secretion (691). The CRF hypophysiotropic neurons from the PVN project to the external zone of the median eminence (ME) and release CRF into a specialized capillary network. The anterior pituitary (or adenohypophysis) is vascularized by hypophysial portal vessels that arise from these median eminence capillary beds. Within the anterior pituitary, CRF interacts with a specific G protein-coupled receptor (CRF-R1) on the corticotrope cell surface, resulting in the stimulation of the synthesis of the ACTH precursor peptide proopiomelanocortin (POMC) and the secretion of ACTH and other POMC-derived peptides (888, 898). Adrenocorticotropin hormone potently induces the secretion of GC from the zona fasciculata of the adrenal cortex. In humans, the major GC is cortisol, but in the rat and mouse, corticosterone is the main steroid product of the zona fasciculata. In a classical endocrine feedback manner, these steroids inhibit the synthesis and secretion of CRF within the hypothalamus and POMC-derived peptides in the pituitary (404, 995). The PVN has an anatomically discrete topography but functionally and phenotypically overlapping organization. The PVN is comprised of two major neurosecretory subdivisions; the magnocellular (mPVN) and parvocellular (pPVN) divisions, as well as more caudal cell groups giving rise to long descending projections to brain stem autonomic structures (738). The mPVN together with the supraoptic nucleus (SON) constitute the magnocellular neurons that are the major cell sources of arginine vasopressin (AVP) and oxytocin released into the general circulation from neurons terminating in the posterior pituitary. The more medially situated pPVN is the major source of hypophysiotropic CRF neurons, which release CRF into the hypophysial portal circulation. The cell groups projecting to autonomic structures contain all three peptides (CRF, AVP, and oxytocin). However, although these subdivisions of PVN are anatomically discrete, the PVN does display considerable peptide phenotype plasticity. The CRF hypophysiotropic neurons also produce a number of additional peptides, most notably AVP (740, 864), which interacts synergistically with CRF to stimulate ACTH secretion (693), and whose synthesis in these neurons can be enhanced during increased pituitary-adrenal activity (738). Furthermore, AVP and oxytocin derived from sources other than the pPVN may also contribute to the pool of ACTH secretagogues in hypophysial portal blood (15, 648, 651). Therefore, although it is generally agreed that CRF arising from the pPVN is the major means of stimulating ACTH secretion, this is not an absolute, with AVP (and possibly oxytocin) secretion from either pPVN or magnocellular neurons contributing to an extent that varies with the nature of the physiological threat. Consistent with the extreme diversity of stressful stimuli that give rise to activation of the HPA axis, the pPVN receives diverse inputs from regions of the brain conveying visceral, somatosensory, auditory, nociceptive, and visual information and also from limbic regions involved in the integration of cognitive and emotional influences (738). These inputs include projections from other nuclei within the hypothalamus (e.g., medial preoptic anterior hypothalamus). Extrahypothalamic inputs include areas both within [e.g., nucleus of the solitary tract (NTS) and other medullary catecholaminergic cell groups] and outside [organum vascularis of the lamina terminalis (OVLT) and subfornical organ (SFO)] the blood-brain barrier (BBB) (738). There are, therefore, multiple levels at which the activity of the HPA axis may be modulated, and indeed, virtually all the regulatory processes described above have been proposed as sites at which cytokines regulate HPA axis activity.
2. Experimental assessment of the HPA axis secretory activity
The measurable end points and experimental methodologies that have been used to examine the secretory activity of the HPA axis in response to cytokines are extremely diverse and shall be considered here briefly. The net result of increased HPA axis secretory activity is the elevated concentrations of ACTH and GC in blood. Temporal measures of immunoreactive levels of these hormones have been widely adopted as means of studying the influence of cytokines on the HPA axis and are the main method suitable for use in humans. Furthermore, the determination in laboratory animals of the effects of various surgical, pharmacological, or genetic manipulations on the plasma hormone response to a given cytokine provides a valuable means to elucidate the anatomic and neurochemical mechanisms involved in the activation of the HPA axis by a particular cytokine. To assess the activity of neuronal components of the HPA axis in response to a particular cytokine, a number of methods have been employed, including the use of electrophysiological recordings and the histochemical determination of the expression of cellular immediate early genes (cIEG), such as c-fos. The demonstration of c-fos as an inducible and widely applicable marker of neuronal activity (558) has afforded a means of mapping neuronal activation in response to a variety of stimuli, including the administration of cytokines. In particular, the stress-induced induction of c-fos mRNA and/or Fos protein within the PVN has been thoroughly examined and validated as a means to identify activation of neurosecretory neurons (152, 436). Another cIEG, NGFI-B, has been used for similar purposes (152, 436). Identification of the peptide phenotype of cells within the PVN expressing cIEG provides a powerful means to identify the activation of particular neurosecretory neurons. However, it should be noted that at present we do not know how the induction of such transcription factors relates to transcriptional activity within the PVN. Indeed, of the three peptides CRF, AVP, and oxytocin, only the AVP gene includes an AP-1 response element that binds Fos-Jun dimers, but all three genes contain potential NGFI-B response elements (152). Functional assessments of the relative neurosecretory rates of peptides from hypophysial PVN neurons have also been perfomed by measuring 1) peptide concentrations directly in portal blood, 2) peptide concentrations in the perfusates from push-pull cannulas or microdialysis probes within the ME, and 3) peptide content of the ME of either normal animals or animals pretreated with colchicine to block axonal transport. Finally, determination of the expression of steady-state mRNA or primary transcript RNA (hnRNA) levels of either CRF or AVP within the PVN have also been well documented. To determine the direct effects of cytokines on particular components of the HPA axis, a number of in vitro methodologies have also been used. Only one cell line has been available to study the action of cytokines on the HPA axis, namely, the AtT20 mouse corticotropic tumor line, which has been used as a model for investigating the direct effects of cytokines on anterior pituitary corticotropes. By far the majority of in vitro studies have utilized static or perfused primary preparations of either hypothalami, anterior pituitaries, or adrenals, with the tissue being intact (whole), in segments or slices, or in dispersed monolayer cell culture. These methodologies produce a more isolated environment in which to define direct actions of applied substances.| |
II. CYTOKINE INFLUENCE ON HYPOTHALAMIC-PITUITARY-ADRENAL AXIS SECRETORY ACTIVITY IN VIVO |
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A. Animal Studies
Numerous studies have confirmed and extended the original findings that the administration of either IL-1 or IL-1 to rats or mice stimulates ACTH and GC secretion, as well as many other indices of HPA activation (see Table 5). In addition, IL-1 has been demonstrated to increase the secretory activity of the HPA axis of chickens (959), sheep (916), baboons (674), and humans (see sect. IIB). Studies addressing interactions between cytokines and neuroendocrine systems in an invertebrate species (snail) have demonstrated the presence of a rudimentary stress system involving CRF-, ACTH-, and bioamine-like molecules in immunocytes (613-616). The snail immunocyte stress system contains, and is responsive to, cytokines, including IL-1, IL-2, and TNF- (612, 615-617). Activation of the HPA axis (or invertebrate equivalent) by IL-1 has therefore been highly conserved throughout evolution in different species and taxa, indicating the importance of this adaptive response to survival (615).
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In mammals, the ACTH response to intravenous IL-1 is usually prompt, commencing within 5-10 min, and of relatively short duration (~1 h). In comparison, the plasma ACTH response to intraperitoneal injection of IL-1 is slower in onset, but usually of longer duration (at least 2 h). Finally, the response to IL-1 administered directly into the brain (intracerebroventricularly) is of intermediate latency and lasts for several hours (usually greater than 3-4 h). The majority of studies have found IL-1 to be more potent than IL-1 in the rat (525, 574, 690, 695). Studies utilizing a panel of monoclonal antibodies have demonstrated that amino acids in the domain 66-85 on the recombinant rat IL-1 molecule are critical for its ACTH-releasing capacity (751). In the rat, IL-1 stimulates ACTH secretion at all stages of postnatal development of both males and females, although the magnitude of the ACTH response depends on age and gender (59, 192, 466, 598, 686).
A single administration of IL-1 not only acutely elevates plasma ACTH and corticosterone concentrations in the rat, but has been demonstrated to produce a long-lasting (at least 3 wk) increase in the coexpression of AVP in hypothalamic CRF neurons and a hyperresponsiveness of the HPA axis (744). Long-term administration of IL-1 to rats enhances CRF- and ACTH-like immunoreactivities in the hypothalamus and pituitary, respectively, increases adrenal weight (573), and elevates plasma ACTH concentrations for at least 7 days (573, 830, 908).
In addition to IL-1, a number of other cytokines have been demonstrated to influence HPA axis secretory activity in experimental in vivo paradigms (see Table 5). Activation of the HPA axis is not restricted to cytokines produced predominantly by myeloid (e.g., monocyte, macrophage) cells (e.g. IL-1), but also by cytokines produced by lymphoid (e.g., T lymphocytes) cells (e.g., IL-2). Where studies have compared the potencies, IL-1 has generally been found to be the cytokine most potent at stimulating ACTH secretion (e.g., Refs. 66, 358, 527, 772). However, this is not necessarily of physiological significance, since when it is the levels of endogenous cytokines that are elevated, the relative concentrations of each cytokine is a major determinant of which cytokine is of greatest influence. For example, during local inflammation, IL-6, which is generally agreed to be a less potent HPA axis secretagogue than IL-1, is elevated to a greater extent and for greater periods of time than is IL-1. Additionally, at least some cytokines act synergistically to enhance ACTH secretion. For example, all hematopoietic cytokines (IL-6, LIF, OM, CNTF, IL-11, and CT-1) enhance ACTH and/or corticosterone secretion produced by IL-1 to an extent greater than can be accounted for by additive effects (52, 639, 1003). Similarly, TNF- synergistically enhances ACTH release produced by IL-1 (907).
A number of studies have produced contradictory data with respect to the effects of various cytokines on HPA axis secretory activity. Although a stimulatory effect of TNF- on the rat HPA axis has not been disputed when the cytokine has been administered peripherally (58, 772, 909), contrasting data have been obtained when TNF- has been administered directly into the brain (intracerebroventricular). For example, although we showed that intracerebroventricular TNF- induces marked elevations in plasma ACTH concentrations in rats (881), a number of other investigators have found little or no effect (673, 772, 909). Such discrepancies may, at least in part, be explained by the use of cytokines of different species origin. In the example cited, we used murine TNF- (881), whereas those reporting little or no effect of intracerebroventricular TNF- on plasma ACTH levels used human TNF- (673, 772, 909). Similarly, studies in mice (51) have shown that plasma corticosterone concentrations are elevated to a greater extent by intracerebroventricular murine TNF- than by intracerebroventricular human TNF-. Species differences have also been noted with IL-6, and the human IL-6R recognizes human IL-6 but not mouse IL-6 (173, 905). The use of human or mouse cytokine preparations has been common due to their wide availability, yet the most commonly used species in the investigation of HPA axis activity is the rat. However, the previous limited availability of recombinant rat cytokines has meant that not many studies have investigated the effects of cytokines in a homologous system (rat cytokine in the rat). When rat IL-1 (574) and rat IL-1 (751) have been tested in rats, the intravenous injection of these cytokines produces a marked elevation in plasma ACTH levels. Recombinant rat cytokines have now become more widely available since the establishment of BIOMED 1 program "Cytokines in the brain" of the European Communities (headed by Dr. R. Dantzer, Bordeaux, France). However, at the time of submitting this review, no studies of the effects on the HPA axis of recombinant rat cytokines generated by this initiative had been published.
Although pharmacological differences of cytokines from different species may seem to complicate interpretation of data, they can actually provide useful pharmacological tools. For example, the difference between the effects of intracerebroventricular mouse and human TNF- on plasma ACTH concentration may reflect the differing pharmacological profiles of the two identified TNF receptors, TNF-R1 and TNF-R2. Murine TNF-R1 has high affinity for either mouse or human TNF-, whereas only mouse TNF- is effective at TNF-R2 (334, 467, 505). Should the same pharmacology be true at rat TNF- receptors, as has been suggested (817), then the increase in plasma ACTH concentrations induced by intracerebroventricular mouse, but not human, TNF- indicates that TNF-R2 is the major receptor isoform involved in cerebral TNF--induced activation of the rat HPA axis.
Although by far the majority of cytokines tested in animal studies have been found to exert stimulatory actions on the HPA axis (see Table 5), two cytokines (IL-4 and IFN-) have been suggested to inhibit HPA axis activity in vivo. The anti-inflammatory cytokine IL-4 dose-dependently inhibits POMC mRNA expression in the anterior pituitary, without affecting CRF mRNA in the pPVN, suggesting a direct inhibitory action at the level of the pituitary (326). Interferon- has also been suggested to inhibit HPA axis secretory activity, since the administration of low doses either intraperitoneally or intracerebroventricularly reduces plasma corticosterone levels and the electrical activity of neurons within the PVN (725, 726, 729). However, higher doses of IFN- given intracerebroventricularly enhance plasma corticosterone levels (726), suggesting that the qualitative effects of IFN- on HPA axis activity are dependent on the dose used.
B. Human Studies
In addition to the numerous studies of the effects of cytokines on the HPA axis of laboratory animals, the clinical trials of a number of cytokines as anticancer strategies have afforded the opportunity to detail their effects on the HPA axis of humans. Such clinical studies have permitted investigation of the effects of cytokines on the HPA axis in a homologous system (i.e., human cytokines in human subjects). Either intravenous or subcutaneous administration of IL-1 (179, 795), IL-1 (176, 179), IL-2 (24, 194, 479-481, 812), IL-6 (519, 520, 809), TNF- (596) IFN- (41, 289, 337, 565, 566, 702, 758), IFN- (596), and IFN- (342, 596, 810) elevates plasma ACTH and/or cortisol concentrations. As in laboratory animals, the stimulation of ACTH secretion produced by cytokines occurs rapidly: within 1 h of intravenous infusion or within 1-4 h of subcutaneous treatment.
A series of experiments (519, 520) in which cancer patients of good clinical performance were examined showed that IL-6 is a particularly potent activator of the HPA axis and that the human HPA axis is remarkably responsive to this cytokine. On the first treatment day, IL-6 (30 µg/kg sc) induced marked elevations in plasma ACTH and cortisol concentrations, with peaks occurring at 1 and 2 h, respectively. Plasma ACTH concentrations returned to basal levels within 5 h, whereas plasma cortisol levels remained elevated for 24 h. By the seventh day of treatment, the ACTH response to IL-6 was markedly diminished, an effect that was probably due to increased negative feedback produced by persistently elevated plasma cortisol levels. The sustained secretory activity of the adrenal was accompanied by gross enlargement of the adrenal glands as assessed by computed tomographic scans. Subsequent studies demonstrated that as little as 0.3 µg/kg (intravenous) is already a maximal dose of IL-6 (520). The magnitude of the plasma ACTH response to a first injection of IL-6 was greater than that reported with the standard tests of pituitary-adrenal function such as injection of ovine CRF or the insulin tolerance test. The relatively mild toxic effects of IL-6 led the authors to propose that IL-6 may provide a new means of testing pituitary-adrenal function (519) and to conduct additional studies in normal human subjects (876). In normal healthy males, IL-6 (subcutaneous) again produced substantial elevations in plasma ACTH and cortisol, with peaks in ACTH and cortisol levels observed at 60-90 and 90-120 min, respectively, and a minimal effective IL-6 dose of 1-3 µg/kg (876).
Overall, human and animal studies agree that many exogenously administered cytokines have marked stimulatory actions on HPA axis secretory activity and suggest that endogenous production of cytokines during homeostatic threats may well play a causal role in the elaboration of the accompanying HPA axis response.
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III. PHYSIOLOGICAL/PATHOPHYSIOLOGICAL CIRCUMSTANCES IN WHICH ENDOGENOUS CYTOKINES PLAY A ROLE IN REGULATION OF HYPOTHALAMIC-PITUITARY-ADRENAL AXIS |
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There are a number of examples of injury, infection, and/or disease that are associated with increased cytokine production and concomitant elevations in HPA axis activity: head trauma (427, 611, 976), cerebral ischemia (stroke) (253, 367, 571), a number of autoimmune diseases (175, 239, 322-325, 347, 455, 539, 654, 959), psychiatric and dementia disorders (165, 302, 510, 896), acquired immune deficiency syndrome (AIDS) (636, 661, 763, 827, 954), and antigenic challenges (69, 72, 73). However, direct and clear evidence for the involvement of particular cytokines in the HPA axis response to such insults has been limited to only a few cases (viral or bacterial infection, local tissue damage and inflammation, and acute physical/psychological stress). Furthermore, although numerous cytokines have been shown to influence the secretory activity of the HPA axis, the direct demonstration of cytokine involvement in physiological or pathophysiological HPA axis responses has been restricted largely to the cytokines IL-1, IL-6, and TNF-. The following sections outline the types of threats to homeostasis that have been demonstrated to elicit activation of the HPA axis via mechanisms that depend critically on the endogenous elaboration of cytokines.
A. Viral Disease
1. Newcastle disease virus
The first direct evidence indicating that IL-1 participates in the HPA axis response to an immune challenge came from studies investigating the neuroendocrine responses to inoculation with NDV (70). Newcastle disease virus is a neutrotropic paramyxovirus which, when administered to rodents, produces symptoms of viral disease without the potential hazard of replication. Within 1-2 h of injection, NDV produces marked elevations in ACTH and corticosterone concentrations in the blood of mice (70, 219-221, 604, 793) or rats (685). The majority of studies demonstrate that the increase in corticosterone is abolished by hypophysectomy (218, 220, 221, 604), indicating the importance of the pituitary in the adrenal response. Furthermore, the ACTH response is completely prevented when rats are passively immunized against CRF (685), indicating that hypothalamic CRF regulates the pituitary ACTH response to NDV. The stimulation of the HPA axis by NDV appears to be produced not by the virus itself, but by mediators released by immune cells exposed to the virus. This is evidenced by the fact that the supernatants of cocultures of NDV with either human peripheral blood leukocytes (HPBL) or mouse spleen cells also elevate plasma corticosterone concentrations. Indeed, intraperitoneal injection of supernatant from NDV plus HPBL produces a fourfold increase in plasma corticosterone concentrations in rats (70). This plasma corticosterone response is prevented by preincubation of the NDV plus HPBL supernatant with a rabbit neutralizing anti-human IL-1 antibody (70). More recent studies have demonstrated that intraperitoneal administration of IL-1ra to mice virtually abolishes the elevations in plasma ACTH and corticosterone concentrations 2 h after NDV (221), confirming the obligatory role of IL-1 in the generation of the HPA axis response to this viral challenge.2. Polyinosinic polycytidilic acid
Polyinosinic polycytidilic acid (Poly I:C) is a synthetic, double-stranded polyribonucleotide commonly used to mimic viral exposure. Injection of Poly I:C, like NDV, produces a rapid (within 1-2 h) activation of the HPA axis (544, 716). Although there have been only a few investigations of the HPA axis response to Poly I:C, it is apparent that CRF is an important mediator of this response, because Poly I:C-induced increases in rabbit plasma cortisol concentrations are abolished by pretreatment with a monoclonal anti-CRF antibody (544). Furthermore, the substantial plasma corticosterone response to Poly I:C observed in normal mice is completely absent in mice deficient in IL-6 (716), indicating that Poly I:C-induced activation of the HPA axis is dependent on the elaboration of the cytokine IL-6.3. Murine cytomegalovirus
Cytomegaloviruses (CMV) are herpes viruses that are a major cause of mortality and morbidity in human transplant recipients, are a serious problem in patients with AIDS, and are the most frequent viral cause of congenital abnormalities. Administration of murine CMV (MCMV) productively infects mice. Recent studies by Ruzek et al. (716) showed that MCMV induces increased levels of IL-12, IFN-, TNF-, IL-1, and IL-6, but not IL-1, in the general circulation at 24-48 h of infection. During this period, there are marked increases in the plasma concentrations of corticosterone and smaller, but statistically significant, increases in plasma ACTH (716). The corticosterone response to MCMV in either normal mice treated with neutralizing anti-IFN- antibodies, or in IFN--deficient mice, is comparable to that in control mice infected with MCMV (716). However, IL-1 and IL-6 appear to play important roles in the activation of the HPA axis. The corticosterone response to MCMV is markedly blunted in either normal mice treated with IL-1ra or in IL-6-deficient mice, without either of these "treatments" having a significant impact on viral load. The elevated IL-6 levels produced by MCMV infection are dramatically reduced when mice are treated with IL-1ra, whereas IL-1 levels are normally elevated in IL-6-deficient MCMV mice (716). Consequently, the authors concluded that IL-6 is the pivotal cytokine in the activation of the HPA axis in response to MCMV and that IL-1 plays a secondary role by contributing to IL-6 production (716).
B. Endotoxin Treatment
Endotoxins are LPS constituents of the outermost part of a Gram-negative bacterial cell membrane that are released upon bacterial lysis. Administration of purified preparations of LPS mimics many of the acute phase responses to Gram-negative infection without actively infecting the host (123). Consequently, administration of bacterial endotoxins to laboratory animals has been the most commonly used model to study the mechanisms underlying the neuroendocrine responses to bacterial infection and sepsis (214, 863, 883).
Although mice and rats are relatively insensitive to LPS in comparison with many other species (including humans), the intravenous administration of LPS to laboratory rodents produces marked elevations in ACTH and corticosterone secretion within 30-60 min. It should be emphasized that, quantitatively, physiological responses to LPS can differ depending on its source and preparation (345). We find that doses of ~5 µg/kg LPS (Escherichia coli serotype O26:B6; lot 20H4025, Sigma Chemical) produce a peak elevation in plasma ACTH concentration of 500-1,000 pg/ml (compared with <20 pg/ml in controls) at 90-120 min after intravenous injection in intact, male rats (889). Similarly, LPS stimulates ACTH and corticosterone secretion in mice, and HPA activation can be observed in both rats and mice after either intravenous or intraperitoneal administration. However, the precise mechanisms through which these two routes of LPS administration influence the HPA axis may be substantially different (144, 925).
In addition to elevated blood levels of ACTH and corticosterone, other indexes of HPA activation have been reported after peripheral administration of LPS. Intravenous or intraperitoneal LPS produces increased pPVN expression of c-fos mRNA or Fos protein and of CRF hnRNA or mRNA (230, 387, 456, 680, 718, 924, 925). Rats in which the PVN has been electrolytically lesioned can mount a detectable plasma ACTH response to an extremely large dose of LPS (2 mg/kg ip), but its magnitude is markedly diminished (227), indicating that the PVN plays a pivotal role in LPS-stimulated increases in plasma ACTH. Indeed, it is clear that CRF is an important mediator of LPS-induced ACTH secretion. This is evidenced by the increased secretion of CRF from the ME after systemic treatment with LPS (292) and by the marked attenuation, or abolition, of LPS-induced ACTH secretion produced by doses of LPS that are either very large (2.5 mg/kg ip) or more moderate (2.5 µg/kg ip or 50 µg/kg iv), respectively (25, 750).
These actions of LPS on the HPA axis in vivo are not due to a direct pharmacological interaction of LPS with HPA axis tissues. Lipopolysaccharide has either no effect or an inhibitory action on either CRF secretion from hypothalamic explants (545, 581, 652) or ACTH secretion from rat anterior pituitary cell cultures (117, 886). It also seems unlikely that LPS enhances the pituitary ACTH response to CRF, since LPS reduces the expression of CRF receptors in the pituitary both in vivo and in vitro (25), and ACTH secretion by rat anterior pituitary cell cultures stimulated with CRF is unaffected by cotreatment with LPS (886). Furthermore, mice (C3H/HeJ strain) that are deficient in their production of IL-1 in response to LPS (373, 759, 856) exhibit markedly reduced elevations in the plasma concentrations of ACTH and corticosterone after intraperitoneal LPS (216), suggesting that IL-1 is an important mediator of the effects of LPS. Lipopolysaccharide is a potent inducer of the synthesis and secretion of a number of cytokines (see sect. IC). In particular, the cytokines IL-1, IL-6, and TNF- are likely candidate mediators of the effects of LPS on neuroendocrine secretion (863).
After administration of LPS directly into the bloodstream, the plasma concentrations of each of these three cytokines are elevated in a regulated temporal manner with TNF- first, then IL-1, and finally IL-6 (174, 189, 290). Not only are the secretions of these three cytokines temporally related, but there is also evidence that they are also causally related. Administration of antibodies to TNF- blunts the secretion of IL-1 and IL-6 in response to LPS (263), whereas immunoneutralization of IL-1 (487) or frequent administration of large doses of IL-1ra (494) abrogates LPS-induced IL-6 secretion. After local injection of LPS (e.g., intraperitoneally or into an experimentally constructed, subcutaneous air pouch), the local concentrations of all three cytokines are also elevated. However, their levels in blood appear to be dependent on the degree of "overspill" into the general circulation, with only IL-6 levels being consistently increased in systemic blood (541, 542, 844, 998). Local administration of IL-1ra at the site of LPS injection inhibits the rise in plasma IL-6 levels (541), again suggesting a causal relationship between the production of IL-1 and IL-6 after LPS. In light of these data, it is surprising that recent experiments performed in mice have shown that the plasma IL-6 response to LPS appears normal in mutant mice lacking either IL-1 (10, 1001) or IL-1R1 (463). Whether this indicates important roles for IL-1 (or IL-1) and the IL-1R2 (or novel IL-1 receptors) clearly warrants investigation.
Comparisons of the time courses of cytokine production and HPA axis activation produced by systemic LPS have produced somewhat conflicting data. The concentrations of TNF-, IL-1, and IL-6 in plasma have been reported to lag behind the rise in plasma ACTH after intra-arterial LPS regardless of LPS dose (290). However, after low doses of LPS administered intravenously, elevations in plasma TNF- coincide with the secretion of ACTH, whereas at higher doses, elevations in plasma TNF- occur after the initial rise in plasma ACTH (223). We find that after LPS (5 µg/kg iv), the TNF- profile in blood precedes that of ACTH, with a time of onset and peak that occurs 15 min before those of plasma ACTH (889).
The importance of cytokines in the ACTH response to LPS was first directly indicated by the pronounced inhibition of this response when mice were pretreated with IL-1 receptor antibodies (690). Further studies showed that destruction of macrophages produced a marked reduction in circulating IL-1 levels in response to a high dose of LPS (2.5 mg/kg iv) and a 40% inhibition of the ACTH response to a small dose of LPS (2.5 µg/kg iv) (195). Either anti-IL-1 receptor antibodies given to mice (640) or IL-1ra injected into rats (223, 752) reduce the ACTH response to LPS admistered intravenously or intraperitoneally. A CNS site of IL-1 action has been suggested by experiments showing that intracerebroventricular infusion of IL-1ra inhibits the increase in CRF mRNA in the PVN of rats 8 h after intraperitoneal LPS (387).
Although the above findings seem to strongly implicate the cytokine IL-1 as a mediator of the activation of the HPA axis by LPS, not all studies support this hypothesis. For example, mice injected with IL-1ra ip, at a dose that inhibited the corticosterone response to either IL-1 or IL-1, failed to inhibit the corticosterone response to LPS (213). Furthermore, the development of genetically manipulated mice, with deficiencies in various components of the IL-1 system, has raised interesting questions regarding the absolute requirement of IL-1 in acute phase responses to LPS. For example, fever in response to LPS is inhibited by either anti-IL-1 antibodies (418, 461, 487) or by IL-1ra (494, 791), clearly implicating IL-1 in the pathogenesis of fever due to LPS. However, mice deficient in IL-1 show only a slightly decreased (10) or even an increased (438) fever in response to intraperitoneal LPS. Similarly, although IL-1 has been implicated in the induction of IL-6 and the cachexia after LPS, neither of these responses are inhibited in IL-1-deficient mice (10, 247, 250, 438, 1001). Studies in IL-1R1-deficient mice demonstrated that IL-1R1 is essential for all the IL-1 mediated signaling events examined (fever, induction of IL-6, induction of E-selectin) (442), but these animals display normal fever and cachexia induced by intraperitoneal LPS (442, 463). Not surprisingly, therefore, investigations of the HPA axis response to LPS in these mutant mice have also failed to confirm a role of IL-1 in the elaboration of this HPA axis response (10, 247, 250).
Investigations of the effect of immunoneutralizing IL-6 and TNF- have also suggested physiological roles for these cytokines in the HPA axis secretory response to LPS. Although inhibition of either IL-1, IL-6, or TNF- abrogates the ACTH response to larger doses of LPS in mice, Perlstein et al. (640) found that only anti-IL-6 an
