Activation of the hypothalamus-pituitary-adrenal axis by bacterial endotoxins: Routes and intermediate signals

Pergamon

Psychoneuroendocrinology, Vol. 19, No. 2, pp. 209-232, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0306-4530/94 $6.00 + .00

0306-4530(93)E0010-S

REVIEW ACTIVATION OF THE HYPOTHALAMUS-PITUITARY-ADRENAL AXIS BY BACTERIAL ENDOTOXINS: ROUTES AND INTERMEDIATE SIGNALS F R E D J . H . TILDERS, l ROEL H . D E R I J K , 1. A N N E - M A R I E VAN D A M , I VALERIE A . M . VINCENT, I KAREL SCHOTANUS, 1 and JEK H . A . PERSOONS 2 Departments of IPharmacology and 2Cell Biology, Research Institute Neurosciences Vrije Universiteit, Amsterdam, The Netherlands

SUMMARY Peripheral administration of endotoxin induces brain-mediated responses, including activation of the hypothalamus-pituitary-adrenal (HPA) axis and changes in thermoregulation. This paper reviews the mechanisms by which endotoxin affects these responses. The effects on theroregulation are complex and include macrophage-dependent hyperthermic and hypothermic responses. Low doses of endotoxin, given IP, activate peripheral macrophages to produce interleukin (IL)-lfl, which enters the circulation and acts as a hormonal signal. IL-I may pass fenestrated endothelium in the median eminence to stimulate corticotropin-releasing hormone (CRH) secretion from the CRH nerve-terminals. In addition, IL-1 may activate brain endothelial cells to produce IL-I, IL6, prostaglandins, etc., and secrete these substances into the brain. By paracrine actions, these substances may affect neurons (e.g., CRH neurons) or act on microglial cells, which show IL-1induced IL- 1 production and therefore amplify and prolong the intracerebral IL- 1 signal. In contrast, high doses of endotoxin given IV may directly stimulate endothelial cells to produce IL-1, IL-6, and prostaglandin-E 2 (PGE2) and thereby activate the HPA axis in a macrophage-independent manner.

INTRODUCTION THE STRESS CONCEPT of Hans Selye was primarily based on stimuli that disturbed the physical integrity of the individual. In his studies, administering toxins to animals, inducing tissue damage, or exposing animals to X-rays played an important role. Selye noted that this type of stimuli resulted in a general and stereotyped somatic response, known as the GeneralAdaptation Syndrome, which involves enlargement of the adrenals, involution of the thymus, lymphopenia, and gastric ulcerations. These changes are associated with increased activity of the hypothalamus-pituitary-adrenal (HPA) axis and played a A d d r e s s c o r r e s p o n d e n c e and reprint requests to: F . J . H . Tilders, Research Institute N e u r o s c i e n c e s Vrije Universiteit, Faculty of Medicine, D e p a r t m e n t of Pharmacology, Van der Boechorststraat 7, 1081 B T A m s t e r d a m , The Netherlands. * Present address: N I M H , Dept. Intramural Res. Prog., Clin. N e u r o e n d o c r i n o l o g y Branch, Bldg 10 R o o m 3S231, 9000 Rockville Pike, Bethesda, Maryland 20892, U . S . A . 209

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F. .I.H. TILLERS ('Id.

crucial role in the development of the stress concept. From these early studies, it appears that conditions that cause tissue damage and inflammation lead to activation of the HPA axis. We now know that injury, inflammation, and infection induce an acute phase response (Dinarello, 1984) which, in addition to activation of the HPA axis, consists of a well orchestrated set of changes in all three major control mechanisms-the nervous system, the endocrine system, and the immune system. The effects on these systems become manifest as changes in behavioral, cognitive, metabolic parameters and in hostdefence responses. The routes by which emotional and physical stressors lead to neuronal and endocrine responses have been studied extensively and a detailed picture of their interrelationships is emerging. More recently, much attention has been focussed on the neuroendocrine responses induced by infection and tissue injury. Many researchers (including ourselves) became intrigued by the mechanisms by which peripheral cells of the (para)immune system that are activated during infection, injury, and inflammation, can influence the activity of the brain and the endocrine system. in particular the HPA axis. In the present paper, we will discuss some of the major hypotheses and findings on this issue, with special emphasis on the contributions that the late Dr. Frank Berkenbosch has made to this field. ENDOTOXINS Bacterial endotoxins are often used in animal models to study the mechanisms involved in inflammation and sepsis. Endotoxins are lipopolysaccharides (LPS) that are present on the outer membrane of Gram-negative bacteria. When shed from the bacterial wall they may remain locally, but nonetheless induce an array of brain-mediated responses including fever (Shalaby et al., 1989), sickness behaviour (Kent et al., 1992), and activation of the HPA axis (Berkenbosch et al., 1992b). In patients with intestinal damage or other massive infections, endotoxins may reach the bloodstream (endotoxemia) resulting in life-threatening situations including septic shock (Martich et al., 1993; Molloy et al., 1993). The pivotal role of endotoxins in central and peripheral responses to Gram-negative bacteria is demonstrated by the fact that peripheral administration of endotoxins to animals and humans, results in peripheral and brain-mediated responses that are very similar to those observed after infection with such bacteria (Martich et al.. 1993; Red1 et al., 1993). Under normal conditions, endotoxins originating from intestinal bacteria are effectively bound to bile acids and are prevented from reaching the bloodstream. However, certain pathologic conditions, for example, obstruction of the bilary duct, multitrauma, and possibly severe physical exhaustion, may result in the appearance of these endotoxins in the circulation and cause symptoms of sickness. Immune cells and macrophages form a diffuse system of migrating cells that operates as an endotoxinsensor and translates the endotoxin signal present locally or in the circulation, into other chemical messages (e.g., cytokines, eicosanoids, nitric oxide) that act locally or affect distant targets (e.g., the brain). In accord with their role as endotoxin-sensors, high affinity endotoxin receptors have been demonstrated on macrophages, lymphocytes, and granulocytes (Morrison et al., 1993). In addition to scavenger receptors, involved in the clearance of endotoxin and bacteria (CD11/18 adhesins), two types of receptors have been described that mediate endotoxin-induced cytokine production. The dominant type on lymphoreticular cells is a 70-80 kDa protein with an as yet unknown molecular structure. The receptor that is

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ROLE OF PRIMARY AFFERENTS The acute responses of ACTH and corticosterone to peripheral administration of endotoxin are mediated by the release of corticotropin-releasing hormone (CRH) from hypothalamic neurons. Thus, in rats treated with chlorpromazine, morphine, and pento-

I-. J.H. .I-Il.DERS

<‘/ (I/.

TABLE I. EFFECTS OF NEONATAL CAPSAKIN TREATMENTON ENDOTOXIN-INDUCED PLASMA ACTH CONCENTRATIONS Plasma ACTH concentrations (pglmb

Treatment

146 I 828 f

Saline Endotoxin (2.5 pgikg

Capban mately wline

1992).

Data

wa\

given

cndotoxin

to the other

concentrations

27 9x+

86 1~ ,J x99 F 299”

IP)

or vehicle 6 weeks,

Capsaicin (50 mglkg SC)

Vehicle

half.

were Irepresent

Rat\

10 ?-day-old

(2.5 gg/kg wcrc

determined mean

ra15 (50 mg/kg

IP)w;hgiven tohalf

decapttated a\

? SEM

described (II

L h-XI.

7.5 h later &ovhere *[J *.

SC’).

After appr"\~-

of each group and plasma

and

At‘TL-I

(VanOrr\r‘~ *II .OS n.

alinc

treated

animal\.

barbital, endoxin fails to elicit an ACTH reponse (Moberg, 1971). Moreover. interruption of the input to the median eminence by anterolateral deafferentation of the basal hypothalamus inhibited the acute ACTH response to endotoxin by more than 80% (Schotanus et al., 1994). In addition, complete inhibition of the ACTH response was observed after passive immunization with relevant amounts of a monoclonal antibody to CRH (Schotanus et al., 1994; Van Oers et al., 1989), which is in line with other observations (Rivier, 1989). It should be noted that not all effects of endotoxins on the HPA axis are mediated by activation of hypothalamic CRH neurons. In contrast to the ACTH response to low doses of endotoxin (pgikg), ACTH responses to high doses (mgikg) appear partly independent of hypothalamic CRH secretion (Elenkov et al., 1992; Schotanus et al., 1994). This may be due to direct effects of endotoxins on the pituitary and adrenal glands as demonstrated in vitro (Koenig et al., 1990; Spangelo et al., 1990). Such peripheral effects of endotoxins may contribute in particular to the late responses seen after high endotoxin doses, but do not contribute much to the fast response to low doses of endotoxin (Schotanus et al., 1993a). It has been hypothesized that endotoxins affect nociceptive afferents in the periphery (Ferreira et al., 1988) and thereby activate the HPA axis. This is based on data showing that destruction of primary afferents inhibits the ACTH response to tissue injury (Amann & Lembeck, 1987; Turnbull et al., 1994). In support of this, neonatal administration of capsaicin, which leads to degeneration of most primary sensory afferents, attenuated the ACTH responses to stressors such as cold exposure, surgery (Donnerer et al., 1992), and hemorrhage (Guarini et al.. 1992). Therefore, we studied the effects of neonatal capsaicin treatment on the endotoxin-induced HPA responses. As illustrated in Table I. treatment of neonatal rats with capsaicin. resulting in a significant decrease in pain sensitivity (hot-plate test), did not attenuate the ACTH response to intraperitoneal administration of a low dose of endotoxin. This observation indicates that activation of primary afferents does not play an important role in endotoxin-induced responses of the HPA axis.

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HPA RESPONSESTO ENDOTOXIN: ROUTES AND SIGNALS

Humoral

Signalling Hypothesis

This hypothesis proposes that factors produced in the periphery under the influence of endotoxins, appear in the general circulation in concentrations that are sufficient to affect distant targets such as the brain, and thereby exert an hormonal action. Because cytokines are produced in response to endotoxins and because administration of cytokines, notably tumor necrosis factor-a (TNFa), interleukin-1 (IL-l) and interleukin-6 (IL-6), into the bloodstream can activate the HPA axis, cytokines are strong candidates for such actions (DeRijk et al., 1991; Ling, 1979; Rivier et al., 1989; Ulich et al., 1991; Wolf, 1974). Although these cytokines may all appear in circulation, they do so in different time domains after endotoxin administration. TNFa is the first cytokine to appear followed by IL-lp and IL-6 (Shalaby et al., 1989; Zanetti et al., 1992). In addition, these cytokines mutually affect each others’ production. TNFa can induce IL-l production and both provoke the production of IL-6 and can act synergistically in this respect (Dinarello et al., 1987; Libert et al., 1990; Shalaby et al., 1989). Although in vitro data show that TNFa mRNA and TNFa can be induced by IL-lp (Dinarello, 1985; Philip & Epstein, 1986), administration of IL-I/3 or IL-la in vivo, does not lead to measurable plasma levels of TNFa (Fisher et al., 1991; Shalaby et al., 1989). In addition, IL-6 can down regulate endotoxin-induced TNFar production in vitro and in vivo as well as the increase in IL-l mRNA (Aderka et al., 1989; Starnes et al., 1990; Ulich et al., 1991). This suggests that IL-6 in addition to its proinflammatory activity also exerts antiinflammatory activity. In view of these interactions, it should be noted that the in vivo administration of any of these cytokines may affect the production of the other cytokines and thereby the interpretation of the results is complicated. As mentioned above, the HPA-response to endotoxin is mediated by activation of hypothalamic CRH neurons. Likewise, TNFq IL-l, and IL-6 also stimulate ACTH secretion through stimulating CRH secretion from hypothalamic CRH neurons (Berkenbosch et al., 1987; Harbuz et al., 1992; Naitoh et al., 1988; Sapolsky et al., 1987; Suda et al., 1990; Watanobe & Takebe, 1992). These observations support the candidacy of all three cytokines to act alone or in concert as mediators of the endotoxin-induced activation of the HPA axis. Which Cytokines Are Humoral Mediators?

In order to become accepted as a humoral mediator secretion, a cytokine has to fulfill the following criteria: 1. 2. 3. 4. 5.

of endotoxin-induced

ACTH

Its concentration in plasma should increase upon endotoxin administration. Administration of the cytokine should activate the HPA axis. Its appearance should precede or coincide with the activation of the HPA axis. Circulating concentrations should be effective for activation of the HPA axis. Blockade of its action should attenuate the effect of endotoxin on the HPA axis.

The first two criteria are met by all three cytokines. Elevated or detectable concentrations of TNFq IL- lp, and IL-6 have been found in plasma after endotoxin administration in various species (DeRijk et al., 1991; McGillis et al., 1987; Schotanus et al., 1993a; Shalaby et al., 1989; Zanetti et al., 1992; Zuckerman et al., 1989). Administration of recombinant preparations of each of these cytokines is followed by a rise in plasma ACTH concentrations (Berkenbosch et al., 1989; Bemardini et al., 1990; Harbuz et al., 1992; Naitoh et al,, 1988; Sharp et al., 1989). When administered IP, their dose-response

214

F. J. H. TILDEHS it rd

curves appear to be shifted towards higher dose ranges as compared to those found after IV administration (Zanetti et al., 1992), which supports the view that the cytokines exert their ACTH-releasing action after entering the blood (Rivier & Vale, 1991; Schotanus et al., 1993a). Comparison of the cytokine-dose ACTH-response characteristics shows that IL-lp is the most potent inducer of ACTH secretion (Besedovsky et al., 1986; Butler et al., 1989; Naitoh et al., 1989) and exerts synergistic effects with TNFa and IL-6 (Perlstein et al., 1991, 1993). The third and fourth criteria, are much harder to fulfill. Especially for IL-lp, the sensitivity of available assays is poor compared to concentrations that are proposed to be biologically active. Therefore, undetectable IL-l levels cannot be readily interpreted. Moreover, it is not always clear what exactly are relevant plasma concentrations (see below). High plasma IL-6 concentrations (0.2-2 pg/ml) have been observed after endotoxin treatment, and IV administration of IL-6 in doses resulting in such plasma concentrations can induce ACTH secretion (Shalaby et al., 1989; Ulich et al., 1991). However, at least following IP administration of endotoxin, the activation of the HPA axis precedes rather than follows detectable increases in plasma IL-6 levels. Various studies have been carried out to abrogate the action of a specific cytokine by the use of antibodies to the cytokine itself, to its receptor or using cytokine-receptor antagonists. In an early study in mice, an antiserum against the IL-l receptor was found to attenuate endotoxin-induced ACTH secretion (Rivier et al., 1989). Although conflicting data have been reported (Dunn, 1992; Perlstein et al., 1993), we demonstrated that the ACTH secretion provoking effect of a maximally effective dose of endotoxin could be blocked by peripheral administration of the IL- 1 receptor antagonist (Schotanus et al.. 1993a). Therefore, IL-1 appears to be a major mediator of endotoxin-induced activation of the HPA axis and of other centrally mediated effects. Others failed to attenuate the endotoxin-induced ACTH response with antisera to TNFa (Dunn, 1992; Perlstein et al.. 1993). Because an antiserum to IL-6 prevented the endotoxin-induced HPA-activation (Perlstein et al., 1993), some researchers have postulated that IL-6 is the prime mediator controlling this response. However, since the recombinant human IL- 1 receptor antagonist did not affect the IL-6 response (Schotanus et al., 1993a; Smith & Kluger, 1992). but blocked the ACTH response to endotoxin, an essential role of circulating IL-6 in this centrally mediated response is most unlikely. Taken together, there is little experimental evidence to support the view that TNF plays an important role as a humoral signal that affects the central limb of the HPA axis. With regard to the role of circulating IL-1 and IL-6, the available evidence is not conclusive, but points towards IL-I as the primary candidate. Cytokines

in

Plasmu

Measurement of IL-ID. Interleukin-1 exists in two forms, IL-la and IL-lb. Despite limited homology in amino acid sequence (26%). they exert similar biological acivities and are known to interact with the same IL-l receptors. Although conflicting data exist, the principal form that is secreted and that enters the bloodstream to act on distant targets appears to be IL-lp, whereas IL-la that is expressed in a membrane-bound form has local actions. If indeed IL-l@ acts as an hormonal factor, orchestrating the neuroendocrine response to injury and inflammation, relevant concentrations should be present in the circulation after endotoxin administration. Considering that some biological responses show medial-effective doses of IL-10 in the order of I-10 pmolil, it is antici-

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215

pated that concentrations as low as 0.1-I pmol/l may exert biological effects in vivo. Accordingly, appropriate IL-l assays should be able to detect IL-I/3 concentrations of at least 1 pg/ml of plasma. We are not aware of any assay with a sufficiently high sensitivity to detect such low levels. Various bioassays have been used for IL-l detection (Hopkins & Humphreys, 1990; Moldawer et al., 1987; Remvig et al., 1991). However, they all suffer from a lack of specificity and respond to other cytokines and hormones. Furthermore, various substances are known to occur in plasma that interfere with the biological activity of IL-l (Dinarello & Thompson, 1991). For instance, a protein has been isolated and identified that binds to IL-l receptors and blocks signal transduction (IL-1 receptor antagonists; IL-lra) (Eisenberg et al., 1990). This naturally occuring receptor antagonist has been cloned and is known to be produced by monocytes and in the brain (Andersson et al., 1992; Arend et al., 1991; Goto et al., 1992; Hannum et al., 1990; Licinio et al., 1991). Thus even by using “specific” bioassays, the results will be influenced by other factors, and unless extensive purification is carried out, the results do not necessarily reflect IL-l,0 concentrations in the circulation. The same interpretation problem exists with competitive binding assays that make use of the IL-l receptor (Zuckerman et al., 1991). Immunoassays can circumvent these specificity problems (Cannon et al., 1990; Hogquist et al., 1991), but immunoreactive IL-l (irIL-1) does not necessarily reflect bioactive IL-I. Recently, we developed a radioimmunoassay for the determination of IL-l/3 in rat plasma by using an antiserum generated in a goat to recombinant human IL-l/3, as described in detail elswhere (DeRijk & Berkenbosch, 1992). By using this assay, we found that IV administration of endotoxin at doses of up to 1 mg/kg to rats, resulted in a dose-dependent increase of circulating irIL-lb levels reaching maximal values of 4-10 &ml after 1.5-2 h. Furthermore, depletion of macrophages, which are the most likely sources of IL- lp (see below), fully prevented the endotoxin-induced appearance of irILI/3 in rat plasma. Immunoreactive IL-lb could not be detected in plasma of untreated rats, and in rats treated with low doses of endotoxin that stimulate ACTH secretion (DeRijk & Berkenbosch, 1992; DeRijk et al., 1991). This is not surprising in view of the limited sensitivity of the assay (100 pg/ml of plasma) and therefore does not demonstrate whether physiologically relevant IL-l concentrations occur under these conditions. Thus, although important information has been collected by our RIA and other immunoassays (Bristow et al., 1991), more sensitive assays are urgently required to unravel the proposed humoral role of circulating IL-l/3 in rats. Measurement of IL-6. The cytokine IL-6 is produced in response to various stimuli by a variety of cell types like macrophages and endothelial cells (Hirano, 1992; Sanceau et al., 1990; Van Damme et al., 1989; Van Snick, 1990). The IL-6 receptor has been identified on various cell dypes like epithelial cells hepatocytes and monocytes (Bauer et al., 1989; Chomczynski & Sacchi, 1987; Snyers & Content, 1992; Taga et al., 1992). Recently, mRNA for the IL-6 receptor has been identified in rat brain (Schbbitz et al., 1992). Measurement of IL-6 is at present carried out with either bioassay or ELISA methods. Frequently used bioassays for human and murine IL-6 are based on the potency of IL-6 to facilitate the proliferation of B-cell hybridoma’s (Frei et al., 1991; Helle et al., 1988). The most popular assay involves B9 cells that had been selected for their sensitivity to IL-6 (Aarden et al., 1987). This assay is sensitive for rat IL-6, has a reasonable specificity, and is not affected by endotoxin, Con A, PHA, PMA, and the

216

I-. J. H. ‘TII

DEHS<‘I crl

human and murine cytokines available to us, with the exception of murine IL-4 (weak effects). Little is known about effects of drugs and hormones, but it should be noted that B9 cells are sensitive to glucocorticoids such as dexamethasone (unpublished observations). Therefore, care should be taken with the interpretation of IL-6 data in plasma samples of animals treated with glucocorticoids or subjected to procedures interfering with the activity of the HPA axis. The high sensitivity of the assay allows the measurement of low but detectable levels of IL-6 bioactivity in the plasma of resting intact rats (Schotanus et al., 1993a; Smith & Kluger, 1992). In addition, endotoxin caused a dose- and time-dependent increase in the plasma IL-6 levels in rats. Similarly, peripheral administration of IL-I also induced timeand dose-dependent increases in plasma IL-6 levels (Schotanus et al., 1993a). Recently, a very sensitive (1 pg/ml) ELISA has been described for human IL-6 (Helle et al. 1991), which is IO-20-fold more sensitive than a commercially available ELISA based on the “antibody sandwich” principle (Genzyme) and a radioimmunoassay (Helle et al., 1991; Teppo et al., 1991). Unfortunately, none of these immunoassays for human IL-6 is suitable for the quantification of IL-6 in plasma of rodents. Role of Macrophages Macrophages and endotoxin-induced peripheral cytokines. Having established that IL-1 and IL-6 can appear in the plasma following administration of endotoxin, the next question concerns the origin of these endocrine or humoral signals. As mentioned above, various cell types have the capacity to monitor endotoxin and respond with the production of these cytokines, including macrophages, lymphocytes, and granulocytes. Most evidence pointing to a crucial role of the macrophage is circumstantial (Oppenheim et al., 1986) and is largely based on the observation that macrophages produce in vitro considerably more cytokines than the other cell types. Recently, a technique has been introduced which allows the study of the exact role of macrophages in the host-defence response. The technique involves liposomes that are loaded with dichloromethylenediphosphonate (van Rooijen, 1989, 1992; van Rooijen et al., 1990). After IP or IV injection of the liposome suspension, liposomes will be ingested by cells of the macrophage lineage. which is followed by fusion of liposomes with lysosomes and the release of the drug. The resulting cell death is probably due to interference of the drug with intracellular calcium metabolism. This so called “macrophage suicide technique” has been shown to result in the elimination of macrophages from several immune organs including the spleen, lymph nodes, liver, and peritoneum. However, macrophages in the lung, testis, and the meninges and choroid plexus of the brain are not eliminated by this treatment. In peripheral organs, macrophage depletion is nearly complete within 24 h, but repopulation with monocytes and macrophage-like cells starts after 5 days. This gives a time window of 2-5 days after the liposome administration, which is suitable for experimentation. By using this approach, we demonstrated that the endotoxin-induced increase in the plasma levels of IL-I was dependent on the presence of peripheral macrophages and therefore most likely originated from these cells (DeRijk et al., 1991). In contrast, the high levels of IL-6 found in circulation following administration of endotoxin (Kluger, 1991; Lemay et al., 1990) are not prevented by depletion of peripheral macrophages, demonstrating that the macrophages that are reached by the liposomes do not contribute much to the plasma IL-6 levels found after high endotoxin doses to rats (DeRijk et al..

HPA RESPONSES TO ENDOTOXIN:ROUTESAND SIGNALS

217

1993). Thus, although macrophages have the capacity to produce both cytokines in response to endotoxin, they are the main source of circulating IL-lp but not of circulating IL-6 in endotoxin-treated rats. The exact cellular source of the humoral IL-6 response to endotoxin remains to be established. Although macrophages that have not been eliminated by the suicide technique may play a role, most evidence points towards endothelial cells or fibroblasts (DeRijk et al., 1994). Macrophages and endotoxin-induced HPA activation. We have used the macrophage suicide technique to study the involvement of peripheral macrophages in endotoxininduced activation of the HPA axis and found that this intervention blocked the fast responses of ACTH and corticosterone to a low dose of endotoxin (DeRijk et al., 1991). This is not due to interference of the liposomes to the integrity of the HPA axis as indicated by normal resting ACTH and corticosterone levels and proper responses to an ether challenge (DeRijk et al., 1991). In contrast to the effectiveness of macrophage depletion in preventing HPA activation by a low dose of endotoxin (2.5 &kg, IP), the effects of a high dose of endotoxin (2.5 mg/kg, IP), known to affect the body temperature, was not prevented by this manipulation (Berkenbosch et al., 1992b). These observations demonstrate that the HPA responses to low, but for the HPA axis maximally effective, doses of endotoxin are indeed mediated by signals produced by peripheral macrophages such as IL-l (Schotanus et al., 1993a). However, high, pyrogenic doses of endotoxin appear to affect other pathways as well (Berkenbosch et al., 1992b; DeRijk et al., 1991; Elenkov et al., 1992). Thus, low doses of endotoxin activate the HPA axis without much effects on other neuroendocrine systems and body temperature (DeRijk et al., 1991), whereas higher doses are required for inducing changes in body temperature, oxygen consumption, behaviour, plasma catecholamines and other neuroendocrine parameters (Dantzer et al., 1992; DeRijk et al., 1991). Macrophages and endotoxin-induced changes in thermoregulation. Fever is another example of a centrally controlled endotoxin-induced change, representing one of the most typical features of the acute phase response to infection, injury, and inflammation. It is generally accepted that fever is caused by endogenous pyrogens, which are produced by macrophages in response to stimuli such as bacterial endotoxin (Atkins, 1984; Dinarello & Cannon, 1988). Cytokines, in particular IL-l, are endogenous pyrogens that can initiate the febrile response. In the brain, the effects of IL-I appear to be mediated by prostaglandins that act on thermosensitive neurons and induce an upward resetting of the thermoregulatory setpoint (Sirko et al., 1989). As a result, an increase in heat production as measured by oxygen production, and a decrease in heat loss occur, resulting in an increase in core temperature (Rothwell, 1990; Wilkinson & Kasting, 1987). To study whether the endogenous pyrogens produced by macrophages are involved in this response, we used the liposome-mediated macrophage suicide technique and found that indeed, elimination of peripheral macrophages in rats prevented the increase in plasma IL-l (DeRijk et al., 1991), and attenuated the increase in body temperature and oxygen consumption to a high dose of endotoxin (DeRijk et al., 1993). Recently, Kluger and co-workers extended the concept of fever induction by stating that the thermoregulatory setpoint is controlled by endogenous pyrogens and antipyretics (Kluger, 1991). Antipyretics are endogenously produced factors that counteract the effects of pyrogens thereby preventing the body temperature from increasing too much. Evidence has been reported indicating that the cytokine TNFa! and the neuropeptides vasopressin and MSH act as antipyretics in the brain (Kluger, 1991).

In addition to thefrequentiy observed increase in body temperature designated as fever, several authors reportedadecrease in body temperature in response to endotoxin (Feldberg & Saxena, 1975; Lytle et al., 1973; Splawinski et al., 1977; Ueno et al.. 1982). The reason for such discordant results is not known. By using the same rat strain. similar housing and testing conditions and an identical endotoxin preparation, we reproducibly observed opposite thermoregulatory responses when these experiments were conducted in different laboratories (DeRijk et al., 1993; DeRijk & Berkenbosch, 1993). While carrying out these studies in Dr. Rothwell’s laboratories, DeRijk et al. found that endotoxin increases body temperature and oxygen consumption. However, in our own laboratories, the same manipulation caused a dose-dependent decrease in body temperature (maximal reduction 2.5-3°C) and oxygen consumption (30-40%) with a nadir after 80-90 min. We speculate that these opposite responses are due to different substrains of rats used in these studies or to possible differences in their immune status. The initial drop in core temperature, as observed in Amsterdam, was followed by a subsequent increase of approximately I “C after 5-6 h. With regard to the initial response, it is of interest to note that oxygen consumption fell to the same levels found in rats under thermoneutral conditions (ambient temperature 27-33°C) (Gordon, 1990). Since endotoxin did not decrease the body temperature of animals tested at a thermoneutral ambient temperature of 3O”C, the hypothermia response seems to be due to decreased heat production rather than to increased heat dissipation. The endotoxin-induced decrease in body temperature and oxygen consumption are inhibited by prior macrophage depletion, indicating that this hypothermic response to endotoxin is mediated by macrophage products. In addition, just as in the case of the endotoxin-induced hyperthermia, indomethacin also prevented the hypothermic response. Taken together, these data indicate that the same mechanisms that mediate the febrile response, mediate the endotoxin-induced decrease in body temperature. This hypothermic response appears to involve antipyretics of central and peripheral origin since peripheral administration of an antiserum to TNFa, reduction of the vasopressin content of the bed nucleus of the stria terminalis (castration), and central administration of a Type-l vasopressin receptor antagonist, all reduced the endotoxin-induced hypothermia (DeRijk & Berkenbosch, 1993). This supports the view that the direction of the endotoxin-induced change in thermoregulatory setpoint depends on a subtle balance between endogenous pyrogens and antipyretics from both central and peripheral origin. What exactly controls this balance remains unclear, but we postulate that subtle differences in the environment and history of the animals and genetic factors may account for differences in themoregulatory responses reported by different researchers. As stated above, our studies and those of others (Dinarello & Cannon, 1988) show biphasic temperature responses to endotoxin. In contrast to the initial hypothermic response, the subsequent hyperthermic response was not affected by depletion of peripheral macrophages. This supports the concept that the initial phase is reguiated by peripheral signals generated by macrophages while the second phase is not (DeRijk et al., 1993). In conclusion, the febrile response to endotoxin consists of an initial phase during which either hypothermia or hyperthermia develops, both of which are dependent on products originating from peripheral macrophages. After a longer time interval, a late hyperthermic response is observed which depends on endotoxin targets other than peripheral macrophages, that may reside in or close to the brain. BRAIN SIGNALLING

HYPOTHESIS

As mentioned before, endotoxin induces a wide range of centrally mediated symptoms including fever, neuroendocrine changes and behavioural changes i.e. hypersomnia,

219

HPA RESPONSES TO ENDOTOXIN: ROUTES AND SIGNALS

depression and anorexia (Hart, 1988). Most if not all of these effects can be mimicked by IL-l. For example, IL-l can induce febrile responses (Busbridge et al., 1989; Kluger, 1979; Opp & Krueger, 1991), suppress reproductive activity (Rivier & Vale, 1989) and thyroid function (Dubius et al., 1988), stimulate HPA-activity (Berkenbosch et al., 1987, 1990; Sapolsky et al., 1987), stimulate slow-wave sleep (Opp et al., 1991), decrease food and water consumption (Chance & Fischer, 1991), and induce sickness behaviours (Kent et al., 1992). These findings strongly support a pivotal role of IL-l in the orchestration of these symptoms. Many of the responses induced by peripheral administration of IL-l, can also be found after central administration of this cytokine (Rothwell, 1991) which has been taken as evidence that circulating IL-l may act in the brain. In support of this, IL-l receptors have been detected in certain brain areas (see below). However, cytokines including IL1 are not believed to pass the blood-brain barrier because they are large hydrophylic peptides. Although carrier-mediated transport has been described for IL-la (Banks & Kastin, 1991), this is unlikely to play an important role because of its limited capacity. Therefore, other mechanisms have to be considered. Several alternative hypotheses have been postulated, including: l l l

activation of IL-1 production in brain cells; action of IL-1 at the level of circumventricular organs; and relay of IL-l or endotoxin by into brain-accessable signals by endothelial

cells.

IL-l Receptors in the Brain For IL-l two distinct receptor types, denoted as Type I and Type II IL-l receptors, have been identified (Chizzonite et al., 1989; Dower et al., 1992). The Type I receptor is present on T cells, fibroblasts, and endothelial cells, whereas the Type II receptor can primarily be found on B cells and macrophages (Bomsztyk et al., 1989; Dinarello, 1991, 1992). In mice, high affinity binding sites for IL-l have been found in the hippocampus, choroid plexus, and pituitary gland (Ban et al., 1991; Haour et al., 1990; Takao et al., 1990). In contrast, in the rat brain, the situation is far from clear. By using hIL-le as a ligand, Farrar (Farrar et al., 1985) found IL-I binding-sites throughout the brain, whereas others have reported specific localization of IL-l receptors in the hypothalamus by using hIL-lfi as a ligand (Katsuura et al., 1988). Despite attempts by various groups, these results could not be confirmed by others. By receptor-autoradiographic techniques, Marquette et al. (1994) demonstrated that the binding of radioiodinated rat IL-lp in the mouse brain showed a regional distribution identical to that of human IL-la. Nonetheless, also with rat IL-lj3 as a ligand, no receptors could be demonstrated in the rat brain, with the exception of the choroid plexus and the pituitary gland. Thus, to date receptors for IL-l/3 have not been detected in rat brain. This may be due to low receptor densities or to properties of the rat IL-l receptor, that are not compatible with the methods used so far. Recently, Type I (Hart et al., 1993) and Type II (Bristulf, unpublished observations) IL-1 receptors of the rat have been cloned. The expected search for IL-l receptor mRNA with species-compatible probes may lead to new insights in the presence and functions of IL-l receptors in the CNS of the rat. IL-l Production in the Brain Both IL-lp protein and IL-lp mRNA have been detected in neurons and in glial cells. Immunocytochemical studies demonstrated the presence of IL-la and IL-l@ in neural cell bodies, axons, and varicose fibers in the forebrain and other brain structures (Breder

F. J. H. TILDERS (II al.

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et al., 1988; Lechan et al., 1990; Molenaar et al., 1993). Synthesis of IL-l in neuronal cells is further supported by in situ hybridization studies demonstrating IL-lp mRNA in neurons of the hypothalamus, hippocampus, frontal cortex, cerebellum, and brainstem (Bandtlow et al., 1990; Berkenbosch et al., 1991). However, it should be noted that the numbers of copies of IL-1p mRNA found in neurons are very low as compared to those found in activated macrophages. We are not aware of evidence demonstrating that neuronal IL-l/? and IL-l/3 mRNA increase in response to peripheral administration of endotoxin or IL-l, and therefore the exact role of neuronal IL-lb in the responses to this challenge remains to be determined. Nonetheless, peripheral administration of endotoxin to mice has been reported to increase IL-la and/or IL-l/3 mRNAs in various brain regions (Ban et al., 1992). In contrast to the apparent indifference of the neuronal IL-l/3 production, glial cells can be activated by endotoxin or IL-1 to produce IL-l/3 in vivo as well as in vitro. Both astrocytes and microglia may contain IL-lb and express its mRNA. In primary cell cultures of neonatal mouse brains, an IL-l-like factor could be detected after incubation with lipopolysaccharide (Fontana et al., 1982). Although such early studies suggested that IL-1 was primarily produced by astrocytes, these results are contradicted by various recent reports demonstrating that only activated microglial cells can produce and secrete IL-l (Frei et al., 1988; Giulian et al., 1986; Hetier et al., 1988; Lee et al., 1993; Yao et al., 1992). In support of this, immunocytochemical studies on cell cultures of mixed glial cells showed that in endotoxin challenged cultures, IL-l@ immunoreactivity appears only in microglia cells but not in astrocytes (Vincent et al., unpublished results). The role of activated microglia in brain-mediated responses to endotoxin is further supported by our immunocytochemical findings (see Figs. 1A and 1B) demonstrating that microglial cells in the brains of untreated rats are not staining for IL-lp, but become positive for IL-lp after peripheral administration of endotoxin in a time- and dose-dependent manner (Van Dam et al., 1992). The importance of this observation is demonstrated by findings showing that intracerebral administration of IL-1 receptor antagonist prevents the hyperexpression of CRH mRNA that normally follows peripheral administration of endotoxin (Kakucska et al., 1993). Other potential sources of IL-l/3 in the brain after peripheral administration of endotoxin are the macrophages in the meninges and choroid plexus and the perivascular cells (Van Dam et al., 1992). Also these cells do not appear to contain IL-lb under resting conditions, but produce IL-1 after peripheral administration of endotoxin. Circumventricular

Organs

The latency of fever induction and the duration of fever is shorter and more pronounced after IV than after ICV administration of endogenous pyrogens (Stitt & Bernheim, 1985). This led to the view that the site of action of cytokines is at brain sites close to the circulation. Accordingly, brain structures, called circumventricular organs, that have a poorly developed blood-brain barrier and fenestrated endothelium, were proposed as possible sites where circulating cytokines have access to the brain. One of these circumventricular organs, the organum vasculosum of the lamina terminalis (OVLT), may play a role in the induction of fever because of its connections with the preoptic and anterior hypothalamic areas where the thermoregulation centers are situated. Thus, circulating IL-l@ may interact with brain structures in the OVLT, where the signal is converted into other signals, for example, PGEz that can affect thermosensitive neurons (reviewed by Coceani et al., 1986).

FIG. 1: Microphotographs of immunocytochemical demonstration of IL-l/3 (A, B) and PGEz (C, D) in the rat cerebral cortex. Adult male Wistar rats were injected with endotoxin (2.5 mg/kg IV) and subjected to perfusion fixation at various time intervals thereafter [(A): 8 h; (B): 24 h; (C): control; (D): 24 h]. Bars = 100 pm (A, B) and 500 pm (C, D). (A) shows IL-l/3 immunoreactivity at the optimal time-point after endotoxin administration, a reaction that has disappeared at 24 h (B). Salinetreated rats do not show PGE2 immunoreactivity in the cortex (C), which can be induced in the microvasculature at 24 h after endotoxin treatment (D). For details see (Van Dam et al., 1992, 1993).

F. J. H. TILDERSet d

0 human

0.3 INTERLEUKIN-1

3 beta

30 (PmOi/mi

1

FIG. 2: Effects of interleukin-l/3

on the release of CRH (open bars) and vasopressin (cross-hatched bars) from the isolated median eminence (ME) of rats. Median eminences of adult male Wistar rats were prepared and incubated in Krebs ringer bicarbonate containing 2 mg/ml glucose and 1 mgiml bovine serum albumin (10 ME/ml medium), and incubated under carbogen in a shaking waterbath. At intervals of 15 min, the medium was poured off and replaced by fresh medium. After 90 min, the medium was replaced by medium containing 0.3,3, or 30 pmol/ml of recombinant human IL-l/3 (4 x 15 min). Exposure to IL-l resulted in a transient increase of CRH release with peak levels during the first 15 min of exposure. Vasopressin secretion was not affected at any of the time intervals studied. The CRH concentrations found in the peak fractions are expressed as percentage of those found in the medium prior to addition of IL-lp. Each dose was tested in quadruplicate.

A circumventricular organ that is of particular relevance for the control of HPA activity is the median eminence. In this brain structure, the nerve terminals of the CRH neurons line the perivascular space around the fenestrated endothelium. Thus, the CRH nerve terminals are de facto exposed to circulating cytokines. Observations of De Souza and collegues indicate that at least in some species, IL-l binding-sites are present in this area (Cunningham & De Souza, 1993). Local injections of IL-l in, or close to the median eminence, induced ACTH secretion whereas injection in other sites were less effective (Matta et al., 1993). Several reports demonstrate that IL-l stimulates CRH secretion from hypothalami of rats in vitro (Hagan et al., 1993; Navarra et al., 1990). However, the preparations used in these studies include both the CRH cell bodies and their nerve terminals. Therefore, it is not clear whether IL-I affects CRH release by actions at the level of the perikarya (nucleus paraventricularis) or at the level of the nerve terminals (median eminence). Recent data indicate that although central and peripheral administration of IL-l both stimulate ACTH secretion, the mechanisms involved may be different since central but not peripheral administration of IL-1 stimulated c:fbs expression in the paraventricular nucleus of rats (Rivest et al., 1992). This leaves open the possibility of a CRH release-inducing action of circulating IL-l at the median eminence. We have approached this question by studying the effects of hIL- 10 on the secretion of CRH from median eminence preparations that contain only the terminals of the CRH neurons. As illustrated in Fig. 2, we found that recombinant hIL- l/3 in concentrations of 0.3 and 3 pmol/ ml induced a fast and transient increase of CRH secretion by 65% and 95%, respectively. whereas 30 pmol/ml was without effect. Furthermore, the release of vasopressin was not affected, which is in accord with the selective CRH release stimulating effect of ILI in vivo (Berkenbosch et al., 1987, 1989; Tilders et al.. 1993). These observations support the hypothesis that interaction of circulating IL- l/3 with CRH nerve endings may contribute to the activation of the HPA axis by peripheral administration of endotoxin.

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Role of Brain Endothelial Cells Endothelial cells of mice are known to exhibit IL-l receptors (Boraschi et al., 1991). Type 1 IL-l receptors as well as their mRNA have been detected in endothelial cells derived from mouse brains (Cunningham et al., 1992). Preliminary data also indicate that brain endothelial cells derived from rats, express functional IL-l receptors and message (Cunningham et al., 1992; Van Dam et al., in preparation). Thus, brain endothelial cells are a very likely target for circulating IL- l/3. Upon interaction with IL- 1, brain endothelial cells may produce and secrete mediators such as IL-l, IL-6, and PGE, into the brain parenchyma. Because IL-l-containing neurons are located at several hypothalamic sites (Breder et al., 1988; Lechan et al., 1990; Molenaar et al., 1993) and at least some IL-l producing neurons appear responsive to IL-l, it has been suggested that IL-l serves as its own intermediate messenger (Breder et al., 1988; Cunningham & De Souza, 1993). We propose that circulating IL-l starts a cascade of events involving IL-l production in endothelial cells that then activates microglial cells to produce IL-I, which in turn may affect ILI- sensitive neurons. Such a mechanism may serve as an intracerebral amplifier of the IL-l signal. The demonstration that IL-l induces production and secretion of IL-l from endothelial cells in vitro (Warner et al., 1987) and the presence of irIL-1 in endothelial cells of porcine brain (Molenaar et al., 1993) support this hypothesis. Interleukin-6, which can also be produced by endothelial cells in response to endotoxin, IL- 1, and TNFa (Jirik et al., 1989), may be responsible for some of the centrally mediated responses including fever (Lemay et al., 1990; Rothwell et al., 1991) and HPA-activation (Navarra et al., 1990). Central administration of antibodies to IL-6 inhibited the effects of IL-l on thermogenesis (Rothwell et al., 1991), indicating a physiological role of IL-6 in signal transfer. This is supported by the presence of IL-6 in the cerebrospinal fluid of rats after peripheral administration of endotoxin (Berkenbosch et al., 1992~) and after brain injury (Woodroofe et al., 1991). In addition to IL-6 mRNA, IL-6 receptor mRNA and IL-6 binding-sites have been detected in bovine and rat brains (Cornfield & Sills, 1991; Schijbitz et al., 1992). In addition to IL-l and IL-6, PGE, is well known to mediate endotoxin-induced fever (Coceani et al., 1986). Prostaglandins appear to mediate several of the behavioural (Crestani et al., 1991) and HPA responses (Katsuura et al., 1988b; Navarra et al., 1990; Rivier & Vale, 1991) to endotoxin and IL-l, since these responses can be blocked by prostaglandin synthesis inhibitors, that is, indomethacin and piroxicam. Recently, peripheral administration of IL-l or endotoxin to rats was found to stimulate the synthesis of prostaglandins in the rat brain (Komaki et al., 1992). By using an immunocytochemical approach involving an antiserum to PGE,, we reported that PGE, appears in the endothelial cells of the microvasculature of the brain after peripheral administration of endotoxin (Van Dam et al., 1993) (see Figs. 1C and 1D). Since receptors for PGE, have been found in the central nervous system of rats (Malet et al., 1982; Matsumura et al., 1992), this localization favors a role of PGE, as an intermediate messenger at least in the late responses to endotoxin. Taken together, these observations strongly support the view that the endothelial cells in the brain play a critical role in translating circulating IL-l concentrations into signals, that is, IL-l, IL-6, and PGE,, that can reach targets beyond the blood-brain barrier. CONCLUSIONS Activation of the HPA axis is probably the most sensitive brain-mediated component of the acute phase response to endotoxin. Increased plasma ACTH and corticosterone concentrations can be found at endotoxin doses that have little effect on other neuroendo-

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FIG. 3: Schematic representation of the mechanisms involved in endotoxin-induced activation of hypothalamic CRH neurons. The segment delineated by the broken line refers to possible mechanisms in the median eminence.

systems, body temperature and behaviour. Corticosterone levels are considered to play an essential role in the control and the tuning of the immune and inflammatory responses. Endotoxins do not activate the HPA axis directly but their effects are mediated by a wide spread system of endotoxin-sensitive cells that include macrophages, immune cells, endothelial cells, and possibly even microglial cells in the brain. These cells respond to endotoxin with the production of different panels of intermediate signals (cytokines, eicosanoids, nitric oxide etc.). It is conceivable that the dose and route of endotoxin administration determines which components of the endotoxin-sensor system are activated and therefore, which intermediate signals are produced. At low doses of endotoxin, in particular when administered IP, macrophages in the peritoneum and liver are the primary targets that are activated to produce large amounts of IL-l in addition to other products. As well as local paracrine or autocrine effects of these products, an unknown fraction of the IL-1 produced by these macrophages enters the peripheral circulation to act as a conventional humoral factor on distant targets such as the brain (Fig. 3). Circulating IL-l is known to activate the HPA axis by stimulating the secretory activity of the CRH neurons in the hypothalamus. Stimulation of CRH secretion involves different routes and mechanisms that may act in different time domains. IL-1 can pass the fenestrated endothelia in the median eminence to act directly on the crine

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CRH-containing nerve terminals lining the perivascular spaces in this circumventricular organ. In addition to this fast and transient response, circulating IL-1 may bind to IL-l receptors on endothelial cells in the brain and initiate a cascade of paracrine and autocrine events by the production and secretion of IL-l, IL-6, prostaglandins, and so on, into the brain (see Fig. 3). In this way, IL-I produced by the endothelial cells may reach and affect IL-l sensitive neurons or microglial cells. Also microglial cells show IL-l-induced IL-l production (Lee et al., 1993) and thus may amplify and prolong the intracerebral IL-l signal. IL-l can also induce the production and secretion of IL-6 and prostaglandin in endothelial cells and both substances are known to activate directly or indirectly hypothalamic CRH neurons. At high doses of endotoxin, in particular when administered IV, endothelial cells are the first elements to encounter the substance. Endotoxin may directly activate brain endothelial cells to produce and secrete intermediate signals into the brain (IL-l, IL-6, PGE,) and thereby start a cascade of events similar to that mentioned above. Thus, although endotoxin given IP and IV both activate the HPA axis, it is conceivable that high doses of endotoxin given IV, may do so in a macrophage independent manner by affecting the central but possibly also the peripheral limbs of the HPA axis. Acknowledgments: We wish to acknowledge our friend, colleague, and teacher, Dr. Frank Berkenbosch for his inspiring role in our studies on neuroendocrine-immune interactions. The authors thank Dr. E.A. Linton, Oxford, U.K. for radioimmunometric measurements of rCRH, Dr. R.M. Buijs for the measurements of vasopressin, Mr. R. Binnekade for his indispensable technical support, and Mr. H. Nordsiek for the reproduction of the figures. A.M. van Dam is supported by a grant from NWA, J.H.A. Persoons by a grant from USF, K. Schotanus by grant 900543-075 from the Dutch Foundation for Medical and Health Research (NWO).

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