Behavioral Effects of Cytokines: An Insight into Mechanisms of Sickness Behavior

[8]

Behavioral Effects of Cytokines: An Insight into Mechanisms of Sickness Behavior Robert Dantzer, Rose-Marie Bluthe, Stephen Kent, and Glyn Goodall

Introduction In his book "Naissance de la Clinique" Foucault (1) describes the important movement that took place in medicine at the end of the eighteenth century and the beginning of the nineteenth century and that, by the introduction of anatomopathology, was to give rise to modern medicine, thanks to the possibility of establishing causal relationships between anatomoclinical features of disease and observed symptoms. However, diseases that could be characterized only by the intense fever that accompanied them were excluded from this movement because of the impossibility of pinpointing their essence in anatomical lesions. The anatomoclinical paradigm became successful because it was built on the concept of specificity, but it did so at the expense of nonspecificity. Nonspecificity was reintroduced to medicine when, fascinated by the commonality of lesions among diseases with different causal factors, Selye (2) began research on the mechanisms of what he called the syndrome of "just being sick." However, his quest led him instead to all the excitement surrounding the myth of stress and, in the process, he forgot about what it means to be sick. Neal Miller, a psychobiologist with many insights, independently rediscovered the concept of sickness behavior (3, 4). While trying to determine the mechanisms of thirst, he was struck by the observation that rats injected with endotoxin, the tool chosen in these experiments to manipulate thirst, were not just no longer interested in water, but were also sick and able to reorganize their behavior in the face of this change in their internal state. More specifically, Miller observed that although endotoxin-injected rats placed in a Skinner box stopped pressing a lever for food or electrical self-stimulation, they increased their response rate when placed in a situation in which they had to press a lever to cause a motor-driven activity wheel, in which they were forced to walk, to be temporarily stopped. Because these effects took some time to appear after injection of endotoxin, Miller went one step further and hypothesized that endotoxin was not acting by itself 130

Methods in Neurosciences, Volume 17 Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

[8] CYTOKINES AND BEHAVIOR

131

but that it triggered the release of a factor X in the blood, which then affected the brain and made the animal feel sick. These fascinating observations remained buried in the literature for two decades, until physiologists interested in endogenous pyrogens were able to join forces with immunologists to elucidate the cellular mechanisms of the acute-phase reaction (5). The acute-phase response is the answer of the organism to disturbances of its homeostasis during infection, tissue injury, neoplastic growth, or immunological disorders. It consists of both a local and a systemic reaction. The local reaction involves phagocytic cells, lymphocytes, fibroblasts, and endothelial cells. Interactions between these different cell types are mediated by soluble factors or cytokines, such as interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor a (TNF-α) and interférons (IFNs). The systemic reaction is mediated by the action of these cytokines on distant target cells and is characterized by leukocytosis, an increased sedimentation rate of red blood cells, activation of complement and clotting cascades, synthesis and release by hepatocytes of acute-phase proteins (e.g., fibrinogen, C-reactive protein, and caeruloplasmin), negative nitrogen balance, decreased serum levels of iron and zinc, activation of the pituitary-adrenal axis, and fever. As pointed out by Kluger (6), fever is not just a symptom but an adaptive response that has evolved to enable living organisms to fight microbial pathogens efficiently. Interfering with the fever process in poikilothermic animals by keeping them in a cold environment or by treating them with antipyretics is detrimental to their survival. In the same manner, shearing the fur of ferrets infected with influenza enhances their sensitivity to the disease. Fever stimulates the immune system and produces a body temperature that is unfavorable for the growth of many microbial pathogens. In addition, reduction in plasma iron and zinc concentrations during fever decreases the availability of these vital elements for growth of microorganisms. The amount of heat needed to increase body temperature and maintain it at elevated values during the febrile process is quite substantial: about a 13% increase in metabolism per 1°C of fever in human beings (6). Because of the high metabolic cost of fever, there is little room for other activities than rest and postures that minimize thermal losses. It is therefore not surprising that the cytokines that are pyrogenic have been found to be somnogenic (7) and to induce lethargia, behavioral depression, and anorexia (8, 9). Because these behavioral changes are similar to the nonspecific symptoms of sickness, it can be postulated that cytokines are the mediators actually responsible for the syndrome of "just being sick."

132

II CENTRAL NERVOUS SYSTEM ACTIONS

Induction of Sickness Behavior by Cytokines Clinical Studies The first evidence in favor of a role for cytokines in sickness behavior came from clinical trials with purified or recombinant cytokines. Although these molecules were thought to have a high potential for enhancing immune defenses in cancer, viral illness, and immunodeficient states, their use turned out to be impractical because of severe general and neurological side effects. For instance, patients treated with IFN-α commonly react with flulike symptoms, including fever, anorexia, fatigue, headache, myalgia, and arthralgia (10). These symptoms culminate in lethargy and withdrawal from the surroundings (11). Repeated injections of high doses of IFN-α into patients with chronic viral hepatitis led to the occurrence of severe neuropsychiatrie manifestations in 10 of 59 cases (17%). These manifestations were extremely variable and fell into three categories: irritability and short temper; extreme emotional lability, depression, and fearfulness; and delirium with agitation, paranoia, and suicidal potential (12). They spontaneously regressed on cessation of treatment, although some patients remained emotionally vulnerable for a few weeks. Similar alterations in mental states have been noted in about half the patients receiving therapy with interleukin 2 and lymphokine-activated killer cells. Of 44 patients with metastatic cancer and exposed to immunotherapy, 15 became agitated and aggressive and 22 experienced severe cognitive changes, in the form of disorientation in time and place (13).

Experimental Studies in Animals Experimental studies in animals have confirmed that systemic or intracerebral injections of recombinant cytokines have profound behavioral effects in addition to their pyrogenic and metabolic activity. The first studies were done by toxicologists and used general activity and food intake as dependent variables. It is only in the past 4 to 5 years that more systematic studies have addressed the mechanisms and significance of the effects of cytokines on behavior. Sickness behavior can be studied indirectly by the conditioned taste aversion (CTA) paradigm. This paradigm is based on the association that animals make between the taste of a new food or drinking solution they have ingested and a subsequent episode of illness (14). In a typical experiment, rats are trained to drink their daily allocation of water during a 30-min presentation of the water bottle. On the day of conditioning, they are given a solution of

133

[8] CYTOKINES AND BEHAVIOR ► Saline

Training 1

Days

FIG. 1 Aversive effects of IL-1 and LPS in rats. Lipopolysaccharide (20 mg/kg) or recombinant rat IL-1/3 (0.1-10 jug/rat) were injected ip after a 30-min session of saccharin presentation on days 1 and 3. Left: Overall mean intake of water during the two last days of training and, for each experimental group, the mean intake of saccharin during the course of conditioning (days 1 and 3) and the mean fluid (saccharin plus water) intake during preference tests (days 5-7). Right: For each experimental group the mean percent preference for saccharin during preference tests on days 5-7. Each experimental group contains six or seven rats. Vertical bars represent SEM [From Tazi et al (15).]

saccharin instead of water and are subsequently injected with a toxic dose of lithium chloride. After recovery from illness, they are presented with the saccharin solution associated with the episode of sickness, either alone (onebottle test) or concurrently with water (two-bottle test). Conditioned animals refrain from drinking the saccharin solution, and the amount of saccharin drunk is an indirect measure of the intensity of the sickness previously experienced. Recombinant rat IL-1/3 injected intraperitoneally (ip) induced a decreased preference for saccharin in a two-bottle test in rats (15). This effect was obtained at doses that decreased body weight (1-10 μg) and was comparable in strength and duration to the one resulting from the administration of lipopolysaccharide (20 mg of LPS/kg from Escherichia coli 026:B6) (Fig. 1). Administration of 50 ng of recombinant rat IL-1/3 into the lateral ventricle

134

II CENTRAL NERVOUS SYSTEM ACTIONS

also induced a marked aversion to saccharin, together with a reduction in fluid intake and a decrease in body weight. Lower doses of IL-1/3 (0.5-5 ng) had no effect (16). Because learned aversions to the available diet develop in rats bearing experimental tumors, the aversive stimulus properties of TNF have been examined. Tumor necrosis factor administration (50 Mg/kg twice a day for 3 days) led to the development of a strong aversion to a novel diet (17). This effect was attenuated by lesions of the area postrema, the site of the chemoreceptor trigger zone for nausea and emesis. In contrast to IL-1 and TNF, IFN-a (800-1600 U/g) did not induce a conditioned taste aversion in mice in a two-bottle test after one or three pairings with chocolate milk (18). Furthermore, the combination of IFN-a with LiCl did not potentiate the LiCl-induced CTA. An important problem with the CTA paradigm is the way it relates to sickness (19). It is commonly assumed that when a treatment induces CTA, it does so because of its sickness-inducing properties. In support of this interpretation, peripheral and central injections of toxic compounds (e.g., lead and mercury) have been found to have potent aversive properties. However, the same is also true for drugs of abuse, such as amphetamine and morphine, which produce CTA at doses that are self-administered (20). There is ample evidence to demonstrate that there is no strict equivalence between illness and the aversive effects of a treatment, as defined by its ability to induce CTA. This means that sickness needs to be inferred from other effects of the treatment under investigation, such as reduction in food and water intake and decrease in body weight. On the basis of these criteria, aversive doses of IL-1 are clearly toxic (15, 16). In terms of subjective experience, changes in internal state induced by aversive drugs are perceived not only along a quantitative continuum but also along a qualitative dimension. Although IL-1 and TNF share many biological properties, they do not necessarily induce the same experience of sickness. However, direct tests of this possibility have not yet been carried out with drug discrimination techniques. In the case of IL-1, there is evidence that the CTA induced by intracerebroventricular (icv) IL-1 is different from the one induced by ip IL-1 (21, 22). Although IL-1 activates the pituitary-adrenal axis in both cases, reexposure to the taste associated with sickness resulted in a significant elevation of plasma corticosterone levels in animals injected with ip but not icv IL-1. Another way of assessing sickness is to look for alterations of ongoing behavior. The choice of the behavioral end points to be used as indicators of cytokine-induced sickness can be guided by what is already known about the nature of fever symptoms. The reduction in body care activities that leads to the scruffy-looking hair coat characteristic of sick animals (8) is not

[8] CYTOKINES AND BEHAVIOR

135

easy to quantify and has therefore not yet been used. Changes in general activity, feeding behavior, and social interactions are easier to assess, using automated recording or direct observation. Proinflammatory cytokines decrease general activity. For instance, mouse IFN-α significantly depressed locomotor activity, head pokes into a food tray, and food intake of mice for up to 24 hr after ip injection of a single dose of 1600 U/g (23). Injections of low doses of rhIL-la (0.1-10 ng) or rhIL-1/3 (1-10 ng) significantly depressed the duration of contacts with novel objects mounted below floor level in a hole-board apparatus divided into nine interconnecting compartments, without altering measures of locomotor activity, such as movements between the compartments or rears (24, 25). The effects of LPS in this last test were different from those of IL-1, because the reduction in exploratory activity was accompanied by a decrease in locomotor activity and in the number of stimulus contacts (26). In addition, mice injected with LPS were observed to remain immobile for many seconds. Depression of social exploration of juvenile conspecifics by adult animals has also been used to assess sickness. It is induced in a dose- and timedependent manner after injection of rhIL-1/3 or LPS to rats (27, 28) and mice (29, 30). Interleukin la has the same effects in mice as IL-lß (29) (Fig. 2). Disruption of food-motivated behavior has been proposed by pharmacologists as a method for assessing sickness induced by injection of naloxone to morphine-dependent rats (31, 32). Peripheral injections of rhIL-1/3 induce long-lasting decreases in the number of opérant responses of rats trained to press a lever for food on a fixed ratio 10 food reinforcement schedule (i.e., 1 food pellet for 10 lever presses) (33). Although there has been little interest in the possibility of interference of sickness with learning ability, preliminary evidence indicates that LPS dramatically affects the learning of an autoshaped opérant response for food whereas it has little or no effect on performance once the response has been learned (Fig. 3).

Experimental Studies in Humans On the basis of earlier work showing that infection with upper respiratory viruses decreases the efficiency with which psychomotor tasks are performed (34-36), Smith et al. (37) injected IFN-α (0.1-1.5 Mu) to 18 volunteers of both sexes. Volunteers injected with the largest dose of IFN were significantly slower at responding in a reaction time task when they were uncertain when a target stimulus would appear. Simultaneously, they displayed hyperthermia and experienced feelings of illness. However, they were not impaired on a pursuit tracking task or syntactic reasoning task. Although the lack of

136

II CENTRAL NERVOUS SYSTEM ACTIONS

A

#

~# on"9 o-o 20 ng x-x 100 ng Δ--Δ 250 ng +1 . D~D 500 ng

Behavior

_150

%

Body weight I 3 * J

ΓΊ *

\v

U «\

1 100 ce σ> *-> CO

Φ

1 50

»

1

\ x' \«

*/

1 \·

t.

0 inj

Δ*

/

f' /

2

/

O)

/

** *

x°

/'''

X' *

1

*

-1

• I. inj

Behavior

.«<

tr-^I^r^

* 2

24

4 6 Time (hr)

B

V · oa

4 6 Time (hr)

·-· 0 ng o--o 20 ng Δ--Δ100 ng +1 o--D500ng

24

Body weight

i

B ioo|

co g) to

£

£°

P

b-"" ! 2

150

c

ca

O)

h-'

50I

I

i.

.t.

mj

2

4 6 Time (hr)

24

2

4 6 Time (hr)

24

FIG. 2 Effects of rhIL-la (A) and rhIL-1/3 (B) on social exploration and body weight in mice. Individually housed male DBA/2 mice were presented with a juvenile conspecific at time 0 (baseline) and the duration of olfactory investigation of this social stimulus was assessed during a 4-min test. They were then injected ip with saline or various doses of the cytokine under investigation and presented again with another juvenile at different time intervals. Body weight was measured at the end of each test session. For clarity of representation, the SEM is presented only for the third point of each curve (n = 8 for IL-Ια, n = 7 for IL-1)8) (**/? < .01, ***/? < .001 compared with baseline value). [From Bluthé et al. (29).]

137

[8] CYTOKINES AND BEHAVIOR 600 υ α> £, 500 jî 400 to

S 300 o. S 200

g σ

-1

10

° o

A-# \ X

O ·

#

■ ° v

2nd INJECTION

P

?

injection

O NaCI · LPS

|

° ' o o- o o w

1

O—O- r> τ

° O 1

1

2

3

4

5

6

7

DAYS

8

DAY 13

9 10 11 12

El· LPS NaCI

FIG. 3 Differential effects of LPS on learning and performance of an autoshaped opérant response for food. Adult male rats were deprived to 80% of their free-feeding body weight and given daily sessions of lever press autoshaping in a Skinner box. Sessions consisted of 36 trials in which a retractable lever was introduced into the box every 45 sec, and remained inserted for 15 sec or until the lever was pressed, whichever occurred first. A 45-mg food pellet was delivered 2 sec following retraction of the lever. The graph shows the cumulative latencies for the thirty-six 15-sec trials as a function of days of training. On day 2, all subjects were injected with either 1 ml of physiological saline/kg (n = 14) or LPS (250 ^g/ml/kg) (n = 7). The difference between groups was significant both over all training days and on each day from day 2 to day 12. On day 13, the 14 rats previously injected with saline received a second injection either of saline {n = 7) or LPS (n = 7). Their latency to respond did not differ according to the treatment, or compared to the latencies of day 12. a control group injected with a placebo precluded the examination of possible effects of the lower doses of IFN, it is noteworthy that the observed effects were similar to the alterations of performance observed in influenza patients. In view of these results, more studies are clearly warranted to examine the behavioral effects of subclinical doses of IFN and other cytokines.

Pharmacodynamic Aspects of the Sickness-Inducing Properties of Cytokines Pharmacokinetics Intravenous injection of rats with 125I-labeled rhIL-1/3 resulted in a rapid distribution of IL-1, with a half-life of 2.9 min (38). Interleukin 1 was eliminated in accordance with first-order kinetics (half-life of elimination, 41.1 min) by degradation primarily in the kidney and liver and excretion via the kidneys. Circulating intact IL-1 was present for up to 5 hr following ip injection and

138

II CENTRAL NERVOUS SYSTEM ACTIONS

was distributed in several organs, including the pancreas. Distribution of IL-1 in this organ was specific to the ip route because it was not observed after subcutaneous (sc) or intravenous (iv) injections. In mice, the reported half-life of the distribution phase of rhIL-1/3 is 5-10 min (39). Tumor necrosis factor a has a similar half-life of distribution, but a much longer half-life of elimination (1.7-11 hr) (40).

Route of Injection In general, peripheral and central injections of cytokines induce the same range of behavioral effects. In the case of social exploration, nanogram amounts of IL-1 jö are required when this cytokine is injected icv instead of the microgram amounts required when it is administered ip (27, 41). Because the time course of the effects of IL-1 on social exploration is similar in both cases, it is tempting to conclude that IL-1 acts centrally to alter social exploration. In contrast, 10 times more IL-1 needs to be injected icv to induce significant decreases in food-motivated behavior (41). In addition, the latency of effect is delayed after icv treatment; the largest effects are observed at 2-4 hr instead of 1 hr (Fig. 4). This suggests that IL-1 does not act centrally to affect food-motivated behavior (cf. Peripheral versus Central Sites of Action). 175-,

-Γ

0

1

2 4 Time (hr)

8

24

FIG. 4 Comparison of the effects of ip and icv IL-1/3 on food-motivated behavior in rats. Animals were injected with rhIL-1/3 ip ( · ) (4 μΒ/rat; n = 5) or icv (A) (40 ng/rat; n = 5) and physiological saline [1 ml ip (O); 1 μΐ icv (Δ)]. Separate groups of rats were used for ip and icv injections. All injections were given immediately after the first test session (time 0). Data are expressed as a percentage of preinjection response rate. (*p < .05, **p < .01, compared to respective control values).

139

[8] CYTOKINES AND BEHAVIOR BEHAVIOR

BODY WEIGHT +2

250L o Ä 200| c o

I ·

S 1501 <-·

-5 +1L o)

r

oL

CO

Φ

•Ξ 100 E

iL

50

oL

-2L 2 Time (hr)

4

6

8

24

Time (hr)

FIG. 5 Comparison of the effects of sc and ip injection of rhIL-1/3 on social exploration in mice. Individually housed male Crl:CD-l (ICR) BR mice, 7 weeks of age, were presented with a juvenile conspecific at time 0 (baseline) and the duration of social exploration was assessed during a 5-min test. Mice were then injected with physiological saline ( · ) (100 μ\, se) (n = 5), rhIL-lß (O) (500 ng/100 μΐ, sc) (n = 5), or rhIL-1/3 (Δ) (500 ng/100 μΐ, ip) (n = 5) and tested 2, 4, and 6 hr later with another juvenile. Body weight was measured at the end of each test session and 8 and 24 hr later. *, Significantly different from saline; + , significantly different from IL-1 injected ip. Because of the importance of the liver in the degradation of IL-1, it is likely that different routes of administration at the periphery lead to quantitatively different effects. Following the observation that se IL-1/3 was more effective than ip IL-Iß on food intake, rectal temperature, and blood glucose in rats (38), the effects of sc and ip rhIL-1/3 on social exploration of mice were compared. Mice injected with 500 ng of IL-1 were more sensitive when the cytokine was injected sc than when administered ip (Fig. 5).

Chronic versus Acute

Injections

Chronic administration of rhIL-1/3 (2 μ-g/day) via a subcutaneously implanted osmotic minipump resulted in the development of tolerance to the anorexic and weight-depressing effects of IL-1 within a few days (43). Tolerance also

140

II CENTRAL NERVOUS SYSTEM ACTIONS

developed to the depressing effect of IL-1 on social exploration (44). There is evidence that tolerance does not develop at the same rate for different behavioral endpoints. Continuous administration of murine IL-la (3 ^tg/day) to rats kept in their home cage reduced eating activity and locomotor activity and increased drinking (45). Locomotor activity remained depressed over the 5 days of treatment. Drinking activity was significantly elevated during the light period and this effect remained constant over the observation period. In contrast, eating behavior returned to control levels on days 4 and 5 during the latter, but not the earlier, half of the dark period. Because of the rapidity of its appearance (1-3 days), tolerance to the effects of IL-1 does not appear to be mediated by the mounting of an immune response to the injected cytokine. Possible mechanisms are represented by induction of IL-1-degrading enzymes, downregulation of IL-1 receptors, or induction of IL-1-soluble receptors or synthesis of the antagonist of interleukin 1 receptors. In contrast to what is seen during continuous administration, repeated injections of IL-1 do not appear to lead to important changes in the magnitude of the observed behavioral response (Fig. 6). The same does not apply to LPS because repeated administration of endotoxin leads to the rapid development of tolerance. In the case of interferons, decreased locomotor activity was observed only after repeated, but not single, administration of murine recombinant IFN-γ to mice (30 /ig/mouse) (46). Although it has not been systematically studied, this sensitization process is similar to what is seen during repeated injections of cytokines in clinical trials.

Mechanisms of Behavioral Effects of Cytokines Role of Endogenous Cytokines The demonstration that LPS induces behavioral effects similar to those of IL-1 in endotoxin-sensitive but not in endotoxin-resistant mice, together with the observation that IL-1 is active in both lines of mice (47), suggests that the secretion of proinflammatory cytokines is responsible for the development and maintenance of sickness behavior in response to LPS. However, the exact cellular targets that are responsible for these effects have not been identified. In the case of the pituitary-adrenal axis, depletion of macrophages by peripheral injection of liposomes encapsulated with dichloromethylene diphosphonate completely abrogated the hormonal response to a subpyrogenic dose of LPS (48). The same strategy has not yet been used for assessing the role of macrophages in the behavioral effects of LPS.

141

[8] CYTOKINES AND BEHAVIOR

$ 150 z O

T\

fee oô 100 LU

>

l·\M L

\ Tx i\

\

\

\

\

\

\

50 h < ce D û

L

o1-

\

\

i 0

/

x

Time (hr) DAY1

\

\ \ \ \ \ \ \ \ \ \ \ \ \ \

J 1

J 1

î

? 1

i

4

0

1 /

x

J

4

Time (hr) DAY 7

FIG. 6 Development of tolerance to the depressing effects of injections of LPS but not IL-1 on social exploration in rats. Rats were injected with physiological saline ( · ) (n = 8), LPS (Δ) (20 mg/kg; n = 4), or rhIL-1/3 (O) (5 jug/rat; n = 8) four times at 48-hr intervals. On the first day of injection (day 1) and on the last day (day 7), their interest toward a juvenile conspecific was measured. Each treatment was administered immediately after the first test session (time 0) and the rats were retested 4 hr later. Note that LPS and IL-1 had similar depressing effects on social exploration on the first day of treatment but that LPS was no longer effective on day 7, in contrast to IL-1.

Another strategy for identifying the mediators that are responsible for the effects of LPS is to use cytokine antagonists acting at the receptor level. Administration of the recombinant antagonist of IL-1 receptors (IL-Ira) at doses that block the behavioral effects of IL-1/3 attenuated the depressing effect of LPS on social exploration and on body weight (28) (Fig. 7). In contrast, the same treatment had no effect on the LPS-induced disruption of food-motivated behavior, suggesting that IL-1 is not the main mediator of these effects (49). Cytokines have the ability to induce the synthesis and release of other cytokines and themselves in a cascade-dependent manner (50). This cascade serves to amplify the local effects of cytokines into a global effect, due to

142

II CENTRAL NERVOUS SYSTEM ACTIONS 1501 o

CD C

g

■*—·

(Ω Q)

>

TT -•Δ

iooL o\

,'T / D ' /o /'/-I-

501

C

---O

oL

^OD

0

6

24

Time (hr)

FIG. 7 Effects of IL-lra on the effects of LPS on social exploration in rats. Rats were injected with either saline or IL-lra, followed by saline or LPS (from E. coli serotype 0127 : B8). Four experimental groups were used: saline (ip) plus saline (ip), n = 5 ( · ) ; saline (ip) plus LPS (250 μg/kg; ip), n = 6 (O); IL-lra (3 mg/rat; ip) plus LPS (250 ^g/kg; ip), n = 5 (Δ); IL-lra (60 μg/rat; icv) plus LPS (250 /xg/kg; ip), n = 7 (D). Injections were administered immediately after the first test session and animals were tested again 2, 4, 6, and 24 hr later. The SEM is represented only for the last time point. *p < .05, compared with baseline values. [From Bluthé et al. (28).]

multiplicity of targets and the overlapping biological effects of cytokines. To test which cytokine is predominant in a given effect, the same pharmacological strategy based on administration of cytokine antagonists can be used. In the case of TNF-a, for instance, pretreatment with IL-lra antagonized the depressive effects of this cytokine on social exploration but only partially attenuated the weight loss, suggesting that TNF-induced sickness behavior is mediated mainly by endogenously released IL-1 whereas metabolic changes are dependent on the release of other cytokines (51).

Peripheral versus Central Sites of Action By their action on several peripheral cellular targets, IL-1 and other proinflammatory cytokines can significantly interfere with spontaneous or acquired behavior. For instance, cytokines induce inflammation and hyperalgesia at their site of injection (52). Pain may also be caused in joints, due to the destructive effects of IL-1 and TNF on cartilage and bone (53, 54). Peripheral administration of IL-1 has potent effects on gastrointestinal functions in the form of inhibition of gastric emptying and reduction of gastric

[8] CYTOKINES AND BEHAVIOR

143

acid secretion (55). Cytokines also act on endothelial cells to cause profound and long-lasting vasodilation and hypotension (56). If the behavioral effects of IL-1 are mediated at the periphery, it should be possible to abrogate them by peripheral but not central administration of the recombinant receptor antagonist molecule. This was found to be the case for the effects of IL-1 on food-motivated behavior (41). Disruption of opérant lever pressing induced by peripheral injection of rhIL-1 ß to rats was fully blocked by peripherally injected IL-Ira but only partially by central injection of this antagonist, at doses that fully abrogated the behavioral effects of centrally injected IL-1 (Fig. 8). In the same manner, the depressing effect of LPS on social exploration was antagonized by peripherally but not by centrally injected IL-lra (28). The possibility that IL-1 and other proinflammatory cytokines act directly in the brain is supported by the observation that tolerance to centrally injected

Food-motivated behavior

Social exploration

^1001

J100

CO

c o

8" 501 ω

50 o Q)

SAL

IL-1 ra

IL-1

IL-1

+

+

SAL

+

IL-1

IL-1 ra i.e.v. IL-1

i.p.

FIG. 8 Differential effect of blockade of central IL-1 receptors on food-motivated behavior and on social exploration. Interleukin 1 receptor antagonist or saline was injected icv into rats trained to press a lever for food on a fixed ratio 10 schedule (24 ^g of IL-lra/rat) or into rats presented with a juvenile conspecific (4 ^g of IL-lra/ rat). This injection was followed by ip rhIL-1/3 (4 ^g/rat for the food-motivated behavior, 4 ^g/rat for social exploration) or saline. Injections were given immediately after thefirsttest session and animals were tested again 1 hr (food-motivated behavior) or 2 hr (social exploration) later. The figure represents percentage of variation with regard to baseline values (*p < .05, ***/? < .001). Note that pretreatment with IL-lra blocked the effects of IL-1 on social exploration but only partially attenuated the effects of this cytokine on food-motivated behavior. [From Kent et al. (41).]

144

II CENTRAL NERVOUS SYSTEM ACTIONS

cytokines has profound behavioral effects at doses that are much smaller than those that need to be injected at the periphery (see Route of Injection, above). The ability of centrally injected IL-lra to antagonize these effects attests to their specificity (41). Because centrally injected IL-lra is also able to fully block the effects of peripherally injected IL-1 on social behavior (Fig. 8) (41), it can be proposed that either the peripherally injected cytokine is able to enter the brain or endogenous IL-1 is produced and released in the brain in response to peripheral IL-1. It is not yet possible to decide between these alternatives (57). Although penetration of IL-1 in the brain has been proposed by Banks et al. (58), the consensus is that IL-1 does not enter the brain but acts at the level of circumventricular organs, where the blood-brain barrier is nonexistent. Interleukin 1 would bind to cells located on the vascular side of the organum vasculosum laminae terminalis, thereby inducing synthesis and release of prostaglandins of the PGE2 series, which would then freely diffuse to nearby neural structures and act directly on them or promote the local synthesis and release of IL-1 and other cytokines (59). Another possibility is that peripheral injection of IL-1 activates substance P-containing primary sensory afférents, which would transmit this information to the central IL-1 compartment (57). There is also evidence for a differential regulation of peripheral and central IL-1 receptors (44). In rats made tolerant to the behavioral effects of IL-1 by continuous infusion of this cytokine via an osmotic minipump implanted sc, acute challenge with IL-1 reversed this tolerance when the injection was given ip but not when it was administered icv. Although it is clear that the behavioral effects of IL-1 and other proinflammatory cytokines can be mediated peripherally or centrally, depending on the behavioral end point, there is clearly a need for a better delineation of the responsible cellular targets. In the case of central targets, a micropharmacological approach consisting of local injections of agonists and/or antagonists would be the best way to address this issue. For example, microinjections of 5 ng of rhIL-1/3 directly into the ventromedial nucleus of the hypothalamus decreased food response to the same degree as injections of 40 ng of this cytokine into the lateral ventricle (42).

Role of Corticotropin-Releasing Factor Because of the potent activating effects of IL-1 on hypothalamic corticotropin-releasing factor (CRF) and the role played by this neuropeptide in IL-1induced thermogenesis (60), the possible involvement of CRF in behavioral

[8] CYTOKINES AND BEHAVIOR

145

effects of IL-1 has been assessed by icv administration of CRF antiserum or a-helical CRF (9-41) (ahCRF), an antagonist of CRF receptors. Immunoneutralization of endogenous CRF in the brain attenuated the anorexic effects of IL-1 (61). In the same manner, icv administration of CRF antiserum blocked the reduction of immobility induced by icv rhIL-1/3 in rats forced to swim in a confined space (62). Central administration of ahCRF was also able to prevent the IL-1 induced reduction of exploratory behavior of mice placed in a multicompartment chamber (25). Although these findings suggest that brain CRF modulates the behavioral effects of IL-1, there is evidence contradicting this conclusion. In particular, the decrease in food-motivated behavior induced in rats by ip injection of rhIL-ljS was not altered by icv injection of either aCRF or CRF (33). When IL-1 was injected centrally, ahCRF did not alter the peak effect but facilitated the return toward baseline (63). The exact factors that are responsible for these differences are still unknown. However, it is clear that the involvement of CRF in the behavioral effects of IL-1 is not a general phenomenon.

Role of Prostaglandins Interleukin 1 and other proinflammatory cytokines are potent inducers of prostaglandin production. Administration of cyclooxygenase inhibitors such as indomethacin at doses that abolish synthesis of prostaglandins attenuates the pyrogenic and anorexic effects of IL-1 (64). Pretreatment with indomethacin or piroxicam blocked the depressing effects of peripherally injected rhIL-1/3 on food-motivated behavior in rats and on social exploration in mice (30). In rats continuously infused with murine IL-la, piroxicam completely inhibited the stimulation of drinking behavior, but had no effect on the reduction in eating activity and locomotor activity induced by the cytokine (65). In the same manner, pretreatment with indomethacin had no effect on the depression of general activity and food intake induced in mice by peripheral injection of IFN-a (66). In the face of these contradictory results, there is clearly a need for further studies on the role of prostaglandins in the behavioral effects of cytokines.

Role of Nitric Oxide The sustained vasodilatation and hypotension induced by IL-1 and other proinflammatory cytokines are mediated by the local synthesis and release

146

II CENTRAL NERVOUS SYSTEM ACTIONS

of nitric oxide (NO) via induction of an NO synthase in both endothelial and vascular smooth muscle cells (67). Besides its potent vasodilatating effects, NO behaves as an effector molecule in immunological reactions and as a neurotransmitter in the central and peripheral nervous system. Administration of agents that block synthesis of NO from L-arginine has been shown to attenuate the dramatic fall in blood pressure that occurs during septic shock or in response to exogenously administered cytokines (68, 69). To test whether NO production is also involved in the behavioral effects of IL-1, mice were pretreated with various doses of iV-nitro-L-arginine methyl ester (NAME), a selective inhibitor of the brain and endothelial NO synthase (70). Administration of high doses of the antagonist (30 mg/kg) potentiated the depressing effects of rhIL-1/3 on social exploration whereas lower doses (5 mg/kg) had no effect. This potentiation was attenuated by Larginine but not by D-arginine (Fig. 9). Administration of the nitro-arginine

SAL SAL SAL (N=6)

SAL L-Arg SAL (N=4)

SAL IL-1 SAL (N=6)

NAME5 NAME 30 NAME 30 NAME 30 IL-1 IL-1 IL-1 IL" 1 SAL SAL L-Arg D-Arg (N=5) (N=5) (N=5) (N=4)

FIG. 9 Potentiation of IL-1-induced changes in social exploration by pre treatment with L-NAME, an inhibitor of NO synthase, and the effect of L-arginine or D-arginine. Each column represents the mean variation measured 4 hr after injection of rhIL-1/3. The number of mice in each group is given in parentheses. *, p < .05 with comparison to saline; +, p < .05 with comparison to IL-1; O, p < .05 with comparison to the (IL-1 plus L-NAME) group. Previous experiments had established that the doses of L-NAME (5 or 30 mg/kg) had no effect on social exploration. [From Bluthé et al. (70).]

[8] CYTOKINES AND BEHAVIOR

147

derivative had no effect on the weight loss induced by IL-l. Although these results suggest that NO production has a protective role in the behavioral effects of IL-l, they need to be complemented by further studies elucidating the effect of IL-l on brain NO synthase and the contribution of the different isoforms of this enzyme to the observed effects.

Opposition of Behavioral Effects of Cytokines by Cryogens Brain vasopressin and α-melanocyte-stimulating hormone have been characterized as physiological inhibitors of fever and are accordingly named endogenous antipyretics or cryogens (59). These peptides are released in the brain in response to endogenous pyrogens and function to suppress fever. They therefore appear to be part of a negative feedback loop regulating the thermogenic effects of cytokines. The possibility that vasopressin counteracts the behavioral effects of IL-l was tested by studying the interaction between vasopressin and IL-l in rats during social exploration (27). Central injection of vasopressin attenuated the behavioral effects of icv rhIL-1/3. Conversely, central injection of an antagonist of the vasopressor receptors of vasopressin, dPTyr(Me)AVP, potentiated the behavioral effects of IL-l. These latter results are important because they suggest that endogenous vasopressin plays a physiological modulatory role in the behavioral effects of IL-l. The vasopressinergic neurons that are responsible for these effects are certainly the ones located in the bed nucleus of the stria terminalis (BNST) and project to the septum. These neurons are highly sensitive to circulating androgens. Castration leads to a dramatic reduction in the content of mRNA for AVP in the BNST neuronal cell bodies (71) and to a reduction in immunoreactive AVP of the terminal areas in the septum (72). These changes are reversed by treatment with testosterone. Castration potentiated the depressing effects of the continuous infusion of rhIL-1/3 on social exploration in rats as well as the effect of an acute central injection of IL-l (27). Furthermore, icv administration of vasopressin was more effective in attenuating the behavioral effects of IL-l in castrated than in intact male rats and, conversely, icv administration of the antagonist of vasopressor receptors was unable to counteract the behavioral effects of IL-l in castrated male rats in which the vasopressinergic innervation of the septum is reduced. Although the stimulatory action of IL-l and other endogenous pyrogens on vasopressin is well established, much more remains to be learned about the way in which vasopressin interacts with the effects of cytokines to understand the mechanisms that are responsible for these effects.

148

II CENTRAL NERVOUS SYSTEM ACTIONS

Conclusion Sufficient evidence is now available to accept the concept that cytokines are interpreted by the brain as internal signals of sickness. Sickness can actually be considered as a motivation, that is, a central state that organizes perception and action. A sick individual does not have the same priorities as a well person, and this reorganization of priorities appears to be mediated by the effects of cytokines on a number of peripheral and central targets. The elucidation of the mechanisms that are involved in these effects should give new insight into the way sickness is represented in the brain (57).

Acknowledgments Supported by INSERM, INRA, and a grant from DRET (RD:90-166).

References 1. M. Foucault, "Naissance de la Clinique." Presses Universitaires de France, Paris, 1963. 2. H. Selye, "The Stress of Life." McGraw-Hill, New York, 1956. 3. J. E. Holmes and N. E. Miller, J. Exp. Med. 118, 649 (1963). 4. N. E. Miller, Bull. Br. Psychol Soc. 17, 1 (1964). 5. P. C. Heinrich, J. V. Castell, and T. Andus, Biochem. J. 265, 621 (1990). 6. M. J. Kluger, "Fever: Its Biology, Evolution and Function." Princeton Univ. Press, Princeton, NJ, 1979. 7. J. M. Krueger, Trends Pharmacol Sei. 11, 122 (1990). 8. B. L. Hart, Neurosci. Biobehav. Res. 12, 123 (1988). 9. R. Dantzer and K. W. Kelley, Life Sei. 44, 1995 (1989). 10. K. Fent and G. Zbinden, Trends Pharmacol. Sei. 8, 100 (1987). 11. F. Adams, J. R. Quesada, and J. U. Gutterman, J. Am. Med. Assoc. 252, 938 (1984). 12. P. F. Renault, J. H, Hoofnagle, Y. Park, K. D. Mullen, M. Peters, D. B. Jones, V. Rustgi, and E. A. Jones, Arch. Intern. Med. 147, 1577 (1987). 13. K. D. Denicoff, D. R. Rubinow, M. Z. Papa, C. Simpson, C. A. Seipp, M. T. Lotze, A. E. Chang, D. Rosenstein, and S. A. Rosenberg, Ann. Intern. Med. 107, 293-300 (1987). 14. J. Garcia, W. G. Hankins, and K. W. Rusianak, Science 185, 824 (1974). 15. A. Tazi, R. Dantzer, F. Crestani, and M. Le Moal, Brain Res. 473, 369 (1988). 16. A. Tazi, F. Crestani, and R. Dantzer, Neurosci. Res. Commun. 7, 159 (1990). 17. I. L. Bernstein, E. M. Taylor, and K. L. Bentson, Am. J. Physiol. 260, R906 (1991). 18. M. A. Segall and L. S. Crnic, Brain, Behav., Immun. 4, 223 (1990).

[8] CYTOKINES AND BEHAVIOR

149

19. R. Dantzer, R. M. Bluthé, and M. Le Moal, Neurotoxicology 9, 471 (1988). 20. P. Goudie, in "Experimental Psychopharmacology" (A. J. Greenshaw and C. T. Dourish, eds.), p. 341. Humana Press, Clifton, NJ, 1987. 21. D. G. Dyck, L. Janz, T. A. G. Osachuk, J. Falk, J. Labinsky, and A. H. Greenberg, Brain, Behav., Immun. 4, 93 (1990). 22. L. L. Janz, R. Brown, L. Zuo, J. Falk, A. H. Greenberg, and D. G. Dyck, Physiol. Behav. 49, 691 (1991). 23. M. A. Segall and L. S. Crnic, Behav. Neurosci. 104, 612 (1990). 24. F. Spadaro and A. J. Dunn, Brain, Behav., Immun. 4, 308 (1990). 25. A. J. Dunn, M. Antoon, and Y. Chapman, Brain Res. Bull. 26, 539 (1991). 26. A. J. Dunn, Y. Chapman, and M. Antoon, Neurosci. Res. Commun. 10,63 (1992). 27. R. Dantzer, R. M. Bluthé, and K. W. Kelley, Brain Res. 557, 115 (1991). 28. R. M. Bluthé, R. Dantzer, and K. W. Kelley, Brain Res. 573, 318 (1992). 29. R. M. Bluthé, P. Parnet, R. Dantzer, and K. W. Kelley, Neurosci. Res. Commun. 9, 151 (1991). 30. F. Crestani, F. Seguy, and R. Dantzer, Brain Res. 542, 330 (1991). 31. M. Babbini, M. Gaiardi, and M. Bartoletti, Psychon. Sei. 29, 142 (1972). 32. V. F. Geliert and S. B. Sparber, J. Pharmacol. Exp. Ther. 201, 44 (1977). 33. R. M. Bluthé, R. Dantzer, and K. W. Kelley, Neurosci. Res. Commun. 5, 149 (1989). 34. A. P. Smith, D. A. J. Tyrrell, K. B. Coyle, and J. S. Willam, Br. J. Psychol. 78, 183 (1987). 35. A. P. Smith, D. A. J. Tyrrell, W. Al-Nakib, K. B. Coyle, C. B. Donovan, P. G. Higgins, and J. S. Williams, Neuropsychobiology 18, 144 (1987). 36. A. P. Smith, D. A. J. Tyrrell, W. Al-Nakib, K. B. Coyle, C. B. Donovan, P. G. Higgins, and J. S. Williams, Psychol. Med. 18, 65 (1988). 37. A. Smith, D. Tyrrell, K. Coyle, and P. Higgins, Psychopharmacology 96, 414 (1988). 38. J. Reimers, L. D. Wogensen, B. Welinder, K. R. Hejnaes, S. S. Poulsen, P. Nillson, and J. Nerup, Scand. J. Immunol. 34, 597 (1991). 39. R. C. Newton, J. Uhl, M. Covington, and O. Back, LymphokineRes. 7,207 (1988). 40. B. L. Ferraiolo, J. A. Moore, D. Crase, P. Gribling, H. Wilking, and R. A. Baughman, Drug Metab. Dispos. 16, 270 (1988). 41. S. Kent, R. M. Bluthé, R. Dantzer, A. J. Hardwick, K. W. Kelley, N. J. Rothwell, and J. L. Vannice, Proc. Natl. Acad. Sei. U.S.A. 89, 9117 (1992). 42. S. Kent, R. Dantzer, and K. W. Kelley, in preparation. 43. N. Mrosovsky, L. A. Molony, C. A. Conn, and M. J. Kluger, Am. J. Physiol. 257, R528 (1989). 44. R. M. Bluthé, R. Dantzer, and K. W. Kelley, C.R. Acad. Sei., Sér. 3, 312, 689 (1991). 45. I. G. Otterness, P. A. Seymour, H. W. Golden, J. A. Reynolds, and G. O. Daumy, Physiol. Behav. 43, 797 (1988). 46. S. B. Weinberger, G. Schulteis, A. G. Fernando, C. Bakhit, and J. L. Martinez, Jr., Life Sei. 42, 1085(1988). 47. F. Crestani, Ph.D. Thesis, University of Bordeaux II (1990).

150

II CENTRAL NERVOUS SYSTEM ACTIONS 48. R. Derijk, N. van Rooijen, F. J. H. Tilders, H. O. Besedovsky, A. del Rey, and F. Berkenbosch, Endocrinology (Baltimore) 129, 330 (1991). 49. S. Kent, R. Dantzer, and K. W. Kelley, Neurosci. Lett. 145, 83 (1992). 50. C. A. Dinarello, Blood 77, 1627 (1991). 51. R. M. Bluthé, R. Dantzer, and K. W. Kelley, Eur. J. Pharmacol. 209, 281 (1991). 52. S. H. Ferreira, B. B. Lorenzetti, A. F. Bristow, and S. Poole, Nature (London) 334, 698 (1988). 53. E. J. Pettipher, G. A. Higgs, and B. Henderson, Proc. Natl. Acad. Sei. U.S.A. 83, 8749 (1986). 54. D. Hamerman, N. Engl. J. Med. 320, 1322 (1989). 55. A. Robert, A. S. Olaffson, C. Lancaster, and W. Zhang, Life Sei. 48, 123 (1990). 56. D. Beasley, Am. J. Physiol. 259, R38 (1990). 57. S. Kent, R. M. Bluthé, K. W. Kelley, and R. Dantzer, Trends Pharmacol. Sei. 13, 24 (1992). 58. W. A. Banks, A. J. Kastin, and D. A. Durham, Brain Res. Bull. 23, 433 (1989). 59. M. J. Kluger, Physiol. Rev. 71, 93 (1991). 60. N. J. Rothwell, Trends Pharmacol. Sei. 12, 430 (1991). 61. A. Uehara, C. Sekiiya, Y. Takasugi, M. Namiki, and A. Arimura, Am. J. Physiol. 257, R613 (1989). 62. S. Del Cerro and J. Borrell, Brain Res. 528, 162 (1990). 63. R. M. Bluthé, F. Crestani, K. W. Kelley, and R. Dantzer, Ann. N.Y. Acad. Sei. 650, 268 (1992). 64. A. Uehara, Y. Ishikawa, K. Okumura, C. Sekiya, Y. Takasugi, and M. Namiki, Eur. J. Pharmacol. 170, 257 (1989). 65. I. G. Otterness, H. W. Golden P. A. Seymour, J. D. Eskra, and G. O. Daumy, Cytokine 3, 333 (1991). 66. L. S. Crnic and M. A. Segall, Physiol. Behav. 51, 349 (1992). 67. S. Moncada, R. M. J. Palmer, and E. A. Higgs, Pharmacol. Rev. 43, 109 (1991). 68. R. G. Kilbourn, S. S. Gross, A. Jubran et al., Proc. Natl. Acad. Sei. U.S.A. 87, 3629 (1990). 69. C. Thiemermann and J. Vane, Eur. J. Pharmacol. 182, 591 (1990). 70. R. M. Bluthé, S. Sparber, and R. Dantzer, NeuroReport 3, 207 (1992). 71. M. A. Miller, L. Vician, D. K. Clifton, and D. M. Dorsa, Peptides (N.Y.) 10, 615 (1989). 72. G. J. De Vries, R. M. Buijs, and A. A. Sluiter, Brain Res. 298, 141 (1984).