Synergistic Effecn of Macrophage Denved Cytokines: Consumption of a Highly Pdatable
Substance and Plasma Corticosterone Alterations
A thesis presented to the Faculty of Graduate Studies and Research. Carleton Cniversity. in
partial fulfillment of the requirements of the degreec of Master of Science in Psycholopy
Carleton Cniversi ty
Ottawa, Ontario
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A bstract
It is becoming apparent that the proinflarnmatory cytokines. interleukin- 1 p (IL- 1 ).
interleukin-6 (IL-6) and tumor necrosis factor-alpha (TTL'F-a). mediate some of the
behavioral and hormonal responses to pathophysiological conditions. In addition to the
illness behavior associated with immune and cytokine challenge (e.g., reduced eating.
exploration, locomotion. the appearance of curled body posture. and soporific e ffects j.
such treamients rnay also provoke anhedonia. Moreover, there is reason to believe that
these treatments may act synergistically in promoting at least some of the actions associated
with endotoxin challenges. The present investigation assessed the potenrial synergistic
effects of macrophage-derive. cytokines on feeding and on plasma conicosterone
concena-arions. Male CD- I mice were rrained to drink chocolate milk. and dose-dependent
dtentions in consumption were measured following the wpmte administration of
inaapentoneal < i.p.) doses of lipopolys3ccharide t LPS). IL- 1 . IL-6 and TSF-a.
Corresponding altentions in plasma comcosterone lsvels were established in naive CD- 1
mice. It wris obsenled that LPS. IL- 1 and TKF-a each reduced chocolate milk
consumption and increrised corticosterone levsls. Based on preliminq findings. sub-
optimal doses of the cytokines were chosen in ordsr to asses' rhs synsrgistic actions of
combinations of IL-1 + IL-6, IL-6 + TSF-a. and IL4 + TXF-a on consurnpticrn and
homone alterations. Cytokine combinarions were adrninistercd in two successive i.p.
injections and consumption of chocolate milk was measured one hour later. Plasma
conicostsronc levels were again measured in a naive goup of mice. The combination of IL-
1 and TSF-cx synergistically reduced consumption of chocolritc milk and increassd
comcostcrone levels. In contrasr, neither the combinations of IL- i + L-6 nor IL-6 + T G -
a induced synergisric effects on behaviour or endocrine funciioning. The data m
discussed in terms of la) the anorexic vs the anhedonic actions of cytokine treatrnent. and
(b) the occurrence and function of synergistic effects af various cytokines.
Acknowledgements
1 would like to express my sincere thanks to Dr. Robert Zacharko and Dr. H p i e
Anisman, without whorn none of this would have k e n possible. I am also exmmely
gateful to Dr. Jerzey Kulczycki who spent so much tirnr analyzing my blood sarnples.
To everyone in the lab. I will always be grateful for your support. your good
humour and your neverending encouragement in rimes of Suess. In paflcular I am indsbted
to Yael Sela who was alwriys there to offer her opinion and her invaluable assistance when
i t was most needed.
To my famil y and friends. 1 appreciate your patience. your understanding and Jour
encouriigement (not to mention your cornputsr! i .
Thia rssearch m.35 supponed by a bIRC p n t .
Table of Contents
Title .................................... .,.. .................................................... L
Recommendation ............................................................................. ii
Abstract ......................................................................................... i i i
Acknowledgements ......................................................................... iv
Table of Contents ........................................................................... v
List of Figures ................................................................................ vi
1 . Introduction .......................... .... ..................................... 1
I I . hfultisystem Interactions .......................................................... 8
III . Cytokine Involvement in Endocnne/CSS Transmitter Arerations ............... 1 1
IV . Purpose ...................... .. ........................................................ Z I
.............................................................................. Experiments 1 - 1 21
Experiments 5-7 .............................................................................. 36
Gsneral Discussion .......................................................................... 45
Refersnces ............................. ,,., .................................................. 60
..................................................................................... Appendiu A 73
Appsndis B ......................... .. ....................................................... 80
..................................................................................... Appendis C X I
...................................................................................... Appendix D 8 2
..................................................................................... Appendix E 83
Appendix F .............................. .. ................................................. 81
Appendix G ..................................................................................... 85
Appendix H ..................................................................................... 86
..................................................................................... Appendis 1 87
List of Figures
Figure 1. Mean ( ~ S . E . M . ) consumption (mis) of a highly pslatable food arnong rnice
that received peripheral lipolysaccharide (LPS) or saline. Various doses of LPS (0.5 pg.
0.75 pg or 1 pg) were adrninistered intraperi toneally to male CD- 1 mice and consurnption
of chocolate milk was measured over a 1 hr. period commencing 90 minutes following
injection. Baseline values were calculated by averaging consumption over the three da?
period irnmediately pnor to testing. Recovery data were collected 24 hr. following testing.
Figure 2. Mean ( ~ s . E . M . ) consumption (mls) of a highly palatable food among mice
receiving peripheral interleukin-lp I L - ) . tumor necrosis factor-alphri (TSF-a) .
interlsukin-6 ( IL-6) or saline. Various doses of IL- 1 (0.025 pz. 0.05 pg. 0.1 pg. 0.2 pa.
or 0.8 ug). T S ï - a i 1 pz. 2 pg. or 4 pz) C or IL-6 (0.1 p g. 0.21 C g. 0.8 pg or 1.6 pg uere
administered inrrapsritonsally to male CD4 mice and cnnsuniption of chocolate milk was
mcasurrd ovcr (i 1 hr. period commencing cither 13 minutes (for IL-] and IL-6i or ?O
minutes (for TSF-a) following injection. Basdine values usrs clilculatrd bu averaging
consurnption o w ~ the threr day p e n d immediately prior to testing. Recovcv data w r e
cnllected 2 1 hr. follou.ing testing.
Figure 3 lrlsan t CS .E.M.) plasma conicostcronc concentrations i pgdl i among micc
that rcceived psripheral lipopolysaccharide (LPS) or sali ne. Various doses of LPS (0.5 pg
or 1 pg) were administered intraperitoneally to male CD-] mice and corticosrcrons
concentrations were measured 2 hr later.
Figure 1. Mem ( ~ s . E . M . ) plasma conicosterone concentr~tions (pg/dll in mice
receiving pcnpherril inisrleukin-l 1 1 ) tumor necrosis factor-alpha t TSF-u i .
interleukin-6 (IL-6). or saline. Various doses of IL- 1 (0.025 pg, 0.05 pg or O. 1 pg). IL-6
(0.1 p g or 0.8 pg), or Th7-a ( 1 pg, 2 pg or 4 pg)were adminisrered inmpentoneally to
male CD- 1 mice and plasma conicosterone concenaations were measured 1 hr later.
Figure 5. Effect of inmperitoneai adminisaation of 0.03 pg IL-1 + 0.1 pg IL-6
(upper panel), 0.1 mg IL-6 + 1 pg TNF-a (middle panel) or 0.025 pg IL- l + 1 pg TW-
a (lower panel) on mean (?s.E.M.) consumption (rnls) of a highly palatable food. Mais
CD-1 mice were injected with the cytokine combinations or 0.025 pg IL-l + Veh. 0- 1 pg
IL-6 + Veh. or 1 pg TSF-a + Veh and consumption of chocolarr milk was rneasured over
a 1 hr. pcriod cornrnencing 1 hr following injection. Baselinc values were calculated b',
avenging consumption over the three day p e n d immediatsly prior ro testing. Recoven-
data were cdlrcted 24 hr. following testing.
Figure 6. Effect of intraperitonenl administration of 0 . 0 3 pg IL- 1 + 0.1 pg IL-6. 0.1
pg IL-6 + 1 pg TXF-a or 0.025 pg IL-1 + 1 pg TSF-" on man ('S.E.M. 1 plasma
comcosieronc concentrations (pgdl). Male CD-I niics wcrc injcctcd wirh the c!.tohne
corntrinations or 0.025 pg IL-1 + Veh. 0.1 pg IL-h - k h . or 1 pg TSF-a înd plasma
corricostcrone levcls H erc measured 1 hr following injection.
Figure 7 . Mean (?s.E.>I.) consumption (in mlsi of (i highly plilarable food h>-
capsaicin- and vehicle-treated rnice following intrapsritoneal 3dministranon of
lipopolysaccharide ( LPS ) or saline. Seo-ncttai CD- I micc uwe uemd uit h subcutaneous
capsaicin or vshicle at Z days of agc. At 60 days of agc hoth groups of micc ucre injscrsd
with 0.5 pg LPS or saline. and consumption of chocol3te milk was rneasured over a I hr.
period cornmenci ng 90 minutes following injection. Baseline values were calculated hy
avenging consumption over the three day p e n d imrnediately pnor to testing. Recovery
data w r e collscted 24 hr. foIIowing testing.
Figure 8. Mean ( ~ s . E . M . ) plasma corcicosterone concenuations (pg/dl) in capsaicin-
or vehicle-aeated mice receiving in~aperitoneal lipopolysaccharide (LPS) or saline. Neo-
n a d male CD- I mice were treated with subcutaneous capsaicin or vehicle at 2 days of age.
At 60 days of age both groups of mice were injected with 0.5 pg LPS or saline and plasma
corticosterone concentrations were measured 2 hr later.
v iii
Introduction
It has become clear that the immune and cenaal nervous systems do not act
independently of one another. Cenaal nervous system (CNS) alterations come to affect
immune functioning, and conversely immune activation rnay promote increased endocrine
and tend neurotransmitter activity (Besedovsky, del Ray, Sorkin, Da Prada, Bum &
Honegger, 1983). Indeed, the view has been offered that the immune system stimulates
CXS acavity. and like environmental stimuli, may be associated with psychopathology. In
pmicular, it is thought diat immune activaaon acts like a saessor to provoke activation of
the hypothalmic-piniitq-adrenal (HPA) endocrine system, and may also elicit variations
of cenaal monoamines (Zalcman, Shanks & Anisman, 1993). As a result, behavioral
alterations may occur ahich are rerniniscent of those elicited by saessors (Dantzer &
Relley, 1989).
The mechanisms by which the immune system influences CNS processes remain to
be deduced. However, several candidates have k e n proposed in this respect. Whde these
systems are not mutually esclusive, it has been suggested that activation of the vatal
system. for example, may play a significant role in CNS-immune communication (Watkins
et al., 1991: Laye et al., 1993). In panicular, it is thought that immune activation by way of
vagal innervation may ulrimately come to excite hypothalamic factors (Gaykema, Dijkstra &
Tiiders, 1995: Fleshner, Goehler, Hermann, Relton, Maier 8: Watkins, 1995). Otha
investigators. however. have emphasized the role of cytokines released from activated
immune cells, panicularly macrophages (Besedovsky, del Rey, Sorhn & Dinarello, 1986).
In particular, antigen-stimulated macrophages release proinfiammatory cytokines
(interleukin- 1. interleukin-6 and nimor necrosis factor-a) which are instrumental in
signding other imrnunological cells, including T helper cells. In addirion, however. these
cytokines rnay gain access to the CNS, albeit in Iimiteù quantities, and among other things.
promote the release of corticotropin releasing hormone (CRH) from the paraventricular
nucleus (Pm-) of the hypothalamus, and either directly or indirectly elicit increased activity
of forebnin monoamines (Anisman, Zalcman & Zacharko, 1993; Lavicky & Dum. 1995).
There have been several reports consistent with the suggestion that cytokines play a
role in affecting CNS processes ( s e Schobitz, De Kloet & Holsboer, 1994; Watkins,
SIairr & Goehler, 1995a for review). Curiously, however, scant attention has k e n devoted
to the behavionl effects of cytokines, other than "sickness behavior" which often
accompanies irnrnunological challenge. In addition. although macrophage activation
promo tes concurrent release of interleukin- 1 (IL- 1 ), in terleukin-6 (IL-6) and tumor necrosis
factm-alpha (TNF-a), most studies that have considered the hormonal, neurotransrnitter
or behaviorai effects of these c ~ o k i n e s have assessed such effects individually. Yet, the
feu. studies thar evaluated the conjoint action of these cytokines have indicated that IL-1 and
IL-6 may act syergistically with respect to HPA functioning (Matta, Weatherbee & Sharp.
1997: Klir. Roth, Szelenyi, McClellan & Kluger, 1993; Perlstein. Whimdl, Abrams.
SIougey 8: Seta. 1993; Zhou. Shanks, Riechman, Liang, Kusnecov & Rabin, 1996). The
purpose of the present investigation is ro detail some of the synergistic effects of these
cytokines. i n particular, administntion of a bacterial endotoxin, such as lipopol ysaccharide
t LPS\. which promotes IL- 1 secretion (Rivier, Chizzonite, & Vale, 1989; Mefford.
.\lasrers. Heyes, & Eskay, 1991; Zanetti et al., 1992), has k e n s h o w to elicit sickness
bchwior. of which anorexia and anhedonia are pnmary symptoms (Hart, 1988: Danner g:
Kellcy. 1989: Plata-Salarnm 8r Borkoski, 1993: Exton, Bull & King, 1995; Yimiiya.
1996: Anisman. Kolckinaides & Merali, 1996). The present investigation xas conducted in
ordsr to determine whether IL-1, IL-6 and TNF-a influence anorexia/anhedonia. and
whether such behavioral effects are subject to the synergistic activity of these cytokines.
Additionally. a prelirninary study was conducted to ascenain whether destruction of vagal
affersnt fibers artmuates the illness behavior provoked by the bacterial endotoxin LPS.
kn0u.n tn mi\-are IL-1. IL-6 and TNF-a (Rivier et al., 1989; Zanetti et al., 1992).
Cvtokines and Vaoal Nerve Influence Immune-Bmin Communication
There is sufficient evidence to suggest that communication between the CNS,
pituitq-adrenal endocrine systern. and the immune systern is multidirectional. Activation
of immune ceiis resulting from pathophysiologicaI stimuli produces a CNS response
which. in tum, modulates the immune system. It is thought that this communication occurs
via alterations in neurotransmitter and hormonal activity (Blalock, 1981; Blalock & Smith,
1985; Berkenbosch, Wolven 8: Denjk, 1991; Dunn, 1990). The immune system,
recognizing that injury or infection has occurred, initiates rneraboiic processes aimed at
eliminating antigenic or pathogenic factors, while the neuroendocrine system ensures that
the menbolic demands of the host are met (Watkins et al., 1995; Blalock, 1991). Typically.
endogenous or exogenous threats to homeostatic mechanisms, which include injury,
bacterial infection or inflammation, give rise to a cascade of irnmunological alterations. In
this context. the host organism's pnmary attempt to restore homeostasis is charactenzed by
rnetsbolic and neuroendocrine changes collectively referred to as the acute phase response
(see Kent, Bluthe. Kelley b. Dantzer, 1992b for review). This response may be mediated
by, arnong orher things, activity of the HPA axis. In fact. it appears that IL-1 is exquisitely
effective in promoting secretion of CRH from the PVN, and is fundamental in the releâse
of pituitary adrenocorticotropin releasing hormone (ACTH) and adrenal corticosterone
(Sapolosby, Rivier, Yamamoto, Plotsky 8r Vale, 1987; Berkenbosch. Van Oers. Dsl Rey.
Tilders &: Besedovsky. 1987; Tumbull & Rivier, 1995). It was proposed, as well, that IL-
1 (and possibly other cytokines) may influence hypothalamic norepinephrine (NE), which
in rum may come to affect CRH release (Dunn. 1988; Dunn. 1990; Lavichy & Dunn.
1995). Additiondly, IL4 ma? also influence mesolimbic SE and dopamine (DA) and
might thus influence affect (Anisman et al., 1993).
In assessing the involvement of immune activation on endocrine and
neurotransmitter processes, numerous investigators have opted to evaluate the effects of the
endotoxin. LPS. which is a subcellular component derived from the outer membrane of
gram-negative bactena Endotoxins, normally present in a healthy host are typically not
found in circulation, but under pathological conditions rnay gain access to the bloodstrem.
As a result, LPS challenge may cause neuroendocrine and behavioral changes similar to
those seen during the acute phase response to infection (Dantzer & Kelley, 1989; Kent et
a1.,1992b).
It is generally believed that LPS does not cross the bld-brain banier, but rather
induces its effects through locaüy mediated signals which are relayed to neuronal targets
(Tilders, DeRij k, Van Dam, Vincent, Schotanus & Persoons. 1993). Peripheral
administration of LPS releases CRH from the hypothalamus, and hence, increases plasma
ACTH and conicosterone Ievels (Kakucska, Qi, Clark, & Lechan, 1993). Inasmuch as the
effects of LPS are attenuated or blocked by an IL- 1 receptor an tagonist (IL- Ira). it is Uely
thsr the effects of LPS are subserved by L-1 (Schotanus, Tilden & Berkenbosch, 1993:
Ebisui et al.. 1991). Moreover. there is reason to suspect that IL-1 may be acting conjointly
w i t h IL-6 and TSF-a to promote the endocrine effects engendered by LPS (Long.
Ortemess. Runkel, Vander & Kluger, 1990; Dunn. 19973; Zanem et al., 1997; Perlstein
er al.. 1993). Such an outcome is, of course, not surprising, given diat LPS fmt promotes
the appearance of TNF-a in the circulation, followed by IL4 and IL6 (Zanem et al..
1992). It remains unclear. however, whether CRH release is a d k c t result of the action of
the cytokines on the brain or is seconda* to endotoxin-induced cytokine-release in the
periphsry.
Recenrly. investigators have reponed that subdiaphngmatic vagotomy cm suppress
CRH and ACTH secrerion ordinarily resulting from LPS-induced HPA acavation
(Gaykema et al.. 1995). These data suggest a putative vagal conmbution to the immune
response follouing endoroxin administration (Laye et al., 1995). The sectioning of vagal
afferenis has also k e n shown to block IL4 induced ACTH secretion (but not
conicosterone secretion) (Kapcala, He, Gao, Peiper lk DeTolla, 1996). providing
additional suppon for the view that cytokines may be influencing the brain via pnmary
sensory afferents. Gaykema et al. (1995) studied c-fos expression in CRH neurons in the
PVN following endotoxin administration. Complete subdiaphragmatic vagotomy blocked
the ACTH response to low doses, and attenuated the response to high doses of LPS in the
rat, while leaving the corticosterone response intact. In addition, Fos expression in CRH
neurons in the PVN was suppressed following subdiaphragmatic vagal uansection. Taken
togeerheer, these studies indicate that immune-brain communication may be mediated by
vagai afferents, possibly through cytokine activation of peripheral sites.
nlness Behaviour: Si ~ n s and Svmptoms
Foilowing immune challenge, animals often exhibit non-specific symptoms of
illness, including fever, anorexia, decreasd water intake, hyperalgesia, increased
somnolence, piloerection, lethargy and reduced social exploration (see Hart, 1988 for
review). While such behaviors were once considered a secondary response to illness, they
are now regarded as an active pan of the host organism's anempt to restore homeostasis
(Dantzer 6r Kelley, 1989). One component of the febrile response includes increased body
remperature owing to s ndogenous pyrogen release. uhich stimulates the immune
response. Other sickness behavion that elevate body tempenture (eg. shivering and
piloerection among others), and reduce the incidence of metabolicdly expensive activities
(eg. somnolence, reduced libido. reduced social exploration), rnay well maximize the
organism's ability to contend with environmental challenges (Kent et al., 1992b). It has
k e n demonsmted rhat beha~iors associated with illness are not secondary to fever. as the
behavioral effects of inflarnrnatov stimuli can be dissociated from the pyrogenic effects of
the treatments. (O'Reilly, Vander & Kluger, 1988; Johnson, Propes & Shavit. 1996)
Evidence that pro-inflanmatory cytokines, such as IL-1, IL-6 and TSF-a, are
involved in the mediation of endotoxin-induced illness has k e n derived from investigations
which demonstrated that systemic administration of IL4 or TNT-a prior to LPS challenge
mitigates the p o p n i c effects of the endotoxin (Long, Monmoto. Nakamon Ji Murakami.
1992; Klir, McClellan, Kozak, Szelenyi, Wong & Kluger, 1995). In addition. the
peripheral administration of IL-1, IL-6 and TM-a results in behaviors reminiscent of
those seen in response to LPS challenge (Shirnomrnura et al., 1990; McLaughlin, Rogan,
Tou, Baile & Joy, 1992; Chance & Fischer, 1992; Goujon, Parnet, Aubert, G d a l i &
Dantzer, 1995). Predictabl y. cytokine receptor antagonists, as weil as antibodies directed
against the cytokines, prevent or limit the effects of LPS on these behaviors (Bluthe,
Danner & Keiley 1992; Plata-Salaman & Ffiench-Mullen, 1992). Taken together, these
studies support the notion that the behavioral components of the immune response to
endotoxin challenge may be mediated by some of the cytokines.
It would be premature to conclude that all sickness behaviors are mediated by an
identical mechanism, or conversely. that only one mechanism can mediate individual
behavinral components of the illness response. Langhans, Savoldelli and Weinganen
(1993). for exarnple, demonsuated that in the rat, peripheral administration of eirher IL-1
or LPS provoked anorexia. While the anorexia associated with LPS resulted from a
reduction in meai hequency, the IL-1 associated appetite suppression was expressed as a
reduction in both meal frequency and meal size. Paracetamol, which inhibits brain
cyclooxygenase, anenuated the IL- 1 -, but not the LPS-induced anorexia, suggesting rtiat
iL-1 affects fieding through a direct acnon on the brain (likely involving the pyrogenic
effects of IL-1). whereas other mechanisms may mediate the effects of LPS on feeding
behavior.
It should be emphasized that the pyrogenic and behavioral effects of the cytokines
do not appear to be mediated exclusively through tend mechanisms. DeRijk, Van
Rooijen, Tilders, Besedovsky, Del Rey and Berkenbosch (1991) reponed that in rats, the
elimination of penpheral macrophages (which secrete IL-1). attenuated the pyrogenic
response to LPS. Klir et al. (1995) found that systemic administration of TNF-a
attenuated LPS-induced fever in rats, whereas central administration had no such effect.
Bluthe et al.. (1992) demonsnated that, in rats, the decreased social exploration and
reduced body weight associated with peripheral LPS injection was attenuated by peripheral
IL- 1 ra, but not by intraventricular IL- 1 ra adminisuation. Taken together, these studies
indicate that peripheral cytokines are intimately involved in the sickness response associated
with immune acavation, but these data do not preclude the involvement of centxal
mechanisms in this respect.
There are several sources of evidence suggesting that ACTH and/or comlcosrerone
may be integrai ro some of the behaviorai/iiIness effects associated with immune activation
(Goujon et al., 1995; Johnson et aL.1996). For instance, while the febrile response
aswciated with endotoxin challenge is likely subserved, in part, by activation of
prostaglandin E (PGE), it appears that endogenous glucoconicoids may also play a role in
this respect (Morrow, McClellan, Conn & Kluger, 1993). It was reponed that pretreatment
of rats with dexamethasone abolished the fever produced by an acute systemic injection of
LPS. Conversel y. adrenalectornized rats showed an increased febrile-response to LPS
administration, which could be inhibited by acute or chronic treatment with dexamethasone
(Coelho. Souza 8: Pela, 1991). Funher, pretreatment with the glucocorticoid antagonist,
RU-38486 (following oral, or innacerebrokrenaicular administration), resulted in elevated
fevers relarive to those in evidence arnong vehicle-treated rats following acute LPS
administration (Morrow et al.. 1993: McClellan, Klir, Morrow 8: Kluger, 1991).
In addition to the potential effects on fever, there is evidence suggesting that
olucocomcoids may be involved in other symptoms of illness which are associated wirh C
LPS treament. Specifically, it was demonstrated that administration of LPS to
adrenalectornized rats provoked sickness behavior (eg. increased body temperature.
reduced social exploration and reduced body weighr) and that such an effect could be
an tagonized by comcosterone implants (Johnson et al., 1996). Moreover. Goujon et al.
(1995) found that in mice, subcutaneous IL- 1 or LPS administration reduced social
exploration. an effect which could be enhanced by adrenalectomy. The sarne effect was
evident among intact animals given subcutaneous injections of RU-38486, indicating that a
reduction in glucocomcoid concenmtion was likely related io the enhanced susceptibili~ to
the behavioral effects of LPS. Indeed, corticosterone implants had a protective effect on the
adrenaiectomized rats injected with IL-1, and partiaily protected mice injected with LPS. In
a similar fashion, Pezeshki, Pohl and Schobitz (1996) demonstrated that LPS
adminisrration in adrenalectomized rats, in doses which ordinarily induce fever and
anorexia, promoted a more profond reduction of locomotor activity relative to intact
endoxtoxin-treated animals. In effect, it seemed that the absence of corticosterone
peminzd greater development of illness behavior. As rnight be expected, the behavioral
effects (with the exception of the reduction in locomotor activity) could be attenuated by
prmeament with corticosterone prior to LPS administration.
An alternative accounting for some of the behavioral effects of immune activation,
including sickness behavior, is derived from the work of Blalock and colleagues (Smith.
Meyer & Blalock, 1982; Blalock, 1984; Blalock &: Smith, 1985). It was proposed that
acrivated lymphocytes secrete an ACT'I-I-like substance which may directly or indirectly
stimulate CNS processes. In fact, it was shown that following hypophysectomy, immune
challenge still increclsed plasma A C ï H levels, possibly reflecting the release of this
hormone h m activated Iymphocytes (Blalock, 1984). Subsequent studies conducted by
Dunn. Powell and Gnskin (1987) suggested that some of the observations derived by
Blalock moy have k e n due to incomplete pituitary ablation, which afforded an ACT'H
source distinct from 1 ymphocyte-ACïH secretion. While incomplete hypoph ysectomy ma'
have k e n a factor in the detection of ACTH release, it is likely that pimit. endocrines
released during immune activation promote CNS effects.
MCLTISYSTEM INTERACTIONS
Svmnnthenc Nsrvous System and the HPA Axis
As indicated earlier, the similarities between the effects of stressors and immune
challenge on endocrine and CNS processes gave nse to the proposition that immune
challenge may be interpreted by the brain as if it were a srressor (Dunn, 1990). C'pon
dctection of a stressor, the sympathetic nervous system is activated, causing rapid
alterations in catecholamine levels in the blood. It has been suggested that the initial
stressor-provoked penpheral NE release may be essential for activating immune processes
(Felten & Felten, 1991). Indeed, it appears that the sympathetic nervous system innervates
a wide variety of organs and tissues, including the primary and secondary lymphoid
organs, as weii as the spleen. Moreover, NE receptors are present on lymphocytes, and
these cells may be activated by factors that provoke sympathetic NE release (e.g, stressors)
(Madden & Livnat, 199 1). Like NE, it has k e n demonsmted that glucocorticoid receptors
are present in the spleen, thymus and lymph nodes, as weli as on the cell surface of
lymphocytes. Activation of these glucocomcoid receptors have the effect of reducing
immune activity (Keller, Schleifer & Deme~opou los , 1991). It has k e n sugpsted that
one of the functions of circulating conicosterone is that of Iimiring the extent to which the
immune system is activated by stressors, hence reducing the potenrial autoimmune effects
thar might otherwise occur (Munck. Guyre. & Holbrook, 1983). In effect. it seems that the
immune systern is inmnsically linked to borh HPA functioning and sympathetic nervous
system acti\+y.
Immune-CSS Communication
Consistent with the notion that immune activation has CNS effects. it has been
s h o w that antigenic challenge promotes catecholamine turnover in the brain and the
periphery. in particular, in rats and rnice, administration of sheep red blood celis (SRBC)
results in changes of NE tumover in the locus coeruleus and hypothalamus (Zalcrnan et al..
199 1). and DA tumover in mesocortical sites (Lacosta, Merali & Anisman. 1995). In
addition, S RBC adminisuarion alters serotonin (5HT) Ievels in the paravennicular and
supraoptic nuclei (Carlson, Felten, Livnat & Felten, 1987). These changes, dong with
increased electrophysiologic activiry in the PVN and in the locus coeruleus (Besedovsky,
Sorkin, Felix & Haas, 1977), corresponded with die immune response being mounted
(Carlson et al., 1987: Zalcman et al., 1991; Lacosta et al., 1995).
Like the effects of SRBC, it was recently demonsaated that neuroendocxine
activation rnay occur as a result of virai challenge or infectious disease. In panicular,
pathogens such as Newcastle disease vims N V ) and influenza vins are associated with
HPA activation (Dunn, Powell, M e i ~ & Srnall, 1989), and increased hypothalamic hiE
release (Dum & Vickers, 1994). Intraperitoneal injection of NDV also increased
hypothalamic concentraaons of tryptophan, the hZ carabolite 3-methoxy 4-
hydroxyphenylethylene_Elycol (MHPG), and the 5-hydrox~yptamine (5-HT) carabolite 5-
hydrox yindoieacetic acid (5-HIAA). In view of the observation that hypophysectomy
blocked elevarions in plasma comcosterone following hDV and LPS challenge, ir seems
that the corticosterone changes were probably not a result of viraI srimulation of the
adrenal. but rather involved HPA activation.
It will be recdled that there are sufficient data to suggest that IL-1 rnay funcnon as
an immunotransmirter to provoke endocrine secretion following immune challenge. Arnong
othcr things. IL-1 is a potent stimulator of CRH release, and hence A C T H and
conicosrerone secretion (Besedovsky et al.. 1986; Berkenbosch et al., 1987: Sapolsky et
al.. 1987: Wehrenberg. Saudan, Corder, Voltz, Giustina & Gaillard, 1994: Meiink
Parsadanirintz. Levin. Lenoir. Roberts & Kerdelhue, 1994). Moreover. central pretreament
with antngonists of IL-1 or CRH limited the A C T H and corticosterone variations induced
by eithsr IL- 1 or LPS trcatment (Dunn. 19922: Schotanus et al., 1993: Ebisui et al., 1994).
Tot unespectedly. the increased paraventricular CRH messenger RNA (mRKA) obsened
following intrapeitoned administration of LPS in rats. could also be abolished by
innûvenricular IL- h a pre-matment (Kakucska et al., 1993). In addition, dexamerhasone
was found to inhibit IL-ha mRNA levels in endotoxin-stimulateci human monocytes, and
inhibired the secretion of IL-lra. These effects could be reversed by the glucocomcoid
antctgonist RL' 38386 (Am et al., 1994).
In addition to these endocrine variations. there is reason to suppose that the effects
of inirnune challenge on exaahypothalarnic hT, DA. and 5-HT may involve cytokines. In
particular, it was dernonstrated that systernic adrninisnation of IL-I promotes severai
distinctive monoamine changes (eg. DA turnover in the prefrontal conex), which are
rerniniscent of the effects associated with suessors (Merali, Lacosta, & Anisrnan, 1997, in
press). Yet, IL-1 and SRBC have distinctly different effects with respect to some of the
centrai neurochemical changes obsenied. In par?icuIar. while SRBC provokes in vivo
accumbal DA release (Lacosta et al. 1995) IL- I does not produce such an outcorne, and IL-
6 reduces DA release (Merali et al., 1997 in press).
CYTOKINES INFLUENCE ENDOCRINE/CNS TRANSMITTERS
Immune activation involves the release of numerous cytokines, including IL- 1, IL-
3, IL-4, IL-5, IL-6, IL-10, IL-I), TNF-a, and interferon-y among othen. Most of these
cytokines have not k e n assessed extensively insofar as their endocrine and behaviorai
effects are concemed (see Berkenbosch, de Rijk, Del Rey 8. Besedovsky, 1990; Schobitz
et al., 1993; Turnbull & Rivier, 1995 for review). It is conceivable that in addition to the
pro-inflarnmatory cytokines, one or more of these other components may act to signal
endocnne/CKS processes. Nevenheless, the amilable data have suggested a prominenr role
for IL- 1. IL-6 and Th- in promoting an interface benveen endocrine and CSS activity.
Accordingly. the present review has k e n lirnited to these cytokines. In discerning potentiai
individual or synergistic effects of this subset of cytokines, one would be remiss if a more
detailed description of their actions were not described.
There are three forms of IL- 1, IL- 1 a, IL- 1P and IL-lra. Although IL- l(3 is the
major secreted fom, IL-la and IL-lp nevenheless appear to have similar effects on
numerous systems (Rothwell lk Luheshi, 1991). Studies in rats suggest, however, that IL-
1P has more prominent effects than IL- la with respect to HPA activation. For instance,
IL-1P is more effective than IL-la in promoting CRH release from the PVN and hence
ACTH release from the pituitary (Harbuz. Stephanou, Sarlis & Lighmian. 1992). In
addirion. the fever and therrnogenesis induced by inmcerebrovennicular (icv)
administration of IL- 1 P, but not IL- la, can be blocked with a centrai injection of a CRF
receptor antagonist (Long et al., 1990; Rothwell & Luheshi, 1994). Likewise, the receptor
antagonist, IL- Ira counteracts the HPA stimulating action of IL- 1 P (Dunn, 1995).
TNF exists in two foms, TNF-a md TNFP. which share approximately 50%
sequence homology. TNF-a (or c a c h e c ~ ) is secreted by a variety of immune cells,
whereas TM+ is exclusively secreted by activated lymphocytes. IL-6 is a B-cell growth
factor that (dong with IL-1) augments T-lymphocyte mitogenesis. Under homeostatic
conditions, the expression of IL-1, IL-6 and Tm-a is ordinarily low in most tissues,
peripheral and cenaal sites, while a marked increase in synthesis and secretion is detected
following inflammation or infection. Like IL-1, elevaaons of IL-6 and TNF-a not only
have paracrine and endocrine-like effects, but also autocrine effects which inhibit their oun
activity . hence lirni ting immune overshoots (Schobitz et al., 1993).
The mechanisrns by which the cytokines signal the brain following infection has yet
to be fully elucidated. Receptors for L- 1, L-6 and TNF-a have been localized in various
brain regions (including the hippocampus and hypothalamus), although it is unclear how
cytokines gain access to the brain. as they are large, lipophobic proteins thar are unlikely to
pass through the blood-brain barier (BBB). It appears. however, that the cytokines may
gain access to the brain via carrier-mediated transport. or possibly via regions lacking a
blood- brain b h e r (eg. the organum vasculosum of the lamina terminalis) (Watkins et al..
1995a). An alternative explmation for the influence of cytokines on behaviorai change is
derived largely fiom the observation rhat illness behviour c m be induced in the absence of
detectable levels of LPS or cytokines in the blood. In effect, cenaal activation may not be
dependent on blood-borne cytokines. Rather. it has k e n suggested that cytokines may
activate peripheral afferent nerves at or near the site of release. It will be recalled, for
exarnple that subdiaphragmatic vagotomy blocks many of the illness responses following
IL- 1, 'Ih'F-a and LPS, suggesting a system of immune-brain communication that is based
on humoral rather than blood-borne factors (Laye et al., 1995, Watkins et al., 1994; BIuthe
et al., 1991, Gaykema et al., 1995).
Alterations in IL-1 levels have been implicated in a number of physiological
changes resulting h m LPS administration. In order to assess the role of IL4 in LPS-
induced pyrogenesis,Denjk, Van Rooijen, Tilders, Besedovsky, Del Rey & Berkenbosch,
1991 and Marquette, Van Dam, Van Rooijen, Berkenbosch and Haour (1994) eliminared
peripheral macrophages through the administration of dichloromethylene diphosphonate
(CEMDP) liposomes. This treamient attenuated the nse in plasma IL-1 concenmtions in
response CO LPS and also abrogated the thexmogenic and fever response to LPS in rats.
These data suggest that peripherally produced IL-1 is involved in the pyrogenic effecrs of
LPS. However, penpheral injection of LPS has been shown to induce IL-l mRNA in rat
brain microglial cells (Buttini & Boddeke, 1995) and induce IL- I activation in ramified
microglia, as well as activated macrophages in the meninges and choroid plexus of the rat
brain (Van Dam. Brouns, Louisse & Berkenbosch. 1992). Thus, there appears to be a
central role of IL-1 in LPS-induced neural activation. Interestingly, depletion of
macrophages also prevented the plasma ACTH and corticosterone responses to su b-
pyrogenic doses of LPS. However, this effect on ACTH and corticosterone was not
evident in response to ppgen ic doses of LPS (Derijk et al., 1991). Evidently, the
pyrogenic acaons of IL-1 cm be disassociated from the ACTH variations following
endotortin treatment.
There are distinct variations in the kinetics of the various cytokines following LPS
administration (de Simoni, Dei Bo, De Luigi, Simard & Forloni. 1995). It has also bern
demonstrated that the pattern of rnRNA induction in rat brain following penpheral LPS
injection is different for each of the cytokines (Gam & Banfai. 1993). i n d i c a ~ g that they
may play temporally linked divergent roles following immune activation. For example,
nF-a Iwels peak approximately one hour following LPS administration. whereas. IL- 1
and IL-6 peak within 3-4 hours (Zanetti et al., 1992; Derijk et al., 1993: Schotanus et al..
1993; Fantuzzi, Di Santo, Sacco, Beniepi & Ghezzi,1995). In addition, de Simoni et al.
(1995) noted that the levels of TNF-a decline more rapidly than those of IL-1 and IL-6,
suggesting that TNF-a may initiate HPA activation following LPS administration, while
IL- 1 and IL-6 susrain the activation once it has k e n triggered
Evidence in favour of the conjoint action of IL- 1, IL-6 and TNF-a in mediating the
endocrine effects of LPS is derived from investigations evaluating the effect of cytokine
antibodies. For instance, the fact that sysvrnic administration of IL- 1 itself induces marked
HPA alterations suggests thar the role this cytokine is apparently more significant than
sustsining a previously excited HPA system. Of course, the possibility cannot be dismissed
that the relative conmbutions of the three pro-inflarnrnatory cytokines are dissimilar with
respect to inirïating vs. maintaining HPA activation. Moreover, recent investigations
involving the synergistic effects of IL-1, IL-6 and TNF-a revealed that these cytokines
mal- interact with respect to the activation of the HPA axis following LPS adminisuation
(Zhou et al.. 1996).
Pçrlstein et al. (1993) reponed that IL-6 antibody pre-treatment prevenred the LPS-
induced ACTH response ordinarily observed 7 and 1 hours after endotoxin adminisuarion.
Prsmatment with ei ther IL- 1 r antibody or TNTF-oL an tibody also blocked the 4 hour A m
response. but did not block the response observed 2 hours after endotoxin treatment. A
combinarion of IL-lr and TNF-a-antibody iirnited the 2 hour ACTH response. In
addition. it was obsen~ed that combined doses of Tb??-a and IL-6, as well as TSF-a and
IL-1. increased production of circulating ACTH to a greater extent than that produced by
these cytokines admnistered separately. Moreover, pretreament with an IL-6 antibody
complctely blocked the conjoint effecü of ThF-a and IL- 1 on the AC3T-I response. Since
TSF* and IL- 1 both stimulate IL-6 release, the data of this investigation suggest bat LPS
directly stimulates IL-6 release 2 hours afier adrninstration, but that TNF-a and IL-l
synergisricrtlly induce the variations of TL-6 observed at the 4 hour interval.
In conaast to the results reporteci by Perlstein et al. (1993), which emphasized the
contribution of IL-6 in mediatïng die endocrine variations associatecl with LPS aeannent,
Schotanus et al. (1993) suggested that this cytokine was not fundamental in the provocation
of the ACTH alterations. In particular, it was dernonstrated that peripheral IL-1
administration increased plasma ACTH, corticosterone and IL-6 concentrations. At low
doses, however, the ACTH and corcicosterone response to peripheral IL1 were not
accompanied by changes in plasma IL-6 concenmtions, indicating that the latter cytokine
was not involved in the ACTH response. In a subsequent snidy. dose-dependent elevations
of plasma ACTH, corticosterone and IL-6 concentrations were seen following peripheral
administration of LPS. The dose required to induce the median IL-6 response was 4 times
higher han the dose required to induce the A m and comcosterone elevations. These
results, dong with the demonstration that the ACTH. but not the IL-6, response to
endotoxin was blocked by peripherd IL-Ira, indicate that IL-6 did not play a prorninent
role in the initiation of the HPA response following endotoxin administration.
Additional evidence that IL-6 is not an essential mediator in the inflammatory
responsc to endotoxin is denved from investigations involving IL-6 deficient mice. Fattori
et al. (1991) found that these mice were able to generate an inflammatory response to LPS
administration. Corticosterone, as well as IL-1 levels in the IL-6 deficient mice were sirnilar
to the values detected in C O ~ D O ~ S following LPS administration. Similady, the reduction in
body weight and food intake following LPS adminstration was comparable in both groups
of animals. Unexpectedly, levels of TNF-a induced by LPS were three-fold higher in IL-6
deficient mice than in controls. which prompted the suggestion that the increased TNF-a
ma' compensate for the absence of IL-6. In effect, it appears that the corticosterone
response following LPS does not require IL-6, and IL-6 is not a necessary component of
the infiammatory response to endotoxin administration.
As indicated earlier, the mechanisms that mediate HPA activation following
pyrogenic doses of endotoxin may be distinct from those that mediate Iow doses of
endotoxin. Indeed, it has k e n demonstrated that LPS induces dose-dependent variations of
glucoconicoids, and that the relative conmibutions of various cytokines Vary as a function
of the LPS dose employed. Ebisui et al. (1994), for instance. found that when smaii
amounts of LPS (0.03 p a g ) were adrninistered intravenously to rats, the maximum
conicosterone response could be almost completely abolished by TNF*-antiserum, but
not by an an IL- 1 receptor antagonist. However. either TNF+-antisenim or IL-lra
anenuated the response when larger arnounts of LPS (10pg/kg) were administered. These
results suggest that, at low doses, ThF-a alone was responsible for mediahg the effects
of LPS on the HPA axis, while IL-I participated in stimulating ACTH secretion following
high doses. Moreover, following LPS administration, rmitment of IL-1 and TSF-a in
the provocation of ACTH release may occur at different times.
Anoirxia i s Infliienced hv Svner~st ic - Actions of C ~ ~ o h n e Treatment
Although it is clear that L I , IL-6 and TSF-a ail participate in the HPA response
to endotoxin. it is difficult to determine which cytokine initiates the HPA response. It will
be recalled that multiple behavioral and endocrine interactions exist between the cytokines.
Sot only do the cytokines act in synergistic ways, depending on the combination of
cytokines activated. but IL- 1, TL-6 and TW-a can al1 modulate the synthesis and secretion
of one anothsr. In view of the relative paucity of data conceming the interactive effects of
cytokines in modulating endocrine functioning. it is not surpnsing that still fewer studies
have foîussd on the behavioral synergisms that occur in response to cytokine challenge.
As indicatzd earlier, anorexia is a prominent behavioral response evident following
immune challenge. and has been assessed following adrninismtion of endotoxins. Dose-
dependent reductions in food intake and body weight have k e n demonstrated following
both peripheral (O'Reilly et al., 1987) and cenaal (hlcCanhy, Kluger, & Vander, 1986)
adminisûation of LPS. It has k e n suggested that the endotoxin-induced reduction in
feeding may be mediated. in pan, by the proinflammatory cytokines. Indeed, IL- 1. IL-6
and TSF-a. al1 of which are induced by LPS administration, suppress food intake (Plata-
Salaman, Oomura & Kai, 1988; Tracey et al., 1988; Socher, Freeman & Mariinez, 1988;
Michie, Sherman, Spriggs, Rounds, Christie & Wilmore, 1989; Fantino & Wieteska,
1993; Langhans et al., 1992; Fanon et al., 1993; Plata-Salaman, 1994). Inasmuch as the
anorexic effects can be induced by either peripheral or central administration of IL- 1 raises
the possibility that this effect is, at least in part, cenmiiy mediated (Shirnommura et al.,
1990, Chance & Fischer, 1991; McLaughlin et al., 1992). Cornmensurate with this view,
peripheral administration of IL- 1 antagonists blocked the anorexic effects of systernic K- 1
(McLaughlin et al., 1992). and centrai administration of IL- Ira attenuated the anorexia
induced by icv IL-1 (Plata Salamas & Ffrench-Mullen. 1992; Plata-Salaman & Borkoski.
1993). Kent. Bret-Dibat. Kelley & Danner (1996) demonstrated that central administration
of IL- 1 profoundly decreased operant responding for food reinforcment, and this effect
could be prevented by central IL- lra preueatment. Interestingl y, however, centrai IL- 1 ra
did not completely block the effect of peripheral IL-1. nor did peripheral IL-Ira block the
effects of centrai IL-1. These results suggest that IL-1 influences both peripheral and
cemal mechanisms to reduce food-motivated behavior.
TW-a and IL-6, it will be recalled, have also k e n s h o w to have a direct action
in die CNS, and it has been suggested diat TNF-a exens its anorecac effects through
centnll y mediated mechanisms. Rogers, Mctigue and Hermann (1 996) demonstrated that
the nauseus influence of gasaic relaxation, cnuld be evoked through unilateral TXF-a
injections into the dorsal vagal complex. In addition, Fantino & Wieteska (1991)
demonstrated that, in rats, central administration of TXF-or resulted in a decrease in food
and water intake, whereas peripheral administration had no effect. Moreover.
elec~ophoreticall y applied TNF-a suppressed the acovity of glucose-sensitive ne wons in
the lateral hypothalamus. (Plata-Slarnan et al,. 1988). Since IL-1 was found to produce
sirnilar effects, it was posited that some of the effects of T ' h W may have ken seconda7
to the induction of IL- 1 .
It has been suggested that IL-6 rnay play a role in the attenuation of anorexia
following ïL- 1 receptor blockade. Oldenburg et al. (1993) investigated the effects of IL-6
and IL-lr antibodies on anorexia in the mouse following sterile rurpentine abcess. The
reduction in food intake and resulting weight loss were attenuated by both L - l r and IL-6
antibody administration. The co-administration of IL-6 and IL-lr antibodies did not,
however, result in a sig~~ificantly greater reduction in the anorexic effects than those in
evidence following the administration of the each separate antibody. Based on the
additional observation that IL- l r blockade anenuated production of IL-6, the investigators
concluded that IL-6 may be the mare direct rnediator of anorexia and weight loss in this
model.
Although there is evidence that the anorexic effects of LPS and IL- 1 are similar to
one another, there are conflicting repons regarding the effects of endotoxin on meal
patterns. For example, O'Reilly et al. (1988) reponed that prolonged exposure to LPS did
not result in tolerance to the anorexic effects, whereas Langhans et al. (1993) demonsmted
that tolerance did deveiop following prolonged LPS exposure. Interestingly, Langhans et
al. also reponed that inaaperitoneal LPS reduced food intake by decreasing rneal
frequency. uirhout affecting meal size. In contrast to this finding, however. Plata-Salaman
(1991) found bat LPS signficantly reduced med size. It is important to note that while
Plata-Salaman (1991) administered LPS centrdly, Langhans et al. (1993) used peripheral
injections to induce anorexia. Thus, it is conceivable that different mechanisms subsenle
the anorexia, with only the central processes affecting meal size.
Although numerous effects of LPS, as already described. have been amibuted to
activation of IL-1, there is evidence that LPS and IL-1 induced anorexia are mediated
through different mechanisms. Mrhereas repeated injections of IL- 1 lead to sensitization of
the anorexic effects, prolonged exposure to LPS lead to tolerance, with no evidence of
cross-tolerance or cross-sensitization between the compounds (Langhans et al., 1993).
Moreover. the cyclooxygenase inhibiror, paracetamol, sipificantly artenuated the IL-1-.
but not the LPS-induced anorexia, suggesting a role for prostaglandins in the IL- 1 mediated
effects.
Vagotomy and Feeding
It will be recalled that several investigaton argued that communication between the
immune system and die CNS rnay occur through the vagal nerve. This proposition was
based on. arnong other things, the finding that subdiaphragmatic vagotomy attenuated the
hormone variations otherwise provoked by immune challenge or bacterial endotoxins
(Gay kema et al., 1995). Severing the vagus nerve eliminates sensory afferent fibres which
c q information conceming the gastrointes~d tract to the centrai nervous system. In
addition, efferent fibres that are involved in motor acrivities of the intestine and stomach are
also severed, which eliminates integrai sources of information conceming intestinal
content, as well as CNS control over the secretory and mocilic functions of the stomach
(Shapiro & Miselis, 1985). Given the important contribution of the vagus to intestinal
processes, it should not be surprising that subdiaphra,patic vagotomy, which involves
severing of the vagal-afferent and efferent innervation, influences feeding in humans and
in infrahuman subjects (Yang, Ratto, Gleason, Bellantone, Crucini 8: Meguid. 1993j.
S uch effects are not dietary dependent, and are readily apparent when dry food or more
palatable liquid diets are provided (Davis, Smith d Kung, 1994; Bernstein. 1995).
Davis et al. (1994) demonsmted that, in rats, the reduced consumption of a liquid
diet following vagotorny could be amibuted to a decline in the rate of licking during the
meal. which was not related to motor dysfunction. Analysis of the microstructure of the
licking response revealed that vagotomized rats had shoner Iicking bouts at the beginning
of the test, and a characteristically defined rapid decline in the licking rate during the test.
This behavioral profile was independent of the type of diet provided (rnilk, or one of 3
concenmtions of sucrose solution) suggesting that neither motivational factors, nor
nutritional content, were particularly relevant in inducing the pattern of behavioral change
in evidence. It was suggested that the decline in the rate of Lickng during the meai was
attributable to an accelerated clearance of fluid from the stomach, which enhanced negative
feedback s ipa l s from the gasmin te s~a l tract ordinarily conveyed to the CNS through
exaavagal pathways.
Recently, it has k e n suggested that the reduction in food uitake following
vagotomy c m be amibuted, at least in part, to peripheral endocrine factors (Rogers et al.,
1996). The dorsal vagal complex @VC) lacks a blood brain bamier, which rendes this site
susceptible to humoral signals such as cytokines and hormones. Since the DVC influences
parasympathetic activity associated with digestive processes, cytokines and ensuing
hormonal changes rnay exen effects on feeding through indirect signals acting on
parasympathetic functioning.
Anorexia and Anhedonia
Regardless of whether cytokines, vagal innervations, or a combination of the two
mediate the behaviod responses to endotoxin challenge, a note of caution must k
inte jested conceming the processes al iged with the anorexic symptoms. When describing
the "sickness behavior" aswiared with immune activation, most investigators have
concluded that compounds such as LPS induce a reduction in feeding and also decrease
rnorivation to consume food. On the one hand. the reduced motivation may reflect
diminished appe tire second. to illness, but on the other hand it may reflecr a diminution in
the rewarding attibutes ordinarily ascxibed to feeding.
It has recently been suggested that activation of the immune system results in
physiological and behaviord changes that resemble the characteristics of depression.
Indeed. i t has been demonstrated that in some cases, depression is associated with immune
activation, characrerized by an increase in plasma levels of cytokines such as IL- 1 and L-6.
and dysreplation of the HPA axis. The loss of appetire, reduced body weight, increased
somnolence and anorexia which follow cytokine or endotoxin adrninistranon in animals are
ail symptoms of depression. In addition, animal models of depression include the
attenuation of sucrose consumption and place-preference paradigms which are used to
study the rewarding properties of food and dnigs (Willner, Towell, Sampson.
Sophokleous & Muscat 1987; Papp, Wilner & Muscat, 1991; Suzuki et al., 1994). In
effect, the view can be taken that the reduced food consumption associated with endotoxin
administration may be a reflection of the potential anhedonic and depressionogenic effects
of this matment Indeed. it was demonsuated that penpheral pretreatrnent with LPS
abolished the reinforcing effects of cocaine in the place-preference paradigm (Suniki et al.,
1994).
Consistent with the view that LPS induces depressive-like effects. Yirmiya (1996)
demonsaated that a single dose of LPS suppressed sexuai behavior in male rats. and
reduced their preference for, and consumption of. a saccharin solution. Furthemore.
repeated treamen t wi t h clinicall y effective an tidepressan t agents (eg. irnipramine)
effectively elirninated the anorexic effects ordinarily induced by LPS treannent.
Purpose
In view of the observation diat the proinflarnrnatory cytokines may have synergistic
actions with respect to hormonal functioning. it was of interest to establish whether these
cytokines would also have such effects on behaviors associated with illness. In this
context. the present investigation was undenaken to determine the individual and conjoint
actions of IL- 1. IL-6 and TNF-a on the consumption of a highly palatable food. as u r l l as
the variations of plasma corticosterone levels. Inasmuch as few srudies have assessed
these cytokine actions, despite their recent chical usage (Meyen & Valentine, 1995). a
dose response curve was determined for LPS and for each of the cytokines, after which the
conjoint actions of suboptimal doses of the cytokines were evaluated.
Experiments 1-4
The initial Experirnents were conducted in order to evaiuate the anorexic effects, as
well as the comcosterone variations induced by LPS, IL-1, IL-6 and TNF-a. Dose-
response curves were established for these compounds, since sub-optirnal doses had to be
detemined to evduate potential synergistic actions of the components. As well, the initial
experiments sought to establish whether the behavioral effects of the treannents paraiieled
their poten tial hormonal actions.
hlaterials and Methods
S ut,-iects
Male CD-1 mice, 8-12 weeks of a g were obtained from Charles River Inc,
Laprairie Quebec. After arriva1 at the facility, mice were housed in groups of four and
acclimated to the laboratory for at lem 2 weeks. Room temperature was rnaintained at 21°
C. lighting uas maintained on a 12 hr Iighddark cycle (lights on at 0800). One week prior
to the beginning of the expenments. mice were separated into individual polypropylene
cages with wire mesh lids and maintained ad libitum on pellet mouse chow (5075 Rodent
aiet Autoclaved. Rdston Purina), and tap water.
Prior to LPS or cytokine administration, rnice were given free access to chocolate
milk (Sealtest. 1% ppartly skimmed) for one hour each da' during the light phase (bonles in
at 10:OO). Bottles were weighed at the beginning and end of the hour in order to detemine
consumprion < weig h ts were convend to volume). Follouling the establishment of a steady
rate of dnnking (3 consecutive days on which consumption varied by less than IO%), rnice
received an acute innaperitoneal (i.p.) injection of eidier vehicle or LPS, al one of several
doses. 90 min prior to receiving the chocolate milk. On the day following dnig
adminismtion. consumption was again measured in order to determine rate of recovery.
Likewise, in Expenments 2-4, following establishment of baseline consumption.
mice received an acute i.p. injection of either vehicie or IL-lP, IL-6 or TNF-a (at one of
several doses) and 45 min (in the case of IL-1 and IL-6) or 30 min (in the case of TM-a)
afterward, consump tion was measured.
D x s
LPS (frorn Escherichia coli serotype 026:B6 L-3755 Sigma) was dissolved in
sterile saline (9%) and administered i.p. 90 minutes pnor to receiving milk. Each mouse
received a single dose of 0.5. 0.75 or 1.0 pg (n=10 per group) AU h g s were
administered in a volume of 0.4 ml. Vehicle animals were injecte. i.p. with 0.1 ml of
sterile saline.
IL- 1 P was kindlp provided by Dr. Craig Reynolds (Biolngicai Response Modifiers
Program, National Cancer Institute, Fredenck, Md. U A . Produced by E.I. Duponr De
Nemours. specific activity 1.8 1 x 1 o4 units/pg). TSF-a ! specific activity=l. 1 x 10 5
units/m_o) and IL-6 (specific activity=7.0 x 103 unirs/pg) wsre obtainrd from R&D
Systems. AU drugs were suspended in srenle saline (9%. IL-1 and IL-6 u-sre
adrninistered i.p. -25 minutes prior to receiving milk. whereris TSF-a utas adrninistsred
i.p. 30 minutes prior to receiving milk. IL4 u a s administered in a single dose of 0.025.
0.05. 0.1. 0.1. or 0.8 pg in=lO per group) in (i volume of 0.1 ml. IL-6 ueas administered
i.p. in a single dose of 0.1. 0.2. 0.8 or 1.6 pg in=10 per group,. TSF-u was
administerrd i.p. in a single dose of 1 . 2 or 4 p g C < n = l O per group ).L'chiclr. animals in di
croups (n=10 psr group) were injected i.p. with 0.4 ml sterile saline. C
In paraIlel eipenments mice rcceived i-p. administrrition of sithsr the LPS'.
iL- 1. IL-6 TXF-a or their respective vehicles. and then either 120 minutes (in the case of
LPS) or 60 minutes afteward (in the case of the cytokinesi. mice uwe decapitarsd and
uunk blood was collectai in tubes containing 10 pl EDTA. The blood was centrifuged a
'Micc in the LPS study wcrc also uscd to cvaluatc the anxiogenic effecis O T endoroxin trcatmcnt. Thus. micc had reccivcd a 5 min cxposurc io a 1 i g h r - d a r k box at 90 min following LPS trcatmcnt. As thc cndotosin \vas withour cffcct O n light-dark pc r fo rmancc . i t was decmcd unnccessary io conduci an a d d i t ional cxpcrimcnt io cvriluatc thc alrcady wcll documcnicd hormonal actions o f LPS.
3500 rpm. for 15 minutes, and the plasma frozen and stored at -70° C. The plasma
corticosterone concenmtions were detemineci, in duplicate, by radioimmunoassay using
kits obtained From ICN Biomedicals 1nc2. These assays were found to yield less than
104Iinm- and interassay variability.
Results and Discussion
Variations of consumption of palatable diet
Figures 1 and 2 show the variations of chocolate milk consumption over a 1 hr
period as a function of the ûeatment rnice received.
LPS
On tesr day. an acute i-p. injecaon of LPS 90 min prior to tesring reduceù
consumption of chocolate milk (See Figure 1 ). A repeated-rneaures analysis of vanance
revealed a significant Treatrnent x Sampling pend intcncrion F(6.71)=6.839. pc.0 1 .
Newman-Keuls multiple cornparisons of the means comprising the simple effects of this
interaction ta = .05) indicated that among vehicle treated mice consumption did not ciiffer
over any of the three sampling periods. In conrnst. al1 doses of LPS provoked a significant
reduction in consumption compared to baseline levels. Consumption of mice retested 24 hr
after LPS treatment rstumed to baseline levels. Betwesn group cornparisons indicated that
on Test da!. consumption in LPS-treated animals al each dosage \seris significantly reduced
relative to vehiclrc treated mice.
Insen Figure i about hers
rL- 1 - IL-1 administration reduced consumption of chocolate milk (See Figure 7-Cpper
panel). and the analysis of variance indicated a significant Cytokine x Sarnpling period
'Owing to ihc cxpcnsc of thc assays. ihe cffccis of only somc of ihe dcses ubcrc dctcrmincd. The spccific doscs for each drug wcrc bascd on pilot studics.
Figure 1. Mean (?s.E.M.) consumption (ml) of a highly palatable food arnong rnice that
received periphenl lipopolysaccharide (LPS) or saline. Various doses of LPS (0.5 pg.
0.75 pg or 1 pg) were adrninistered inuaperitoneally to male CD-] mice and consumption
of chocolate mik was measured over a 1 hr. period cornmencing 90 minutes following
injection. Baseline values were calculated by averaging consumption over the three day
period irnmediately prior to testing. Recovery data were collected 24 hr. following tesring.
LPS dose ------- Saline
8
Test I
Recovery
interaction F( 10.108) = 5.435, pc.0 1. Multiple comparisons within groups revealed that
mice treated with 0.05, 0.1, 0.2 or 0.8 pg of IL-1 45 minutes pnor to testing reduced
consumption of chocolate mik relative to baseline levels. Consumption in al1 animals teste.
retumed to baseline levels 24 hr following treatment. Among vehicle ueared rnice or those
that received the lowest IL- 1 dose (0.025 pg) consumption did not Vary over the sampling
penods. Between group comparisons indicated that following cytokine neatment. mice in
the 0.05 - 0.8 pg groups also consumed significandy less chocolate milk than saline mated
animals.
----------------------------------*-
Insen Figure 2 about here
TSF-a
Analysis of varirtnce of chocohte milk consumption in the TSF-a ueatsd mice
yidded a significant C-iokine x Sampling p e n d interaction. F( 6. 72) = 3.978. p < - O 1 .
.MulnpIe cornpuisons of the simple effects comprising the interaction indicated that among
the vehicle rreatd rnice consumption did not diffsr over the 3 mnpling penods. In
connast. u i t h i n each of the three n F - a groups a signifierint dsclinc irom basclinc values
uas evidsnt on the da) of cytokine treritmcnt. followed by a significant increass 21 hr latcr.
Betwsen-group çoniparisons confirmai that the groups did not diffsr li t baselins or 24 hr
after cytokine treatmenr. The TNF-a was found tn rèduce consumption of the chxo1;ite
milk relative to saline-ûeated mice. but this diffrrence on1 y reachsd statistical significrtnce
among niice treated with the 1.0 pg dose (see Figure .-Middle plinel).
L-6 - In contras to the effects of IL-1 and TSF-a. systemic administration of IL-6 dia
n a significan tly affect consumption (Sce Figure 7-Lowsr panel J. Although ii small
decreass in chocolats milk intake was seen on the day of rreatrnent in mice receiving cither
0.1 or O.? pg of IL-6. this decrease did nor approrich statistical significiince. Indeed. at the
Figure t Mean (~s.E.M.) consumption (mi) of a highly palarable food among mice
receiving penpheral inrerleukin- 1 P (IL- 1 ), turnor necrosis factor-alpha (TNF-a).
interleukin-6 (IL-6) or saline. Various doses of IL- 1 (0.025 pg, 0.05 pg, 0.1 pg. 0.2 pg.
or 0.8 pg--Upper panel). TNF-a (1 pg. 2 pg, or 4 pg--Middle panel). or IL-6 (0.11 g . 0 . 3 ~ g, 0.8 pg or 1.6 pg--Lower panel) were administered intrapentonedly ro male CD- 1
mice and consumption of chocolate mik was measured over a 1 hr. penod comrnencing
either 45 minutes (for IL-1 and ILK-6) or 30 minutes (for ThF-a ) following injection.
Baseline values were calculated by averaging consumption over the three da) period
immediatcly pnor to testing. Recoverp data were collected 21 hr. following rcsting.
doses Saline 0.025 ug 0.05 ug
.------.. a........ 0, 1 "g - m-- 0.2 ug - 0.8 ug
- 7 I
BaseIine Test Recovery
TNF doses
Baseline Test Recovery
IL-6 doses ---a--- Saline
0.1 ug . . . 0.2 ug - 0 8 u g - 7 6 u g
I
Baseline Test Recovery
higher doses tested (0.8 pg and 1.6 pg), a modest increase in consumption was detected
relative to baseline levels. Rates of consumption arnong vehicle treated mice did not differ
over the three sampling periods.
Surnrnarizing, the results of Experiments 1 -4 indicate that systernic administration
of IL-1, TNF-a, and LPS (which induces the synthesis and release of IL- 1, IL-6 and
RIT-a) dose dependently suppressed consumption of a palatable substance. In contrast,
IL-6 administration was wirhout effect. Although these findings are consistent with
previous repons of cytokine- and/or LPS-induced anorexia (Van der Meer. Sweep,
Pesman, Born & Hermus, 1995; Plata-Salaman, 1995; Sonti, ilyin B; Plata-Salaman.
1996), the possibility remains that the behavioral effects observed in the present
investigation may not simply reflect an anorexic response. Indeed, it has recendy been
argued that ihe administration of LPS (Yirmiya, 1996) and cytokines (Anisman et al.,
1996) results in behavioral alterations that may reflect anhedonia, and in the case of LPS
the behavionl changes were antagonized by chronic administration of an antidepressant.
Thus, it ought to be considered that the reduction in consumpaon of a palatable diet
following LPS or cytokine administration may k chancreristic of an anhedonic as well as
an anoresic response.
Hormonal Variations
Figures 3 and 4 show the variations of plasma comcosterone levels as a funcnon
of the treatment rnice received.
LPS - Consistent with earlier repons (Mefford et al., 199 1 ; Dunn, 1992a; Johnson et al.,
1992), treatment with LPS was found to dose-dependently increase plasma comcosterone
concentrations (See Figure 3). Analysis of variance revealed that corticosterone levels
varied as a function of the LPS Treatment, F(2, 23) = 47.763, p c.01, and testing.
Newman-Keuls multiple cornparisons indicated that at a i l doses of LPS tested. plasma
co~costerone levels were significan tly increased relative ro saline-treated animals (a =
-05).
Insen Figure 3 about here
------------------------------------
rL- 1 - When administered i.p. one hour prior to testing, the behaviorally active doses of
IL-1 increased plasma corticosterone levels in a dose-dependent manner (See Figure 4-
Upper panel). Analysis of variance revenled a signifimnt effect of Treatment F(3.36.~=10.3.
pc.01 and multiple cornparisons indicated that in mice treated with 0.05 and 0.1 pg of IL- 1
corticosterone levels were significantly increased relative ro saline-treaied animals.
Although nearment with 0.025 pg of IL-1 produced a modrst increase in plasma
conicosterone levels relative ro saline-treated mice. this increasc did nor approach statisticd
sipificsnce.
TXF-a
.As in the case of the IL-1 treatrnrnt. anlilysis of variance indicated thltt TSF-u
administered one hour prior ro tesring sign ificantly incrcaxd plaïniri comcosrerons Isvels
F(3.36)= 16.087. pc.0 1. Multiple compnisons revealed that plasma conicosterone Ievels in
mice treated with 1 pg of TNF-a were significantly incrsased whcn compÿred to mice
rreated with saline. 1. or 2 pg of TNF-a. The lower doses. however. yielded
comcosterone levels that did not differ From those of control miîe (Sre Figure l-AMiddls
panel).
Figure 3. Mean ( + S .E.M.) plasma corticosterone concentrations (pg/dl) among mice
that received peripheral iipopoIysaccharide (LPS) or saline. Various doses of LPS (0.5 pg
or 1 pg) were administered inaaperitoneally to male C D 1 rnice and comcosterone
concentrations were measured 2 hr later (* p 4 1 relative to conrrols 1.
Saline
Figure 4. Mean ( ~ s . E . M . ) plasma conicosterone concentrations (pg/dl) in mice
receiving peripheral interleukin- 1 P (IL- 1 ), tumor necrosis factor-alpha (TNF-a).
interieukin-6 (IL-6) or saline. Various doses of IL- 1 (0.025 pg. 0.05 pg or O. 1 pg-Upper
panel), TNF-a (1 pg. 2 pg or 4 pg-Middle panel), or IL-6 ( 0 . 1 ~ g or 0.8 pg-Lower
panel) were administered intraperitoneall y to male CD- 1 mice and plasma comcosterone
concenuarions were measured 1 hr later (* pc.05 relative to controis 1.
Saline 0.025 ug 0.05 ug 0.1 ug
TNF
U A
In view of the observation that no significant behavioral effects of IL-6 were
detected, only two doses were chosen in order ro assess the effects of the cytokine on HPA
activation. Consistent with earlier reports (Dunn. 19%; Zalcman et al., 1994), the
analysis of variance confmed that there was no difference betwecn plasma comcosterone
levels in mice veated uith O. 1. or 0.8 pg of IL-6 and saline-ueatd mimals (See Figure 1-
Lower panel).
Taken together. it appean thût LPS, IL4 and TKF-a. each of which influenced
consumption. also had the effect of increasing plasma caticosterone concentration S . 1 n
concrast. matment with IL-6. which failed to affect food consumption. likrwise was
without effect on plasma corticosterone concentrations. I t is of considerabIe interest that in
each expsnment. behaviorally active doses of LPS and the cytokines wrre associated with
increases in HPA activity. Morsover. those doses of TSF-a and IL-6 which did not
significantly alter consumption uwe also uithout effsct on plasma cnnicosterone lrvcls. Ir
is conceivablç that rhe correspondence ktween the changes in consumption of a pdarribls
substance and the HPX sffects inducsd by LPS or cytokine adminisrarion rsflects a
similarit) in rhe mrchanism(s) that undsrlie both rhe anoresic and the hormonal responses
to periphsral cytokine cidrninistntion.
Esperiments 5-7
As indicated sarlier. thers is reason ro expsci that the pro-intlammaiory cytokines
may have jyergistic effects with respect to the behavioral and homonal vrinlitions.
Expcriments 3-7 assessed the syergistic actions of these cytok ines. S ub-opiimîl doses ( i n
tems of behavior and endocnne effects) of the cyrokines were chosen to assess the
synergistis effect of cwadminisuation of IL-1 and IL-6. IL-I and TSF-a. and IL-6 and
TKF-a on consumption of a palatable subsrance and plasma conicostzrone levels.
Table 1
Dnig Treaments Administered in Experiments 5-7
Ex~eriment 5 Ex~erirnent 6
Veh + Veh Veh + Veh
IL-1 + Veh L-6 + Veh
Veh + IL-6 Veh + TPLF-a
IL-1 + IL-6 IL-6 + TNF-a
E x m m e n t 7
Veh + Veh
TM-a + Veh
Veh + IL- 1
TNF-a + IL- I
Figure 5. Effect of inmperitoneal administration of 0.025 pg L I + O. 1 pg IL-6
(Upper panel), 0.1 mg IL-6 + 1 pg TNF-a (Middle panel) or 0.025 pg IL- 1 + 1 pg
TNF-a (Lower panel) on mean ( ~ s . E . M . ) consumption (mls) of ü highly palatable food.
Male CD- 1 mice were injected with the cytokine combinations or 0.075 pg IL- 1 + Veh. 0.1
pg IL-6 + Veh, 1 pg TM-a + Veh or Veh + Veh, and consumption of chocolate milk was
measured over a 1 hr. p e n d commencing 1 hr following injection. Baseline values were
calculated by averaging consurnption over the three day period irnmediately prior to resting.
Recovery data were collected 24 hr. following testing.
---a--- Veh + Veh * IL-1 + V e h ......... ~h..-.-. IL-6 + Veh
Baseline Test Recovery
---O--- Veh A Veh - IL-6 + Veh . ..........,... .... TNF + Veh
IL-6 - TNF
Baseline Test Recovery
---O--- Veh - IL-1 .........+........ .,. TNF - 11-1
Veh Veh Veh TNF
Baseline Test Recovery
vehicle mated mimals. In contrast to the IL-1 marnent, the andysis of variance revealed
that neither the main effect of IL-6, nor the interactions involving this variable approached
significance.
IL-6 +TNF- ci
The combination of O. 1 pg IL-6 + 1 pg TM-a did not act synergisrically to reduce
chocolate rnilk consumption (See Figure 5-Middle panel). A repeated masures ANOVA
revealed a significant TSF-a x Sarnpling p e n d interaction F(2,72) = 5.807, pc.01, and
multiple cornparisons indicated that on Test day injection of 1 pg TNF-a + Vehicle
significantly reducai the consumption of chocolate mi.& relative to baseline levels and
vehicle treated mice. Treatment with IL-6 did not influence consumption, and likewise the
interaction benveen T'\T-a and IL-6 was not significant.
IL4 + TYF-a
Figure 5 (Lower panel) shows the consumption of chocolate mik as a function of
the IL- 1 and TSF* treatments. Analaysis of variance of the consumption scores indicated
a significant interaction betwecn IL- 1. TNF-a and Sampling period, F C , Z ) = j . 175,
pc.0 1 . Mulnple cornparisons of the simple effects comprising this interaction confmrd
that arnong vehicle treated animals consumption was stable over sampling periods. As
expected. the low doses of IL-1 and TNF-a did not provoke changes of consumption.
However. the combination of these treatments markedly reduced consumption of chocolate
milk rdanve to baseline scores as well as in comparison with that of animals that received
only one of the ueaments. Regardless of the trament mice received. the consumption
exhibited 23 hr afteward was comparable to the baseline scores.
The results of the present investigation indicated that the co-administration of IL-6
with either IL4 or TNF-a was no more effective in suppressing the consumption of
chocolate rniik than the administration of IL4 or Th'F-a alone. Of course. the possibility
cannot be excluded hat a synergism would have appeared had other doses or àme frarnes
k e n assessed. It is panicularly interesting, however, that the combinaaon of sub-effective
doses of IL-1 and TNF-a markedly suppressed consumption in a synergistic fashion.
These results are consistent with recent repons indicating a synergistic effecr between IL- 1
and TBF-a on feeding in rats (Yang, Koseki, Meguid, Gleason & Debonis, 1991: Van
der Meer et al., 1995). The latter studies suggested that 1 - 1 and TNF-a act
synergistically to provoke anorexia, as reflected by the suppression of feeding in rats given
free access to lab chow.
Hormonal ~ a r i a t i o n s
Figure 6 shows the variations in plasma corticostcronr lewls as a function of the
re;itmttnt miie receivsd.
Insen Figure 6 about hue
------------------------------------
IL- 1 - IL-6
A n of variance of plasma sonicostcrone Icvels fcillowing the s e
~idministriiticin 01 0.025 p g of IL- 1 t O. 1 pg of IL-6 rsvcaled thrit the effects of IL-h did iior
intsrxt wir h thox of IL- 1. In contrasr. ueritmsnt w i t h IL- 1 significrintly incrcrissd
circulrtring mriicostsrone concentrations. Fi 1.36 = 79-82. p c . 0 1 . relarive to control
animlils. Tnrimim u i t h IL-6 was ineffective in affectin- the levsls of conicosterons (sec
Figure 6-Cpper p:ineI)
IL-6 + TSF-a
Trentnisnt wi th TSF-a increased plasma corticosterone levels. Fc 1. 36 > = 19.70. p
<.01. umhersris IL-6 was again found to be wirhout sffsct. As wsll. IL-6 uearment did not
augment the ricrions of TSF-a (See Figure 6-Middle panel). Thus. it sèems that the
Figure 6. Effect of intraperitoneal administration of 0.025 pg IL- 1 + 0.1 pg IL-6
(Upper panel). 0.1 pg IL-6 + 1 pg TNF-a (Middle panel) or 0.025 pg IL- I + 1 pg TNF-
a (Lower panel) on mean ('s.E.M.) plasma conicosterone concentrations (pg/dl). Male
CD4 mice were injected with the cytokine combinations or 0.025 pg IL- 1 + Veh. O. 1 pg
IL-6 + Veh, 1 pg TNF-a , or Veh + Veh, and plasma conicosteronr levels were
measured 1 hr following injection (*synergistic increase relative to conrrol animais pc.05).
Veh IL- t IL-6 IL- 1 + .. - -
Veh Veh Veh IL-6
i'eh IL-6 TNF iL-6 - * - L'en Ver -. -
Ver. l . 4 r
combination of these nvo cytokines was not more effective in provoking an increase in
corticosterone levels than the administration of TNF-a + Vehicle.
IL- 1 + TNF-a
As observed in the behavioral study, the co-administration of 0.025 pg IL- 1 and 1
pg T h l resulted in a synergistic increase in plasma conicosterone levels (See Figure 6-
h w e r panel). Analysis of variance reveded a significant IL-I x TNF-a interaction. Ft 1 .
36)= 4.01. pc.01. The separare adminisuanon of either IL-1 or TSF-a also significantly
increased levels of comcosterone (F(1,36)= 21.126, pc.01 and F( 1.36)=9.598. p<.0 1
repectively) relative to saline-treated animals. however. multiple cornparisons indicated that
when the cytokines were administrred jointly. the levels of conicostcrone exceeded those
seen in mice that received either treatment alone (a=.05).
It appsars that in rnice treated wi th low doses of IL4 and TSF-a. synergistic
effects may be induced wi th respect to consumption of a highl! palatrihlc substance and
circulatiq corricosterone levels. In conrrast. neithsr i h r combinarion of IL- 1 and IL-6. nor
that of IL-6 and T\T-a. was associared ufirh such synsrgisrns. 11 i3 imponani to note rhrit
the altsrations in consumption of a palatable food obsen~ed in the khavioral studirs appear
to correspond ro the hormonal variations. hevious investigators h w c reponed a synsrsism
between IL- 1 and TSF-a on HPA activity (Van der Meer sr al.. 19951. A s u s the case in
the present investigation. the reducrions in food intcike in the Van der .Meer study
corresponded to the increascs in corticosterone Ievels. Whils i t is premrirurs ro conclude
that the behaviorai and homonal variations observed are crtusally linked. it is interesting to
note thnt over the course of the three da' infusion p e n d in the Van der Meer study. borh
feeding and HPA activity retumed to badine levels at the snmè time.
General Discussion-
Sumerous mechanisms have k e n elucidated uhich anempt to describe
development of anoresia. including modifications of neurotrxmittsr levels. altsrations in
hormone and/or peptide levels. and modulation of hypnthalarnic or grisnointestinal
hormone andor peptide levels, and modulation of hypothalarnic or gasmintestinal
functioning. Feeding can be disrupted by satiety s ipals (e.g., leptin, CCK, bombesin)
(Gibbs, 1985; Smith, Jerome & Norgren, 1985; Edwards, Ladenheim & Ritter, 1986;
Corwin, Gibbs & Smith. 1991; Pekyrnounter et al., 1995; Campfield, Smith, Guisez,
Devos & Burn, 1995; Halaas et al., 1995; Sarraf et al., 1997; Plarnadon & Merali, 1997),
but it should not be surpnsing to find that this behavior is also infiuenced by factors
unrelated to satiety per se. For instance, it is well documented that feeding is reduced by
illness (Kent et al., 1996), avenive stimulation (Griffirhs, Shanks, & Anisman, 1992). and
psyhological factors such as srress and depression (Willner, 1985 ). At Ieast some of
these events rnay reflect biologically adaptive responses. For instance, the reduced feeding
assnciated with aversive events may be important so that organisms in stress situations
focus on eliminating the aversive stimuli, rather than becoming involved in food gathering.
Similarly. it has k e n suggested that the anorexia associated with illness is essential to
direct energ). resources to immune functioning, nther than to diven energy to
oasrrointesrinal processes (Han, 1988; Kent et al., 1992b). Thus, it was suggested that C
alterarions in cytokine levels promote various illness behaviors, which may include the
khaviorally adaptive response of diminished feeding (De Kloet. Oitzi 8: Schobitz, 19%:
Kent er al., 1996).
Esperimental parad ips involving both humans and infrahuman species have
demonsuated that acute or chronic administration of cytokines, such as IL-1, IL-6 and
TSF-a can result in anorexia (Plata-Saiaman, Oomura & Kai, 1987: Socher et al., 1988;
Chance & Fischer. 1991; Plata-Salaman & Borkoski, 1993: Myers et al., 1991).
Consistent with these observations. the results of Experiments 1-4 indicated that (a) in the
CD- 1 mouse peripheral administration of LPS, which induces IL- 1, IL-6 and TNF-a
release from macrophages, effectively provoked dose-dependent reductions in the
consumption of a palatable food, and (b) acute i.p. administration of IL-1B or TNF-a
provoked a dose-dependent reduction in the consumption of a palatable food, whereas IL-6
mament was without effect.
The finding that i.p. IL-6 did not suppress feeding is consistent with previous
reports that demon strated that perip heral administration of IL-6 is associated with modes t
reductions in consumption even with doses approaching phamiacological levels (Plata-
Salaman, 1988). Thus, although cenual IL6 matment is reliably associated with anorexia
(Plata-Salaman, 1988; Schobitz et al., 1995), the effect of peripheral administration is
much less pronounced even at high doses (Espat et al., 1996). The lack of effect in the
present investigation may therefore be amiburable to the relatively low doses used.
However, it is also significant that in the present investigation consumption of a highly
paiatable substance presented during the light cycle was assessed, whereas in the Plata-
Salaman study, fiee feeding volume was detemined noctumally. Thus, while Plata-
Salarnan might have been assessing anorexia, consumption in the present investigation mai
have k e n an index of anhedonia. rather rhan anorexia per se. Of course, the procedures
used in the present investigation do not permit dissocianon of the anorexic and the
anhedonic effects of the cytokine treatments and such a conciusion is highly provisional.
It will be recalled that LPS induces the release of severai cytokines, which have the
potential to operate synergistically in determining hormonal and behavioral changes.
Indeed. it was demonsuated that I L 1 and IL-6 had interactive effects in the provocation of
corticosterone (Zhou et al., 1996) and ACTH secretion (Perlstein, Mougey, Jackson Br
Neta. 199 1; Matra et al., 1992). while IL-1 and TNF-a synergistically decreased b l d
glucose levels (Vogel, Henricson 8: Neta, 199 1). and induced anorexia (Yang et al .,
1991). In the present investigation L I and IL-6 were not found to have synergistic actions
with respect to chocolate milk consumption, nor did the combination of TNP-a and IL-6
have effects beond those elicited by TNF-a alone. However, the reduction of
consumption in mice that received the combination of low doses of IL-1 and TNF-a was
appreciabiy greater than the additive effects of the two treatments. In effect, it seemed that
these cytokines acted synergistically in suppressing palarable food consumption. It is of
considerable interest that previous investigations (Yang et al., 1994. Van der Meer et al..
1995) reporthg the synergistic effects of IL-I and TNF-a on food consumption found
that the most significant decline in food intake (Purina rat chow) occmed during
nocturnally associated ingestion intervals. The reduction in food intake diurnaily, although
significant, was less conspicuous. In the present investigation, the palatable diet was
provided during the light-phase, since it was of interest to establish the effects of the
cytokines on consumption of a highly palatable food in sated animals. Under such
conditions IL-I and TNF-a stdl interacted synergistically to produce a profound
suppression of consumption, a finding consistent with the proposition that the matment
lcad to an anorexia independent of effects on satiety per se.
It must be underscored bat in the present investigation a limited number of test
doses were used. and consumption of chocolate rnilk was assessed fiom 1-2 hr following
adminisrration of the cytokines, and plasma comcosterone was assessed 1 hr after
administrarion. It is certainly conceivable that synergistic actions of other cytokine
combinations would have k e n detected at other doses or at other post-adminisuao'on
in te~als . In fact. Zhou et al. (1996) found that a 10 ng inmvenous (iv) dose of bodi IL-1
and IL-6 s ~ c r ~ s t i c a l l y increased ACTH secretion in rats 30 minutes following
administration. followed by a return to baseline levels by 60 minutes. Ceadminismtion of
100 ng of IL- 1 and IL-6 also produced maximal effects 30 minutes after administration.
although at 60 ACTH levels were sri11 significantly elevated. Matra et al. (1992)
have like~eise reponsd that when injected iv, 100 ng of L I and IL-6 produced a maximal
effect o n ACTH levels which occurred soon after administration (20 min). Further to this
point. Pcrlstein et al. (1991) found that when adrninistered i.p., 10 ng of IL-la combined
with 1 p g of IL-6 produced a synergistic increase in the eariy ACTH ïesponse (30-60
minutes later). while at 1-3 hr pst-injection the ACTH response was similar to that elicited
by IL- 1 alone. In effect. it seems that the synergistic actions of the two treatments may not
plasma ACTH level occumd 120 minutes following the coadrninismtion (i.p.) of 1 pg
ThF-a and 10 ng IL- la, whereas the combination of 1 pg TNF-a with 1.35 k g of IL-6
produced a peak synergistic effect on ACTH levels from 30-60 minutes foilowing
administration. In light of these time-dependent synergis tic effects of the cytokines on
ACTH variations. it is premature at this juncture to conclude that cytokine combinations
other han IL-1 and TNF-a do not engender synerpstic actions with respect to either
palatable food intake or hormonal variations.
Hormonal changes associated with the administration of LPS and die cytokines
indicate that. in almost every expenment, elrvations of conicostsrone levels wcurred
contemporaneously with the anorexic and/or anhedonic effects. Indeed. in Expenments 1 -
3, the doses of TSF-a. IL4 and LPS which prrduced significan! reductions in
consumption of a palatable food. were also associatcd u i t h significrinr increase, of
circulxing corticosterone levels. Moreover, those doses of TSF-a anci IL-h which did not
producc sign i ficmt reductions in chcrolate milk consurnption u-cre also wi t hout effecr on
conicosterons l s \ds . Ir might k noted ar this juncture thsr d r h o u g h wiritioni; of HP.-\
activir>, have typically bsen associatsd with a\wsi\.s sL.snr5. ~ippetitive stimuli ma!
influence CRH. ACTH and corticosterone IsveIs N c Intosh. Kent. k: Merali. 1996 t . and
cnnvsrsely alterations of HPA homonai activir) mriy impact on fesding (Srrack.
Sebasrian. Schwanz 6: Dallmcin. 1995). Thus. hm i z reasoii ro aupposs rhat an
association esists, a lki t not necessarily a causal one. k turen the etTscts of the cytokines
on anorexiri rand/or rinhedonia) and on HPA functioning.
Ir ma? be cidded parenthrtically at this juncturc that Gclin. Irloldauer. Iresjo and
Lundholm ( 1993) reponed that in mice uith intact adrenal glands. TSF-a and IL- 1 4 5 0
and 10 ng/d~day. respectively) did not influence food consumption. Howevcr. in
adrenalectoniizrd animals, these rreatrnents reduced consumption significantl y. and a
funher dccline was provoked by the combination of the trerttments. In effect.
adrenslcctom) potentiated the anoresic respon w to the cytokines. Interestingly .
funher decline was provoked by the combination of the treatments. in effect,
adrenalectomy potentiated the anorexic response to the cytokines. Interesùngly,
pretreament with pharmacologicai doses of hydrocortisone (2.5 mg) abolished the anorexic
response to the individual or cornbined doses of IL-I and TNF-a. Taken together, these
data suggest that the anorexic effect of TNF-a and IL- 1 (or their combination) is probably
not a direct result of the corticosterone changes induced by the treatments. Clearly, if
hormonal factors were solely responsible for the anorexia, then adrenalectomy should have
antagonized the effecrs of the cytokines.
As in the behavioral smdy, there was no evidence of an increase in plasma
corticosterone levels following peripheral IL-6 adminisnation. Although previous studies
reponed increases in ACTH levels following central IL-6 marnent (e.g., Matta et al..
1 W6), it appears that peripheral IL-6 aeatment, at least at the doses selected in the present
investigation. does not have a comparable influence on corcicosterone concentranons. It
should be underscored that in rodents, IL-6 does not appear to stimulate HPA activity to the
sarne degree as IL- I or TNF-a (Matra et al., 1992: Dunn, 1992a; Perlstein et al., 1993:
Van Haasteren et al., 1991). Indeed, only moderate increases in ACT'H or comcosterone
levels are seen after administration of large (i.e., 100 ng icv) doses of IL-6. The results of
the present investigation are thus consistent with studies showing that low/moderate doses
of IL-6 are insufficient ?O produce significant changes in levels of corticosterone.
While it does appear that IL-6 cm directly activate the HPA axis, there is some
evidence that it may be more important for sustaining, rather than initiating, KPA activation
as a result of inflammation (Zhou et al., 1996). Moreover, Fatton et al. (1 994) suggested
activation of the KPA axis c m and does occur even in the absence of IL-6. The!;
demonsaated that IL-6 deficient rnice are capable of mounting a normal inflarnmatory
respnnse to systemic tissue danage generated by the i.p. administration of LPS. Both wild-
type and IL-6-deficient mice showed similar induction of comcosterone and rnild anorexia
following LPS, suggesting that the physiological contribution of IL-6 to the KPA response
in the case of systemic inflammation is negligible.
Commensurate witb such findings. it was demonstrated in the present investigation
that the increases of comcosterone following the c~admuiistration of either IL4 and IL-6
or TNF-a and IL-6, were not appreciably greater than those evident following
administration of either IL-1 or T N F u It is possible that a synergistic effect would have
k e n observed following the administration of higher doses of the cytokines, or if
endocrine measures had been taken at a different time following marnent. Nevenheless,
results such as these suggest thst peripheral mamient with a low dose of IL-6 did not
activate unique corticosterone secretagogues in addition to those influenced by the separate
adminisnation of IL- 1 andor TW-a (Matta et al., I992).
The CO-administration of IL-1 and TNF-a, as indicated earlier, produced a
synergistic increase in plasma corticosterone levels measured one hour following treament.
These results were not unexpected, given the dramatic synergisric effects of the two
cytokines on the behavior measured in this, and previous investigations (Long et al., 1992:
Sonti et al., 1996; Bluthe et al., 1993). It wiil be recalld that Van der Meer et al (1995)
reponed that the co-adminismtion of peripheral L-1 and TNF-a acted synergistically in
inducing anorexia, and increasing plasma ACTH and corcicosterone levels in rats. Thus,
the results of the present investigation support the suggestion that low doses of peripheral
cytokines can act synergistically to influence HPA activity and feeding in the mouse. Given
the overlapping biological functions of IL- 1 and TNF-a it is not surprising that these two
cytokines can act in a synergistic fashion, although the reason for the existence of two such
similar, and apparendy redundant, mechanisms is not clear. Perhaps, as suggested by Van
der Meer et al. (19951, the two systems CO-exist in order to increase the host's sensitivity to
subtle inflanmatory stimuli and to maxirnize the ability to respond to such threats in an
appropriate rnanner.
Although the CO-administration of IL4 and TNF-a has been shown to have
synergistic effects on a number of behavioral (e.g., reduction in food intake and mia l
exploration) and metabolic measures (e.g., increased plamsa corticosterone levels,
depression of blood glucose levels). it must be emphasized at this point, that the synergistic
actions of cytokine combinations do not necessarily extend to a l l behaviors or hormonal
actions (Vop l et ai., 1991: Perlstein et ai., 1993; Bluthe et al., 1994; Van der Meer et al.,
1995). It would be premature to conclude. for instance. b a t the lack of a synergistic effect
of the combination of IL- 1 and IL-6 on food consumption or plasma comcosterone levels
would preclude a synergistic effect on social exploration or b l d glucose levels.
Moreover, as indicated earlier, it would be essentiai to evaluate the hormonal and
behavioral actions of the cj~okine combinations across a wide range of pst-administration
in tervals.
The view was offered that cytokine-induced anorexia was a cennally mediated
effect. since icv administration of low doses of cytokines (in the nano,gmn range)
effectively reduced feeding (Plata-Salaman et al., 1987; Chance & Fischer, 1991; Myers et
al.. 1991: Plata-Salaman, Sonti, Borkoski, Wilson & Ffrench-Mullen, 1996). Reductions
in food intake were not seen following periphçral administration of cytokines, unless
rehnvely high doses (in the microgram range) were administered (McLaughlin et al.,
1992,). Funhcr, the ami-pyretic, indomethacin, blocks both the anorexia and fever
provoked by cytokine or LPS neatment, a finding consistent with the position that such
effects are mediated by a common, centrai mechanism (Langhans, Hariacher & Schmer
1989: Crestani, Seguy 8: Dantzer, 1991). Moreover, the anorexic effects of cenûally
administered cytokines could be blocked by pre-treatmenr with the appropnate receptor
antagonists appfied icv, supporting the contenrion that the suppression of feeding was
mediated by direct action in the CNS (Plata-Salaman & Ffrench-Mullen, 1992).
It was recently shown. however, that peripherai mechanisms may also contribute to
cytokine-induced anorexia. In particular, Kent et al. (1996), demonstrated that food-
motivated behavior in rats was reduced to a comparable extent following either i.p. or icv
IL- 1 beamen t Peri p herall y admùiistered IL- 1 required a much larger dose to d u c e food-
motivated beh avior, but decreased the latency to reduced responding relative to that seen
following icv administration. 'Ihus these data provisiond y suggest that peripheral
mechanisms may also be involved. Consistent with this conclusion, it was demonstrated
that the anorexic effects of i.p. L - 1 were blocked by pretreatment with an i.p. (but not an
icv) injection of IL-lra. Kent et al. (1992a) showed that cytokine-induced anorexia could
be dissociated from the febnle response. by using central IL- lm to block the pyrogenic. but
not the anorexic effects of icv IL-1 treament. Moreover, Bret-Dibat, Kent, Couraud,
Creminon & Dantzer (1994) reported that in addition to reducing food-motivated behavior,
peripheral LPS rreatment increased levels of substance P, neurokinin A and calcitonin gene-
relatai product in spinal cord afferents. It would thus appear that both penpheral and
central mechanisms subsente the anorexia induced by cytokines.
It has k e n suggested that peripheral cytokine signalhg to the cenaal nervous
system is mediated by the vagal afferent fibres (Bluthe et al., 1991; Laye et al.. 1995:
Gaykema et al.. 1995; Kent et al., 1995). In particular, subdiaphragmatic vagotomy has
been shown to attenuate or block the behavioral effects of i.p. administration of either LPS
or IL- 1 (Bluthe et al., 1994: Bluthe, Michaud, Keiley Lk Dantzer 1996a: 1996b). Moreover,
such a marnent attenuated the increased plasma ACTH and corticosterone secretion
ordinarily observed following i.p. administration of either IL- 1 or LPS (Gaykema et al..
1995; Fleshner et al., 1995: Kapcala et al., 1996). The alterations in ACTH secretion
following subdiaphragmatic vagotomy appear to be specific to immune-related stressors, as
hypoglycemic stimulation of ACTH secretion was unaffected by sectioning of the vagus
(Kapcala et al., 1996). It is important to note as well, that the effects of vagotomy on
ACTH and comcosrerone levels do not appear to reflect a generalized reducnon in HPA
responsivi ty , but instead are likel y relared specifically to immune activation. In particular,
vagotomy does not affect increases in plasma corticosterone levels produced by elecaic
shock (see discussion in Watkins, Maier & Goehler, 199%). Moreover, while
subdiaphragrnatic vagotomy completely blocks PVN c-fos induction foilowing cenaal or
peripheral LPS administration, it potentiates PVN c-fos induced by footshock in rats (Wan,
Wetmore, Sorensen. Greenberg & Nance, 1994).
The findings of the aforementioned investigations have led to the suggestion that the
influence of subdiaphragmatic vagotomy is Limited to those functions which are mediate.
by the transmission of messages from peripheral cyokines to the brain (Watkins, Maier &
Goehler, 1995b). Indeed, Bluthe et al. (1996a; 1996b) demonsaated that vagotomy
anenuates the behavioral effects of peripheral, but not subcubcutaneous, innavenous, or
central administration of IL- 1. Presumably, sectioning of the vagus prevents the activation
of vagal afferents ordinarily stimulated by peripheral cytokines. Thus. such a matment
would be espec td to preclude the essential factor rhat would othenvise lead to increased
brain cytokine levels that ultimately mediate ilIness responses, including fever and
anorexia. It should be emphasized that while the vapus may be an imponant route for
cytokine-brain communication, such innervation does not preclude the existence of alternate
communication pathways for immune signalling of the CKS.
In addition to a potentid role for immune-brain communication, vagal afferent
fibres have also been implicated in the mediation of feeding. For example. it was reponed
that selscrive sectioning of va@ afferents. but not efferents. biocked the satiating effects of
peripherdly administered CCK (Smith. Jerome, Cushin, Etemo & Simansky, 198 1 ; Smith
et a.. 1985). South and Rirter (19881, and Bluthe et al. (1996a; 1996b) subsequently
demonstrated that direct application of capsaicin to subdiaphrapatic vagal afferents also
attenuated CCK inducrd suppression of food intake. Likewise, capsaicin influenced
variations of feeding ordinarily provoked by bombesin neatment (Kent & Merali. 1996:
SlcIntosh & Merali; 1996). It was funher demonstrated that, in rnice, vagotomy attenuated
the decrease in responding for food reward following peripheral administration of IL-1 or
LPS (Bret-Dibat et al.. 1993; Kent et al., 1996). Such findings are not unexpected in view
of the observation that vagal afEerent fibres project to the paravenaicular nucleus of the
hypothalamus, which appears to be an integml area in the mediation of feeding. It should
also be considered that the activity of glucose sensitive neurons in the lateral hypothalamus
(LH) is suppressed following iontophoretic application of IL-1 and TNF-a, whereas
glucose-sensitive neurons in the venuornedial hypothalamus are excited following
the application of IL-1 (Plata-Salaman et al., 1988; Kuriyama, Hori, Mori & Nakashima,
1990). Alterations in the activity of both the LH and VMH have k e n associated with
changes in the rnicrosmicnire of feeding behavior, including decreases in meal fRquency
and meal size. as well as increases in post-prandial intermeal intervals (Plata-Salaman,
1997). Similar microsmictural alterations in feeding patterns are evident following
subdiphragmatic vagotomy (Davis et al., 199-2; 1995).
In view of the proposed relationship between immune activation of vagal afferents
and feeding, it was hypothesized that destruction of these afferent fibres would attenuate
the reduced consumption of a palatable f ' induced by i.p. LPS marnent. As outlined in
Appendix A, capsaicin administered to neonatal mice su bcutaneously selectively destroy s
approximately 9 5 5 of C fiber afferents (Holzer, 199 1). Accordingly, it was expected that
neonatal capsaicin administration would antagonize the anorexia otheruise induced by LPS
(See Appendix A). The results of this supplemeniary expriment failed, however, to
support this hypothesis. The consumption of chocolate mik by rnice ueated wth capsaicin
did not differ significantiy from that of vehicle treated animals following an i.p. injection of
LPS. In addition, there were no significant differences in corticosterone levels between
either g o u p of mice.
Several possibilities, in addition to non-involvement of vagal afferents in post-LPS
feeding, exist to account for the observed outcorne. The most parsimonious explanation for
the lack of behavioral differentaàon among capsaicin and vehicle treated mice is indequate
vagal intemption. In the present investigation, capsaicin neated neonatal mice exhibited
mild respiratory distress and cutaneous vasodilation for a bnef p e n d immediately
following h g administration. In conrrast, here was no evidence of eidier physical or
respiratory discomfon among vehicle treated mice. Nonetheless, the results of the eye-wipe
test conducted at 30 days of age (data not shown). which is used as an index of afferent
fibre destruction, failed to reveal any ciifferences in the response of either the capsaicin or
vehicle treated anirnals. It is therefore conceivable that the failure to provoke effects on
LPS-induced feeding and corticosterone levels rnay be amibutable to incomplete destruction
of the afferent fibres following capsaicin administration.
An altemarive explanation for the apparent lack of effect among capsaicin mated
rnice is that the reduction in consumption of chocolate mik induced by LPS was rdated to
activanon of central mechanisms, which precluded the appearance of putative peripheral
effects of capsaicin. This hypothesis is not a likely one since the low dose of LPS
employed (0.5 pg) would have primarily influenced peripheral mechanisms. Indeed,
preliminary studies revealed that this dose of LPS elicited only a moderate decline in the
consurnption of chocolate rnilk in othensise unueated mice, accompanied by a modest
increase of corticosterone levels. Funhermore, Dunn (1992b; Dum & Chuluyan, 1994:
Lavicky % Dunn, 1995) demonsrrated rhar when LPS was administered i-p., the minimal
effective dose which increased cerebral catecholarnine metabolisrn was 1 pg. with the peak
effect occumng between 2-8 hours following dmg administration. Thus, it is likely that a
0.5 pg dose of LPS would not be sufficient to mgger cenaally mediated changes in HPA
activity.
The possibility that capsaicin did not effectively disnipt vagal afferents cano t be
readil y dismissed. As indicated earlier, injection of capsaicin resulted in imrnediate
behavioral changes consistent with vagotomy. Nevenheless, it should be ernphasized that.
as adults, these mice did not show a disturbed corneal sensitivity as assessed by the
eyewipe tesr. Interestingly, a very ment study conducted by Bret-Dibat et al. (1997) also
reveded that. in contrast to surgical vagotomy. capsaicin did n a block the decline in food-
morivated behavior ordinarily produced by LPS or IL- 1 treatment. In this study a senes of
7 i.p. capsaicin injections (75 rng/kg), administered on successive days, was used to
destmy C-fiber afferents in adult mice, and the effectiveness of the capsaicin mamient was
verified by alterations in comeal and pain sensitivity, and by attenuation of CCK-induced
satiety. It has likewise been reporred (Tilders et al., 1994) that capsaicin treatment in rats
did not attenuate the ACTH response to a 2.5 mgkg i-p. dose of LPS. Clearly, the findings
of the present investigation concerning the lack of an effect of capsaicin on chocolate rnilk
consumption and plasma corticosterone levels are consistent with both of these reports. It is
possible, as suggested by Bret-Dibat et al. (1997), that the signaihg pathway ùiat
ûansmits information regarding penpheral cytokines to the brain is not exclusively
comprised of capsaicin- sensitive C-afferen ts.
The question remains as to whether the reduction in consumption of a palatable
food, such as the chocolate milk, represents an anorexic effect, or would be more
appropriately considered a reflecrion of anhedonia. It will be recailed that several
differences can be identified between the feeding behavior assessed in this investigation and
feeding measured in other laboratones ( e g , Yang et al., 1994; Van Der Meer et al., 1995:
Plata-Salaman et al., 1996; Sonti et al., 1996; Bret-Dibar et al., 1997). In the present
investigation, mice were given ad libitum access to lab chow. The chocolate milk was
only presented dunng the light-cycle, shortly after the lights were tumed on. Diumal
associated feeding would be expected to be minimal following the relative hyperphagia
which occurs nocturnally (Plata Salaman et al. 1996; Sonti et al., 1996; Yang et al., 1996).
Indeed, mice would not be expected to be 'hungry'. Despite these factors, sated mice
consumed a substanrial amount of chocolate milk during a one hour pend , implying a
strong motivation to consume a palatable substance. Indeed, it has been argued that the
consurnption of palatable diets under some circumstances (e.g., following stressor
exposure) may represent an ided method of assessing anhedonic effects associated with
such treannents (Willner et al., 1987; Papp et al., 1991). While not dismissing the
potenaal effects of cytokines on anorexia, it is possible that the reduction in consumption of
chocolate milk observed in the present investigation following the administration of LPS,
Th- and IL- 1 may be a reflection of a decrease in sensitivity to rewards, similar to that
observed following stressors.
A very recent snidy conducted by S wiergiel, Srnagin & Dunn (1 997), assessed the
effect of infection with influenza virus, LPS and IL- 1 on the ingestive behavior of rnice. In
this study, both intake of food pellets and the consumption of sweetened condensed milk
were measured following acute i.p. injections of LPS or IL-1, or intranasal innoculation
with influenza virus. It was observed that LPS, IL-1 and infection with a lethal dose of the
v i n s were associated uith a reduction in both food pellet intake and consumption of milk.
In contrast, exposure to sublethal doses of influenza virus caused a lesser reduction in food
pellet intake. but was without effect on milk consumption. Thus it would zppear that mice
infested with sublethal doses of influenza virus were more motivated to drink the highly
palamble rnilk than to eat food pellets. B a s d on these results. it might be argued that the
reduction in food pellet intake reflects anorexia, while the reduction in the consumption of
condensed milk reflrcts anhedonia. Indeed. in the same study, pretreatment with
indomerhacin significcintly attenuated the rduction in both food pellet and milk intake
caused by IL- 1 and LPS. Iii conmst, in mice infected with a lethal dose of influenza virus.
chronic indomethacin attenusted the reduction in food pellet intake. but was without effect
on the rsduction of rnilk intake.
Xnhedonic-like behaviors have previously k e n demonsmted following the
adniinistrrttion of cytokines or LPS. Peripheral LPS administration markedly reduced
hedonic behsvior in male rats. as reflected by a decrease in consumption of saccharin
snlurion. and a suppression of sexual behavior (Yirmiya, 1996). Anisman et al. (1996),
showed that, in rats, peripheral IL-2 t r e a m n t dismpted responding for rewarding brain
stimulrition. accompanied by decreased DA release from the nucleus accurnbens. Dopamine
nctiviry in this ai-ea has k e n implicated in motivationai disturbances associated with goal-
dirscted responding to positively reinforcing stimuli. In connast to the effects of IL-2.
however, rats treated with IL-1 showed proficient responding for rewarding brain
s tirnulahon, and normal accumbal DA efflux, although there w as evidence of increased
accurnbd 5-FIT utilization in these mimals. Interestingly, peripherat administration of IL-6
(which had no influence on feeding in the present investigation), also dismpted accurnbal
DA efflux, although it did not affect responding for rewarding brain stimulation (Anisman,
Kokkinidis, Borowski, & Merali, 1997). Together, these data suggest that the effects of
IL-1 are distinct from those of IL-2 and IL-6. Furthemore, the behavioral and
neurochernical profiles associated with IL4 administration are not consistent with
treatments beiieved to have anhedonic action (e-g., stressors, neuroleptics). In effect, when
coupled with the data of the present investigation, it would appear iikely that the actions of
IL-1 would be more comfortably interpreted in terms of the potential anorexic effecrs of
such a treamient. Of course. this does not imply that the effects of TNF-a are independent
of anhedonic effects. Indeed, it is conceivable that creatments such as LPS, which promote
release of IL-1 and TKF-a, suppress feeding owing to both anorexic and anhedonic
effects. It remains to be determined whether sy stemic administration of ThTF-a influences
responding for rewarding stimuli that do not have an appetitive component (e-g., rewarding
brain stimulrtrion).
Finally. it ought to be underscored that the potential hormonal and behavioral
consequences of cytokine administration is only a newly evolving research area, and
understandably . the available data are limited. Comple te dose response Cumes, and time-
response curves, have not been reponed with respect to the behavioral actions of the
cytokines. As a result, data such as those of Anisman et al. (1997) must be considereù as
provisional, at best, pending more detailed elabration of cyokine actions. Inasmuch as the
conclusion of the present investigation is denved, in part, from the Iirnited literarure
available, the notion that the cytokine-induced feeding effects involve anorexic rather than
anhedonic effects, must likewise be consmed as tentative.
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Appendix A
Experiment 8
In order to further investigate the involvement of vagal afferent fibres in the
mediaaon of the behaviord and endocrine effects following LPS-indu& immune
activation, capsaicin was used to eliminate afferent fibres in neonatal CD4 rnice. As
indicated earlier, subdiaphragmatic vagotomy severs both afferent and efferent vagal fibers.
Thus, the effects of this neamnt on LPS-induced immune or endocrine variations may
refelect the actions of the endotoxin on messages to the CNS or, altematively, the effects of
negative fedback mechanisms on endocrine or behwioral processes (Tiiders et al.. 1993).
L.nlike vagotomy, neonatd treatment with capsaicin has k e n s h o w to selectively eliminate
afferent vagal fibers, while sparing efferent fibers (Holzer. 1991). Thus. effects of this
matment on LPS-induced immune, endocrine or behaviod effects cm be ascribec? more
direclly to the interference of messages from the penpheral to central nervous system.
Experimenr 8 was conductrd to assess the effects of capsaicin-elicited vagal disturbances
on the behavioral and hormonal changes exened by LPS ûeatment.
Materials and hiethods
Suhiects
Teonatal male CD-I rnice fiom an in-house breeding colony were used. Mice were
injectrd subcutaneously with capsaicin at 2 days of age and then retumed to their mother.
Follouing weaning (at 21 days) mice were separated into groups of 4 and housed under the
conditions previousl y described. At 60 days of age mice were individually housed for one
week prior to tesring.
Dni ps
Capsaicin was suspended in 108 Tween 80, 90% distilled water and 10% ethanol
i9YZ). Mice were administered an acute 50 mgkg subcutaneous injection of capsaicin or
vehicle (Tween 80, distilled water. 95% ethanol). This procedure, which has been used by
Dr. 2. Merali, has k e n shown to eliminate up to 95% of vagal afferent fibres (Nagy.
Iversen, Goeden, Chapman, & Hunt, 1983).
Method
At 60 days of age mice in the capsaicin and vehicle groups (n=12 per group) were
separately housed for one week pnor to k i n g trained in the chocolate miUr paradigm, and
as in Experiment 1, tested follouing mtment with LPS or saline. On the day of the test.
mice were aven a single intraperitoneai injection of 0.5 pg LPS or saline 90 minutes pnor
to receiving rnilk, and consumption was measured and compared to baseline levels as
previously described. Consumption was measured again 21 hours following the test period
in order to determine rate of recovery. In a parallel study the effects of capsaicin on the
plasma corticosierone changes elicited by the endotoxin were assessed. The dose of LPS,
based on Experiment 1, was one that produced submâuirnal effects on behavior and
hormone factors. thus precluding the possibilit y of a lack of effect owing to a dose that was
too high.
Results and Discussion
Yariations in consumption of a palatable food
Analysis of variance of chocolate milk consumption indicated that rhere was a
significant LPS x Sampling period interaction F(2,88)=11.082, pc.01, as well as a
significant main effect of LPS marnent F(1,41)= 10.9 16, pc.0 1. In contrast, neirher the
main effect nor any of the interactions involving capsaicin treatment reached statistical
significance. As seen in Figure 7, in both vehicle- and capsaicin-ûeated p u p s there was
a significant decline in consumption of chocolate milk, relative to control animals,
following a single i.p. injection of 0.5 pg of LPS 90 minutes pnor to testing. In both
capsaicin- and vehicle-mated groups, the rate of consumption of control mice was similar
across sampling periods. Post-hoc multiple comparisons, in fact, confmed that there was
no difference in the reduccion of rnilk consumption by capsaicin-treated mice that received
Figure 7. Mean (?s.E.M.) consumption (in mls) of a highly palatable food by
capsaicin- and vehicle-neated mice following inwperitoneal administration of
lipopolysaccharide (LPS) or saline. Neenatal CD-1 mice were rreated with subcutaneous
capsaicin or vehicle at 2 days of age. At 60 days of age both groups of mice were injected
with 0.5 pg LPS or saline, and consumption of chocolate milk was measured over a 1 hr.
pend comrnencing 90 minutes following injection. Baseline values were calculated by
averaging consumption over the three day period immediarely prior to testing. Recovery
datawere collected 24 hr. following testing.
I
Baseline 6
Test Recovery
Figure 8. Mean ( ? s.E.M.) plasma romcosterone concentrations (pg/dl) in capsaicin- or
vehicle-treated mice receiving inwperitoneal lipopolysaccharide (LPS) or saline. Seo-natal
male CD-1 mice were ueated with subcutaneous capsaicin or vehicle at 7 days of age. At 60
days of age b t h groups of rnice were injected with 0.5 pg LPS or saline and plasma
cortjcostemne concennations were measured 2 hr later.
Appendix B
ANOVA S ummary Tables for Experimen t 1
Experirnent 1 : Lipopolysaccharide (LPS) Dose Response Curve (chocolate milk consump tion)
Two-Factor repeated Measures AVOVA
Source df Sum of Squares hlean Square F-test pvalue
Dose (A) 3 12.726 4.242 1.37 -2674 Subj. W. Groups 36 1 1 1.446 3.096 Days (B) 2 18.649 9.324 27.155 .O00 1 AB 6 14.07 2,354 6.829 .O001 Enor 72 24.723 -343
LPS Dose Response Curve (plasma conicosrerone concenaanons)
Source df Sum of Squares Mean Square F-test p-value
Dose (A) 2 3 154.662 1577.33 1 47.763 .O00 1 Error 21 693.501 3 3 .O24 To ta1 23 3848.162
Appendix C
ANOVA Surnmary Tables for Experiment 2
Experiment 2: Interleukin- 1 (IL-1) Dose Response Curve (chocolate milk consumption)
Two-factor Repeated Measures ANOVA
Source df Sum of Squares Mean Square F-test
Dose (A) 5 18.73 1 3.746 1.169 Subj. W. Groups 51 173.005 3.204 Days (BI - 7 27.321 13.661 38.495 AB 10 19.286 1.929 5.435 Error 108 38.326 ,355
p-value
IL- 1 Dose Response Curve (plasma corticosterone concentrations)
Source df Sum of Squares Mean Square F-test p-value
Dose (A) 3 1439.669 379.89 10.3 .O00 1 Error 36 1677.333 46.593 Total 39 31 17.002
Appendix D
ANOVA Summary Tables for Experiment 3
Experiment 3: Tumor necrosis factor-alpha (ThTF-a) Dose Response Curve (chocolate mi& consumption)
Two-factor Repeated Measures ANOVA
Source df Sum of Squares Mean Square F-test p-value
Dose (A) 3 3.299 1.1 .42 -74 Subj. W. Groups 36 94.341 2.62 1 Days (B) - 3 1 1.222 5.61 1 19.082 . O00 1 AB 6 10.547 1.758 5.978 .O00 1 Error 72 21.171 2 9 4
TSFa Dose Response Curve (plasma corticosterone concentrations)
Source df Sum of Squares Mean Square F-test
Dose (A) 3 1030.2 15 343 -405 16.087 Error 36 768.48 2 1.237 To ta1 39 1798.695
p-value
. O00 1
Appendix E
ANOVA Summary Tables for Experiment 4
Experiment 1: Interleukin-6 (IL-6) Dose Response Curve (chocolate milk consumprion)
Source df Sum of Squares Mean Square F-test p-value
Dose (A) 4 18.60 1 4.65 1.678 -1717 Subj. W. Groups 45 124.7 1 3 2.77 1 Days (B j 2 2.103 1.05 1 4.098 ,0198 AB 8 2.321 2 9 1.131 .3506 Error 90 23.088 -257
IL-6 Dose Rssponse C w e (plasma conicosterone concenaations)
One- f x tor ASOVA
Source df Sum of Squares Mean Square F-test
Dose i.4) - 3 43.022 23.0 1 1 3 3 5 Error 2 1 864.34 41.164 Total 23 908.367
p-value
S936
ANOVA S ummary Tables for Experiment 5
Experiment 5: Interleukin- 1 (IL- 1) + Interleukin-6 (IL-6) Ceadminismtion (chocolate rnilk consumption)
Three-factor Repeated Measures ANOVA
Source df Sum of Squares Mean Square F-test
S N L - 1 (A) 1 Sd/IL-6 (B) 1 AEl 1 Subj. W. Groups 36 Samplingperiod(C) 2 AC 2 BC 2 mc - 7 C x subj W. Groups 73
p-vaiue
IL- 1 + IL-6 Co-administration (plasma corticosterone concentrations)
Source df Sum of Squares Mean Square F-test p-vaiue
IL-1 (A) IL-6 (B) AB Error
Appendix G
ANOVA Surnrnary Tables for Experirnent 6
Experirnent 6: Interleukin-6 (IL-6) + Tumor necrosis factor-alpha (TNF-a) C e adminismtion (c hocolate milk consumpaon)
Three-factor Repeated Measures ANOVA
Source df Sum of Squares Mean Square F-test
SaML-6 (A) 1 SaVTSF- a (B) 1 AB 1 Subj. W. Groups 36 Sampling period (C) 2 AC - 3
BC - 7 ABC 2 C x suhj W. Groups 72
IL-6 i TSF- a Co-administration (plasma corticosterone concenûations)
T w - factor .ASOVA
Source df Sum of Squares Mean Square F-test
IL-6 (.A i I 604.506 604.506 19.795 TSF- a ( B j 1 15.055 15.055 -493 AB 1 61.951 61.95 1 2.029 Error 36 1099.359 30.538
pvalue
p-value
Appendix H
ANOVA Sumrnary Tables for Experiment 7
Experiment 7: Interleukin- 1 (IL-1) + Tumor necrosis factor-alpha (R'JF- a) Co- adminis~ation (chocolate ndk consumption)
Three- factor Repeated Measures ANOV A
Source df Sum of Squares
Sal/ TNF- a (B) 1 3.37 AB 1 .O3 8 Subj. W. Groups 36 74.231 SampIing penod (C) 2 4.455 AC - 7 6.236 BC 2 12.638 ABC - 7 3.601 C x subj W. Groups 72 25.016
Mean Square F-test
IL- 1 + D7- a Co-administration (plasma corticosterone concenûations)
Two-factor ASOVA
Source d f Sum of Squares Mean Square F-test
IL-1 (A) 1 969.23 969.24 21.126 TW- a (B) 1 330.365 440.365 9.958 AB 1 183.783 183.784 4.006 Error 36 165 1.63 45.879
p-value
p-value
Appendix I
ANOVA Summary Tables for Experiment 8
Experiment 8: Effect of nw-natal capsaicin treaanent on response to Lipopolysaccharide (LPS) administration (chocolate rnilk consumption)
Three-factor Repeated Measures ANOVA
Source df Sum of Squares Mean Square F-test p-value
VeNCap (A) SU LPS (B) AB Subj. W. Groups ~ampling period (C) AC BC ABC C x subj W. Groups
Effect of neo-natal capsaicin treatrnent on response to LPS administration (plasma comcosterone concenuations)
Two- factor AYOVA
Source df Sum of Squares Mean Square F-test p-value
VeNCap (A) 1 17,541 17.541 -4 13 S296 SaVLPS (B) 1 3432.968 3432.986 80.802 .O00 1 AB 1 .O03 .O03 .O0006 .9938 Error 16 679.78 42.486
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