y 197 (2008) 29–36www.elsevier.com/locate/jneuroim

Journal of Neuroimmunolog

Lipopolysaccharide and a social stressor influence behaviour, corticosteroneand cytokine levels: Divergent actions in cyclooxygenase-2 deficient

mice and wild type controls

Shawn Hayley ⁎, Emily Mangano, Michael Strickland, Hymie Anisman

Institute of Neuroscience, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6

Received 1 November 2007; received in revised form 11 March 2008; accepted 20 March 2008

Abstract

Administration of the endotoxin, lipopolysaccharide (LPS) diminished motor activity and increased plasma corticosterone as well as circulatinglevels of interleukin-1β (IL-1β), IL-6, tumor necrosis-factor-α (TNF-α) and IL-10. Among cyclooxygenase-2 (COX-2) knockout mice thebehavioural, corticosterone and cytokine variations promoted by LPS were moderately (home cage activity, corticosterone, TNF-α) or largely (IL-6)reduced. However, if mice were exposed to a psychosocial stressor (social disruption associated with grouping mice with novel cage-mates after aperiod of isolation) coupled with LPS treatment, then the effects of the COX-2 deletion were absent, or there was a synergistic or additive elevationapparent (e.g., in the case of TNF-α, IL-6 and corticosterone). Evidently, COX-2 deletion may have either pro- or anti-inflammatory actions,depending upon the psychosocial context in which immune activation occurs.© 2008 Elsevier B.V. All rights reserved.

Keywords: Cytokine; Interleukin; Stress; Endotoxin; COX-2

1. Introduction

Stressors enhance central monoamine utilization and stimu-late the release of hypothalamic pituitary adrenal (HPA)hormones (Anisman et al., 2001; Dunn et al., 2004; Hayley etal., 2005). These outcomes are not only elicited by neurogenicand psychogenic stressors, but have also been observed inresponse to psychosocial stressors (Dronjak et al., 2004; Gandhiet al., 2007; Isovich et al., 2000; Tidey and Miczek, 1996). If astressor is sufficiently sustained, then neurochemical processesmay become overly taxed (allostatic overload; McEwen, 2001;Schulkin et al., 1994), rendering the organism more vulnerableto pathological outcomes. In this respect, in addition toneurochemical and hormonal variations, chronic stressorexposure may also elicit disturbances of neuroplasticity,including reductions of neurogenesis and impaired neuronal

⁎ Corresponding author. 1125 Colonel By Drive, Ottawa, Ontario, CanadaK1S 5B6. Tel.: +1 613 520 2600x6314; fax: +1 613 520 4052.

E-mail address: shayley@ccs.carleton.ca (S. Hayley).

0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.jneuroim.2008.03.015

dendritic branching (Duman et al., 2001; Sapolsky, 2004; Manjiet al., 2000). Ultimately, disturbances of neurochemical andneuroplastic processes may contribute to the onset and/orprogression of depressive and anxiety disorders, and may evenaffect the course of neurological conditions (Hayley et al., 2005;McEwen and Magarinos, 2001).

Agents that activate the inflammatory immune system, such asthe bacterial endotoxin, lipopolysaccharide (LPS), or the viralanalogue, Poly I:C, have been proposed to act as systemicstressors (Herman and Cullinan, 1997; Hayley and Anisman,2005; Anisman et al., 2007). Moreover, systemic immunestressors may interact with more traditional stressors, therebyaugmenting each other's central actions. For instance, we reportedthat the neurochemical alterations provoked by Poly I:C weresynergistically increasedwhen administered tomice that had beenexposed to a social stressor, in this instance one that involvedreadjustment of group status when isolated mice were reintro-duced to previous cage-mates (Gandhi et al., 2007). Similarly,mice that were subjected to a social stressor and were treated withinterferon-α (IFN-α), exhibited appreciably enhanced plasma

30 S. Hayley et al. / Journal of Neuroimmunology 197 (2008) 29–36

corticosterone, circulating IL-6, TNF-α and IL-10 levels relativeto animals exposed to either the cytokine or the stressor alone(Anisman et al., 2007). Cytokine-stressor synergisms may bedependent upon the nature of the stressor, since rodents thatreceived IL-6 in conjunction with a novelty stressor displayedsynergistically enhanced HPA activation, but no such effect wasobserved when the stressor involved restraint (Zhou et al., 1996).

It is possible that common inflammatory factors activated byboth immune and non-immune stressors may contribute to theirindividual, additive and synergistic effects. In this regard,stressors, such as restraint, as well as the cytokines, includingIL-1β, IL-6 and TNF-α, were shown to provoke expression ofthe inflammatory enzyme, cyclooxygenase-2 (COX-2) (Tanakaet al., 2007; Lacroix and Rivest, 1998; Dhir et al., 2006;Madrigal et al., 2003; Choudhury, 2004). Enhanced COX-2expression following a number of pro-inflammatory challengesresults in the production of, among other things, theprostaglandin, PGE2, which promotes febrile and behaviouralalterations (Hinz, 2000a,b; Harris et al., 2002; Rummel et al.,2006). Indeed, COX-2 inhibition attenuated the febrile andanxiety-like responses, as well as locomotor and memorydisturbances provoked by exposure to a variety of neurogenicstressors (Dhir et al., 2006; Li et al., 2001). Similarly, COX-2inhibition prevented the ACTH and corticosterone elevations,as well as the anorexic effects of LPS (Johnson et al., 2002;Gadek-Michalska and Bugajski, 2004). Although less informa-tion is available concerning the role of COX-2 followingpsychogenic stressors, inhibition of the enzyme using one ofseveral antagonists (naproxen, rofecoxib, valdecoxib) dimin-ished the motor, anxiety and cognitive behavioural changes andaccumulation of oxidative factors with the cortex of rodentsfollowing immobilization (Madrigal et al., 2003; Dhir et al.,2006; Kumari et al., 2007). Yet, the selective COX-2 antagonist,NS-398, did not significantly influence the ACTH andcorticosterone effects of a social crowding stressor (Bugajskiet al., 2003). However, the COX-2 inhibitor, celecoxib,augmented the clinical efficacy of the antidepressant, reboxetine(Muller et al., 2006) and also modulated the behavioural andimmune responses in an olfactory bulbectomy model ofdepression (Myint et al., 2007). In effect, stressors and systemicimmune challenges may promote neuroinflammatory cascadesthrough actions upon COX-2 receptors found on the blood brainbarrier (BBB) or within the brain parenchyma (Rivest et al.,2000; Blais and Rivest, 2001), thus promoting behavioural andneurochemical alterations.

The present investigation sought to assess whether COX-2genetic deficiency attenuated the effects of acute LPS treatmentupon cytokine and corticosterone levels, as well as onbehavioural activity. Moreover, given the more potent effectsof LPS administered on stressor backdrop (Gibb et al., 2008),we also assessed whether the effects of the COX-2 deficiencywould influence the conjoint actions (additive or synergistic) ofLPS and a psychosocial stressor. The psychosocial stressorcomprised subjecting mice to a rapid change in social housingconditions as this appears to have greater ethological validitythan most laboratory stressors (Gandhi et al., 2007; Anisman etal., 2007). The fact that LPS and the psychosocial stressor

synergistically enhanced cytokine levels, as well as behaviouraland neurochemical activity (Hayley et al., 2005; Gibb et al.,2008), supports the notion that inflammatory factors might beimportant for the conjoint actions of these challenges. In effect,we hypothesize that LPS+stressor exposure will augmentcytokine levels leading to activation of COX-2 and subsequentbehavioural and inflammatory effects, as were previouslyshown to be associated with cytokine-induced central COX-2expression (Dunn et al., 2006; Blais et al., 2005).

2. Materials and methods

2.1. Animals

Male C57BL/6 wild type and C57BL/6 mice possessing acyclooxyganase-2 knockout (COX-2 KO) mutation, originallyfrom Jackson Laboratory, were bred within our facility andserved as experimental subjects at 10 weeks of age. Animalswere housed on a twelve hour light–dark cycle (light cycle:7am–7pm) in a climate-controlled environment (23 °C,relative humidity 60%), and were permitted free access towater and mouse chow pellets. All procedures and housingconditions were carried out under protocols approved by theCarleton University Animal Care Committee (CUACC), inconjunction with the Canadian Committee on Animal Care(CCAC).

2.2. Housing manipulations and LPS treatments

At 3 weeks of age, mice were housed 4 per cage. At10 weeks of age, half of the wild type and COX-2 knockoutmice remained group housed in their home cages while theother half were individually transferred to a new home cage andremained single housed for 2 weeks. On the test day (2 weeksfollowing housing manipulations), half of the single housed andhalf of group housed mice received a single injection of LPS(10 μg (mice ranged in weight from 25 to 27 g), i.p.; Serotype026:B6, Sigma-Aldrich, ON) and the remaining animalsreceived vehicle (0.9% saline, i.p.). Immediately after theinjections, the 2 week isolated mice were placed in a new cagefor 1 h with three novel cage-mates. During the 1 h test period,the group housed mice remained undisturbed in their homecages. Thus, mice of both genotypes were subjected to eitherunchanging standard group housing or a shift from grouphousing to 2 weeks of single housing followed by transfer tonovel group housing. Thus, the experiment comprised a 2(genotype)×2 (housing condition)×2 (endotoxin treatment)with n=6–8 for each of the eight treatment groups.

2.3. Behavioural assessment

Immediately following the social disruption, mice wereassessed for locomotor activity using MicroMax® photosensorbeam break cages (MKRO model; Accuscan Instruments,Columbus, OH, USA). All animals' were initially permitted30 min to habituate to the test situation after which home cageactivity levels were recorded for 1 h.

Fig. 1. Home cage activity was assessed over a 1 h period among COX-2knockout mice and their wild type counterparts that received either saline (whitebars) or LPS (10 μg, i.p; black bars) followed by exposure to unchanging or anovel housing condition (psychosocial stressor). Specifically, after receiving thesaline or endotoxin injections, mice that had been singly housed for 2 weekswere transferred to a novel cage with 3 new cage-mates (isolated-group),whereas group housed animals (group; 4 per cage) remained undisturbed in theirhome cages. Thus, the saline or endotoxin treatments were administered in thecontext of standard unchanging or disturbed housing conditions. ⁎ pb0.05,relative to the respective saline treated mice.

31S. Hayley et al. / Journal of Neuroimmunology 197 (2008) 29–36

2.4. Corticosterone determination

Mice were rapidly decapitated following assessment of homecage activity and trunk blood collected and centrifuged at15,000 rpm for 10 min. Supernatants were stored at −80 °C unituntil corticosterone and cytokine analysis commenced. Plasmacorticosterone levels were determined, in duplicate, using acommercially available radioimmunoassay (RIA) kit (ICNBiomedicals, CA). Assays were conducted in a single run,precluding inter-assay variability, and the intra-assay variabilitywas less than 8%.

2.5. Pro- and anti-inflammatory cytokine analysis

A multiplex immunoassay was used for concurrent determi-nations of several cytokines from individual plasma samples.The Luminex 100 is a suspension based bead array system thatuses xMAP technology. Essentially, sets of microspheres(5.6 μm beads) are internally dyed with different ratios offluorophores, with each conjugated to a different capture probe(cytokine specific antibody). Following incubation, a classifica-tion laser identifies the particular cytokine bound and a secondreporter laser quantifies the signal present.

A Beadlyte Mouse Multi-Cytokine Detection System 2 kit(Upstate, Cell Signalling, Lake Placid, NY, Cat # 48-004) wasused in conjunction with the Luminex 100 system for cytokinedeterminations. A mouse diluent kit (Cat # 43-007) was used todilute plasma supernatants and a serial dilution series performedto cover a range of standards (from 0–5000 pg/ml). Then, 25 µlof Beadlyte Cytokine Assay Buffer was placed in each well anda gentle vacuum applied, followed by addition of 25 µl ofplasma diluent and 25 µl of plasma to each well. The filter platewas then incubated on a shaker for 20 min and vortexed. Tofinalize the initial reaction, 25 µl of bead solution was added toeach well and the plate covered and vortexed. Plates were thenplaced on a shaker and incubated overnight in darkness at 4 °C.After incubation, plasma samples were washed twice using75 µl of Beadlyte Cytokine Assay Buffer and 25 µl of Biotinconjugated Beadlyte Anti-Mouse Cytokine was added to eachwell and then incubated for 1.5 h at room temperature.Thereafter, 25 µl of diluted Beadlyte streptavidin-PE wasadded to each well, covered, vortexed, and incubated for 30 minon a plate shaker. Finally, 25 µl of Beadlyte stop solution wasadded, samples were then resuspended in 125 µl of sheath fluid,and read in the Luminex 100 instrument.

2.6. Statistical analysis

Data for cytokine, corticosterone and behavioural changeswere analyzed by 2 (genotype)×2 (housing condition)×2(endotoxin treatment) between groups analyses of variance(ANOVA). In some cases samples were lost due to error orextreme variability (N5 standard deviation from both the groupand overall mean), hence the degrees of freedom varied slightlyacross the dependent measures. In all instances follow-up t testswere conducted using Bonferonni corrections to control forfamily wise error at α=.05.

3. Results

3.1. Behavioural measures

As shown in Fig. 1, total home cage activity levels varied as afunction of the Housing condition×LPS treatment,F(1,39)=9.26,pb0.001. The follow-up tests confirmed that LPS reduced motoractivity, as did the social stressor (psb0.05) and together thesetreatments additively reduced home cage locomotion.Although the effects of LPS were more pronounced in thenon-stressed mice, this was clearly due to a floor effect in thestressed animals precluding a still greater reduction of activityfollowing the LPS administration. The interaction between theLPS treatment, Stressor condition, and Genotype was notsignificant. However, as seen in Fig. 1, in the absence of astressor (i.e., in the continuously group housed mice) theeffects of the LPS treatment were greater in the wild type mice(activity scores being reduced by 71%), whereas in the COX-2knockouts the LPS only elicited a 32% reduction of activity,relative to their saline injected counterparts. These findings arein keeping with the a priori hypothesis that the effects of LPSon basal motor activity would be attenuated in the knockoutmice, yet, the social stressor appeared to affect behaviourindependent of COX-2.

3.2. Corticosterone variations

As shown in Fig. 2, levels of plasma corticosterone variedsignificantly as a function of the housing stressor×geneticbackground interaction F (1,43)=7.84, pb0.01, such that COX-2 deficient but not wild type mice displayed elevated levels ofthe hormone in response to the social stressor. Corticosterone

Fig. 3. Circulating levels of IL-1β (mean±SEM) were determined, using amultiplex assay, in COX-2 knockout and wild type mice exposed to theendotoxin and psychosocial stressor treatments. Group housed mice remained intheir home cages after receiving the saline (white bars) or LPS (black bars)treatments, whereas animals that were previously isolated were transferred to anovel cage with new cage-mates (isolated-group) immediately after theinjections. ⁎ pb0.05, relative to the respective saline treated mice, ⇟ pb0.05relative to saline treated groups housed mice.

Fig. 2. Mean±SEM plasma corticosterone are depicted for COX-2 deficientknockout mice and their wild type counterparts following administration ofsaline or LPS (10 μg, ip). Mice received the LPS treatment in the context of thestandard unchanging grouped housing (group) or immediately prior to beingintroduced to a novel cage with 3 new cage-mates following 2 weeks of isolatedhousing (isolated-group). ⁎ pb0.05, relative to saline treated group housed mice⇟ pb0.05, relative to group housed wild type mice that received LPS, as well asCOX-2 knockouts that received both the LPS and stressor treatments.

32 S. Hayley et al. / Journal of Neuroimmunology 197 (2008) 29–36

levels were also markedly increased by LPS administration,irrespective of housing condition and genotype F(1,43)=60.56,pb0.01. However, among the group housed mice, the COX-2knockout was associated with a blunted response to LPS, suchthat corticosterone levels were reduced in these mice (by ~34%)compared to wild type mice that received the endotoxin undergrouped housing conditions (pb0.05, Fig. 2). Although thethree way interaction between the stressor, LPS treatment andgenetic difference was not significant, it was clear that thecombined LPS and stressor treatments additively increasedcorticosterone levels only in the COX-2 knockout mice(pb0.05; Fig. 2). Thus, it appeared that although COX-2deletion diminished the corticoid response to LPS underunchanging grouped housing, it enhanced HPA responding tothe social stressor alone, as well as its additive effects whenadministered with the endotoxin.

3.3. Cytokine concentrations

The LPS treatment increased circulating IL-1β levels(F1,39=12.34, pb0.001), and concentration of the cytokinealso varied as a function of the Genotype×Social housinginteraction (F1,39=4.51, pb0.05, Fig. 3). In this regard, thesocial stressor (i.e., transferring mice from isolated to grouphousing) increased the IL-1β levels in the COX-2 knockoutsbut had no such effect in wild type animals. Although theinteraction with LPS was not significant, follow-up tests wereconducted based on a priori predictions regarding the effects ofthe endotoxin. These comparisons confirmed that among thewild type mice, transfer from isolated to grouped housing didnot significantly enhance the effects of LPS relative to that seenin the grouped condition, but levels of the cytokine now did

exceed that of saline treated animals. Among the knockoutmice, the levels of IL-1β were essentially below the detectionlevel in the absence of LPS. The LPS treatment increased thelevel of IL-1β in the knockouts, although the level was stilllower than in their wild type counterparts. When the knockoutswere exposed to the social stressor, however, the levels of IL-1βrose so that the circulating cytokine concentration wasequivalent to that of the wild type mice. This was the case inthe saline and the LPS-treated mice (Fig. 3). In effect, it appearsthat although the COX-2 mice exhibited a diminished IL-1βresponse to LPS, the influence of the genotype was eliminatedamong animals exposed to both the psychosocial stressor andthe LPS treatment.

The levels of circulating IL-6 and TNF-α varied as a functionof the Genotype×Social housing×LPS treatment interaction(F1,39=4.96, 4.10, psb0.005). The follow-up comparisonsrevealed that LPS increased IL-6 and TNF-α levels in the wildtype mice, but this effect was not further modified by the socialstressor (Fig. 4). Although the levels of IL-6 and TNF-α wereelevated in the grouped housed COX-2 knockout miceadministered LPS (pb0.05), this effect was diminished relativeto that evident in the similarly treated wild type mice However,COX-2 knockout mice that received the LPS in the context ofthe social housing stressor displayed markedly higher levels ofthe cytokines. Indeed, circulating IL-6 levels following LPSamong the stressed knockouts were as great as in the wild typemice, and in the case of TNF-α, the effects of the LPS amongthe knockouts exceeded that seen in the wild type mice(pb0.05). Thus, COX-2 deletion appeared to antagonize theindependent actions of LPS, but engendered a synergistic

Fig. 5. Circulating levels (mean±SEM) of IL-10 were determined in COX-2knockout and wild type mice exposed to the endotoxin and psychosocial stressortreatments. Group housed mice remained in their home cages after receiving thesaline (white bars) or LPS (black bars) treatments, whereas animals that werepreviously isolated were transferred to a novel cage with new cage-mates(isolated-group) immediately after the injections. ⁎ pb0.05, relative to therespective saline treated mice.

Fig. 4. Circulating levels (mean±SEM) of IL-6 (left) and TNF-α (right) were determined in COX-2 knockout and wild type mice exposed to the endotoxin andpsychosocial stressor treatments. Group housed mice remained in their home cages after receiving the saline (white bars) or LPS (black bars) treatments, whereasanimals that were previously isolated were transferred to a novel cage with new cage-mates (isolated-group) immediately after the injections. ⁎ pb0.05, relative to therespective saline treated mice, ⁎ pb0.05, relative to wild type mice that received LPS treatment in the context of the isolated-group housing manipulation.

33S. Hayley et al. / Journal of Neuroimmunology 197 (2008) 29–36

elevation of circulating IL-6 and TNF-α when the endotoxinwas administered in the context of the social stressor.

In contrast to the aforementioned pro-inflammatory cyto-kines, less robust alterations were apparent for the anti-inflammatory cytokine, IL-10, with the only significant effectbeing that LPS treatment increased levels of this cytokine(F1,37=4.88, pb0.05). As shown in Fig. 5, this was the caseirrespective of genotype or stressor administration.

4. Discussion

Consistent with previous reports (Hayley et al., 2004; Huanget al., 2007; Konsman et al., 2000), LPS provoked a markedreduction of home cage locomotor activity and increased plasmacorticosterone levels. As well, circulating levels of the pro-inflammatory cytokines, IL-1β, IL-6, TNF-α as well as the anti-inflammatory cytokine, IL-10, were elevated by LPS challenge.As reported following pharmacological inhibition of COX-2(Cernak et al., 2002; Harris et al., 2002; Deak et al., 2005),among the COX-2 knockout mice housed under standardgrouped conditions, the effects of LPS on motor activity,corticosterone, IL-1β and TNF-α levels were moderatelyattenuated, and the elevation of IL-6 was largely diminished.Interestingly, however, the LPS injected COX-2 mice that werealso exposed to the stressful housing manipulation displayed themost marked cytokine and corticosterone elevations, such thatthe LPS and social stressor treatments synergistically enhancedIL-6 and TNF-α levels and acted to additively elevate plasmacorticosterone. These data raise the possibility that endogenousCOX-2 may have contributed, at least partially, to thebehavioural, corticoid and pro-inflammatory cytokine variationsprovoked by LPS alone. However, in the context of the socialstressor, the role of the COX-2 may have switched to being aninhibitory one acting to restrain the combined impact of the LPS-stressor challenge. One technical consideration that deservesattention is the fact that blood was only obtained at a single time

point (2.5 h) following LPS treatment, and it is uncertainwhether a different profile of corticosterone and cytokinechanges might have occurred at earlier times. In this regard, ithas been reported that elevated corticosterone is apparent within30–45 min following LPS, whereas the cytokine changesgenerally appear some time later, with TNF-α typicallyoccurring first (30–90 min), followed by IL-6 (60–120 min)and IL-1β (120 min) (Barber et al., 1993; Chen et al., 2000;Mekaouche et al., 1996).

As already alluded to, the altered motor activity observedfollowing LPS administration alone in wild type mice was morelimited in the COX-2 knockouts, supporting the view that thisinflammatory enzyme contributes to the behavioural effects of the

34 S. Hayley et al. / Journal of Neuroimmunology 197 (2008) 29–36

endotoxin (Jain et al., 2002; Satyanarayana et al., 2004).However, caution should be exercised in the interpretation ofthe behavioural findings since only a single test of home cageactivity was performed and ideally assessment of open-field,sucrose consumption and other tests would provide a morecomplete behavioural description. For instance, the actionsassociated with LPS and the COX-2 deletion may be related tomalaise (sickness behaviours) ordinarily associated with theendotoxin (Dantzer et al., 1999; Hayley et al., 2001). Alter-natively, given that COX-2 contributes to toxin-induced neuro-degeneration of the basal ganglia, and hence may be involved inthe subsequently observed motor impairments (de Meira et al.,2006; Teismann et al., 2003), it is also possible that COX-2deletion may have served as a buffer against motoric changeselicited by LPS, through mechanisms unrelated to malaise, suchas neuronal alterations involving the basal ganglia.

It has been reported that COX-2 may have a stimulatoryeffect upon corticoid activity, such that COX-2 antagonistslimited the plasma corticosterone elevations associated withLPS or cytokine administration (Aguilera, 1998; Bale and Vale,2004; Zhang and Rivest, 2000; Cover et al., 2001; Gadek-Michalska and Bugajski, 2004; Zhang and Rivest, 2000).Similarly, COX-2 deletion in the present investigation had amodest (34%), albeit significant, attenuating effect on corticos-terone levels among group housed mice that received LPStreatment. However, in the present study, we found that anabsence of COX-2 enhanced the corticosterone variationsprovoked by the psychosocial stressor administered alone, aswell as its additive effects with LPS. In contrast, others reportedthat COX-2 inhibition had no influence on the corticosteronerise elicited by CRH when administered either alone or in thecontext of a crowding social stressor (Bugajski et al., 2003).However, this previous study involved acute administration of apharmacological COX-2 antagonist (NS-398), whereas lack ofthe enzyme from birth might be expected to have moreprotracted effects, as well as possible compensatory changes inalternate inflammatory factors (as discussed in an ensuingsection). Moreover, in contrast to the stimulatory effect thatCOX-2 often has upon corticosterone release, the enzymenormally has inhibitory actions with respect to severalinflammatory factors, including the chemokine, monocytechemoattractant protein (MCP-1), as well as the innate immunereceptors, CD14 and Toll-like receptor-2 (TLR2) expression(Blais et al., 2005). As well, given that administration of thenon-selective COX antagonist, ketorolac, augmented the abilityof IL-1β or TNF-α to provoke central MCP-1 expression (Blaiset al., 2005), raises the possibility that enhanced expression ofchemokines, such as MCP-1, might have contributed tocorticoid variations associated with the psychosocial stressor.

It had previously been reported that if mice were exposed to apsychosocial stressor and then treated with Poly I:C, IFN-α orLPS, the behavioural, corticosterone and pro-inflammatoryresponse to LPS was markedly enhanced (Anisman et al., 2007;Gandhi et al., 2007; Gibb et al., 2008). In the presentinvestigation, which involved a slightly different procedure, thiscorticosterone synergy was not apparent in wild type mice andonly an additive effect was apparent in COX-2 knockouts.

Specifically, in this instance mice were exposed to thepsychosocial stressor and received the LPS treatment at thesame time. Thus, it may be that the synergy between thetreatments is most notable when the stressor preceded the LPStreatment (e.g., 1 h as in our previous studies), and hencecorticosterone release elicited by the stressor would already havebeen instigated at the time of LPS treatment. In effect, theinteraction between the two treatments might either reflect therelative influence of LPS given the temporal dynamics regardingcorticosterone release, or it may be that time-dependent variationsof other factors were responsible for the synergy. Alternatively, itshould be noted that C57BL/6 mice were used in the presentinvestigation (given that this is the genetic background of COX-2knockouts), whereas the CD1 strain was used in the previousstudies that revealed synergistic corticoid variations among micethat received the psychosocial stressor and immune treatments(Anisman et al., 2007; Gandhi et al., 2007). Hence, straindifferences, especially since C57BL/6 was reported to be a morestressor hardy mouse strain (Hayley et al., 2001), may account forthe differing results between these studies.

Despite the fact that COX-2 deletion antagonized the actionsof LPS alone on circulating cytokines, when the endotoxin wasadministered in the context of a psychosocial stressor, the levelsof the pro-inflammatory cytokines were markedly elevated.Indeed, in the case of IL-6 and TNF-α, the levels of thesecytokines in the knockouts exceeded that evident in wild typemice that had received both the stressor and the LPS treatments.In effect, it appears that COX-2 deletion promotes anti-inflammatory actions following LPS challenge; however, lackof the enzyme may have pro-inflammatory consequences whenthe endotoxin is administered in the context of a psychosocialstressor. Essentially, we posit that COX-2 deficiency mightengender a compensatory up-regulation of certain endogenousinflammatory factors that might be preferentially affected bystressor treatment. In this regard, COX-2 deletion was reportedto enhance levels of several inflammatory mediators, includingleukotrienes, and although lack of the enzyme blunted theinflammatory response to several viral and bacterial infections(e.g. influenza, LPS), the response to other challenges, includingHelicobacter pylori and allergen sensitization was actuallyenhanced in COX-2 deficient mice (Carey et al., 2005; Nakataet al., 2005; Li et al., 2006). As well, a compensatory elevationof COX-1 activity also occurred in COX-2 knockout mice(Bosetti et al., 2004). Alternatively, other processes unrelated toinflammation might have been altered in the COX-2 knockoutsand contribute to the observed stressor-LPS synergy. Indeed,previous reports indicated that COX-2 inhibition synergisticallyenhanced the impact of other challenges, including the analgesicactions of morphine and the apoptotic effects of IFN-α (Pinardiet al., 2005; Nakamoto et al., 2006).

In summary, although COX-2 inhibition diminished thebehavioural and pro-inflammatory consequences of LPS, whenCOX-2 deficient mice received the endotoxin in the context of apsychosocial stressor, a synergistic elevation of pro-inflamma-tory cytokine levels was provoked. Given that the depressive-like consequences of immunotherapy or exposure to infectiouspathogens may stem from synergistic interactions between these

35S. Hayley et al. / Journal of Neuroimmunology 197 (2008) 29–36

immune insults and concomitant stressors being experienced(Anisman et al., 2005), these data may be pertinent to thedevelopment of strategies to minimize the adverse effects of thecombined treatments.

Acknowledgements

S.H. and H.A. are Canada Research Chairs in Neuroscience.This work was supported by funds awarded to S.H. from theNatural Sciences and Engineering Research Council (NSERC),as well as a New Investigator Award from NARSAD. Thetechnical assistance of Geoffrey Crowe and Ming Yang isgreatly appreciated.

References

Aguilera, G., 1998. Corticotropin releasing hormone, receptor regulation and thestress response. TEM 9, 329–336.

Anisman, H., Hayley, S., Kelly, O., Borowski, T.B., Merali, Z., 2001.Psychogenic, neurogenic and systemic stressor effects on plasma corticos-terone and behavior: mouse strain-dependent outcomes. Behav. Neurosci.115, 443–454.

Anisman, H., Merali, Z., Poulter, M.O., Hayley, S., 2005. Cytokines as aprecipitant of depressive illness: animal and human studies. Curr. Pharm.Des. 11, 963–972.

Anisman, H., Poulter, M.O., Gandhi, R., Merali, Z., Hayley, S., 2007.Interferon-α effects are exaggerated when administered on a psychosocialstressor backdrop: cytokine, corticosterone and brain monoamine variations.J. Neuroimmunol. 186, 45–53.

Bale, T., Vale, W., 2004. CRF and CRF receptors: Role in stress responsivity andother behaviours. Annu. Rev. Pharmacol. Toxicol. 44, 525–557.

Barber, A.E., Coyle, S.M., Marano, M.A., Fischer, E., Calvano, S.E., Fong, Y.,Moldawer, L.L., Lowry, S.F., 1993. Glucocorticoid therapy alters hormonaland cytokine responses to endotoxin in man. J. Immunol. 150, 1999–2006.

Blais, V., Rivest, S., 2001. Inhibitory action of nitric oxide on circulating tumornecrosis factor-induced NF-kappaB activity and COX-2 transcription in theendothelium of the brain capillaries. J. Neuropathol. Exp. Neurol. 60, 893–905.

Blais, V., Turrin, N.P., Rivest, S., 2005. Cycloxygenase 2 (COX-2) inhibitionincreases the inflammatory response in the brain during systemic immunestimuli. J. Neurochem. 95, 1563–1574.

Bosetti, F., Langenbach, R., Weerasinghe, G.R., 2004. Prostaglandin E2 andmicrosomal prostaglandin E synthase-2 expression are decreased in thecyclooxygenase-2-deficient mouse brain despite compensatory induction ofcyclooxygenase-1 and Ca2+-dependent phospholipase A2. J. Neurochem.91, 1389–1397.

Bugajski, J., Gadek-Michalska, A., Bugajski, A.J., 2003. Effect of cyclooxygenaseinhibitors on the CRH-induced pituitary-adrenocortical activity duringcrowding stress. J. Physiol. Pharmacol. 54, 99–108.

Carey, M.A., Bradbury, J.A., Seubert, J.M., Langenbach, R., Zeldin, D.C.,Germolec, D.R., 2005. Contrasting effects of cyclooxygenase-1 (COX-1)and COX-2 deficiency on the host response to influenza A viral infection. J.Immunol. 175, 6878–6884.

Cernak, I., O'Connor, C., Vink, R., 2002. Inhibition of cyclooxygenase 2 bynimesulide improves cognitive outcome more than motor outcome followingdiffuse traumatic brain injury in rats. Exp. Brain Res. 147, 193–199.

Chen, G., McCuskey, R.S., Reichlin, S., 2000. Blood interleukin-6 and tumornecrosis factor-alpha elevation after intracerebroventricular injection ofEscherichia coli endotoxin in the rat is determined by two opposing factors:peripheral induction by LPS transferred from brain to blood and inhibition ofperipheral response by a brain-mediated mechanism. Neuroimmunomodula-tion 8, 59–69.

Choudhury, G., 2004. Linear signal transduction pathway involving phospha-tidylinositol 3-kinase, protein kinase Cepsilon and MAPK in mesangial cellsregulates IFN-γ-induced STAT1α transcriptional activation. J. Biol. Chem.279, 27399–27409.

Cover, P.O., Slater, D., Buckingham, J.C., 2001. Expression of cyclooxygenaseenzymes in rat hypothalamo-pituitary-adrenal axis: effects of endotoxin andglucocorticoids. Endocrinology 16, 123–131.

Dantzer, R., Gheusi, G., Johnson, R.W., Kelley, K.W., 1999. Centraladministration of insulin-like growth factor-1 inhibits lipopolysaccharide-induced sickness behavior in mice. NeuroReport 10, 289–292.

de Meira Santos, Lima M., Braga Reksidler, A., Marques Zanata, S., Machado,Bueno H., Tufik, S., Vital, M.A., 2006. Different Parkinsonism modelsproduce a time-dependent induction of COX-2 in the substantia nigra of rats.Brain Res. 1101, 117–125.

Deak, T., Bellamy, C., Bordner, K., 2005. Protracted increases in core bodytemperature and IL-1 following acute administration of LPS: implicationsfor the stress response. Physiol. Behav. 85, 296–307.

Dhir, A., Padi, S.S., Naidu, P.S., Kulkarni, S.K., 2006. Protective effect ofnaproxen (non-selective COX-inhibitor) or rofecoxib (selective COX-2inhibitor) on immobilization stress-induced behavioral and biochemicalalterations in mice. Eur. J. Pharmacol. 535, 192–198.

Dronjak, S., Gavrilović, L., Filipović, D., Radojcić, M.B., 2004. Immobilizationand cold stress affect sympatho-adrenomedullary system and pituitary-adrenocortical axis of rats exposed to long-term isolation and crowding.Physiol. Behav. 81, 409–415.

Duman, R.S., Nakagawa, S., Malberg, J., 2001. Regulation of adult neurogenesisby antidepressant treatment. Neuropsychopharmacology 25, 836–844.

Dunn, A.J., Swiergiel, A.H., Palamarchouk, V., 2004. Brain circuits involved incorticotropin-releasing factor-norepinephrine interactions during stress.Ann. N.Y. Acad. Sci. 1018, 25–34.

Dunn, A.J., Swiergiel, A.H., Zhang, H., Quan, N., 2006. Reduced ingestion ofsweetened milk induced by interleukin-1 and lipopolysaccharide isassociated with induction of cyclooxygenase-2 in brain endothelia.Neuroimmunomodulation 13, 96–104.

Gadek-Michalska, A., Bugajski, J., 2004. Role of prostaglandins and nitricoxide in the lipopolysaccharide-induced ACTH and corticosterone response.J. Physiol. Pharmacol. 55, 663–675.

Gandhi, R., Hayley, S., Gibb, J., Merali, Z., Anisman, H., 2007. Influence ofpoly I:C on sickness behaviors, plasma cytokines, corticosterone and centralmonoamine activity: moderation by social stressors. Brain Behav. Immun.21, 477–489.

Gibb, J., Hayley, S., Gandhi, R., Poulter, M.O., Anisman, H., 2008. Synergisticand additive actions of a psychosocial stressor and endotoxin challenge:circulating and brain cytokines, plasma corticosterone and behavioralchanges in mice. Brain Behav. Immun. 22, 573–589.

Harris, S.G., Padilla, J., Koumas, L., Ray, D., Phipps, R.P., 2002. Prostaglandinsas modulators of immunity. Trends. Immunol. 23, 144–150.

Hayley, S., Anisman, H., 2005. Multiple mechanisms of cytokine action inneurodegenerative and psychiatric states: neurochemical and molecularsubstrates. Curr. Pharm. Des. 11, 947–962.

Hayley, S., Merali, Z., Anisman, H., 2001. Central monoamine activity ingenetically distinct strains of mice following a psychogenic stressor: effectsof predator exposure. Brain. Res. 892, 293–300.

Hayley, S., Crocker, S.J., Smith, P., Shree, T., Jackson-Lewis, V., Przedborski,S., Mount, M., Slack, R., Anisman, H., Park, D.S., 2004. Mice lacking thetumor necrosis factor-α or Fas receptors are resistant to MPTP induced deathof dopaminergic neurons. J. Neurosci. 24, 2045–2053.

Hayley, S., Poulter, M.O., Merali, Z., Anisman, H., 2005. The pathogenesis ofclinical depression: stressor- and cytokine-induced alterations of neuroplas-ticity. Neuroscience. 135, 659–678.

Herman, J.P., Cullinan, W.E., 1997. Neurocircuitry of stress: central control ofhypothalamo-pituitary-adrenocortical axis. Trends. Neurosci. 20, 78–84.

Hinz, B., 2000a. Prostaglandin E(2) upregulates cyclooxygenase-2 expression inlipopolysaccharide-stimulated RAW 264.7 macrophages. Biochem. Bio-phys. Res. Commun. 272, 744–748.

Hinz, B., 2000b. Salicyclate metabolites inhibit cyclooxygenase-2-dependentprostaglandin E(2) synthesis in murine macrophages. Biochem. Biophys.Res. Commun. 274, 197–202.

Huang, Y., Henry, C.J., Dantzer, R., Johnson, R.W., Godbout, J.P., 2007.Exaggerated sickness behavior and brain proinflammatory cytokine expres-sion in aged mice in response to intracerebroventricular lipopolysaccharide.Neurobiol. Aging May 30; [Electronic publication ahead of print].

36 S. Hayley et al. / Journal of Neuroimmunology 197 (2008) 29–36

Isovich, E., Mijnster, M.J., Flügge, G., Fuchs, E., 2000. Chronic psychosocialstress reduces the density of dopamine transporters. Eur. J. Neurosci. 12,1071–1078.

Jain, N.K., Patil, C.S., Kulkarni, S.K., Singh, A., 2002. Modulatory role ofcyclooxygenase inhibitors in aging- and scopolamine or lipopolysaccharide-induced cognitive dysfunction in mice. Behav. Brain Res. 133, 369–376.

Johnson, P.M., Vogt, S.K., Burney, M.W., Muglia, L.J., 2002. COX-2 inhibitionattenuates anorexia during systemic inflammation without impairingcytokine production. Am. J. Physiol. Endocrinol. Metab. 282, E650–E656.

Konsman, J.P., Luheshi, G.N., Bluthé, R.M., Dantzer, R., 2000. The vagus nervemediates behavioural depression, but not fever, in response to peripheral immunesignals; a functional anatomical analysis. Eur. J. Neurosci. 12, 4434–4446.

Kumari, B., Kumar, A., Dhir, A., 2007. Protective effect of non-selective andselective COX-2-inhibitors in acute immobilization stress-induced beha-vioral and biochemical alterations. Pharmacol. Rep. 59, 699–707.

Lacroix, S., Rivest, S., 1998. Effect of acute systemic inflammatory responseand cytokines on the transcription of the genes encoding cyclooxygenaseenzymes (COX-1 and COX-2) in the rat brain. J. Neurochem. 70, 452–466.

Li, S., Ballou, L.R., Morham, S.G., Blatteis, C.M., 2001. Cyclooxygenase-2mediates the febrile response of mice to interleukin-1beta. Brain Res. 910,163–173.

Li, G.Q., Xia, H.H., Chen, M.H., Gu, Q., Wang, J.D., Peng, J.Z., Chan, A.O.,Cho, C.H., So, H.L., Lam, S.K., Hu, P.J., Liang, Y.J., Lin, H.L., Berg, D.E.,Feng, Z.H., Langenbach, R., Wong, B.C., 2006. Effects of cyclooxygenase-1 and -2 gene disruption on Helicobacter pylori-induced gastric inflamma-tion. J. Infect. Dis. 193, 1037–1046.

Madrigal, J.L., Moro, M.A., Lizasoain, I., Lorenzo, P., Fernández, A.P.,Rodrigo, J., Boscá, L., Leza, J.C., 2003. Induction of cyclooxygenase-2accounts for restraint stress-induced oxidative status in rat brain.Neuropsychopharmacology 28, 1579–1588.

Manji, H.K., Moore, G.J., Chen, G., 2000. Clinical and preclinical evidence forthe neurotrophic effects of mood stabilizers: implications for thepathophysiology and treatment of manic-depressive illness. Biol. Psychiatry.48, 740–754.

McEwen, B.S., 2001. Plasticity of the hippocampus: adaptation to chronic stressand allostatic load. Ann. N.Y. Acad. Sci. 933, 265–277.

McEwen, B.S., Magarinos, A.M., 2001. Stress and hippocampal plasticity:implications for the pathophysiology of affective disorders. Hum.Psychopharmacol. 16, S7–S19.

Mekaouche, M., Siaud, P., Givalois, L., Barbanel, G., Malaval, F., Maurel, D.,Assenmacher, I., Ixart, G., 1996. Different responses of plasma ACTH andcorticosterone and of plasma interleukin-1 beta to single and recurrentendotoxin challenges. J. Leukoc. Biol. 59, 341–346.

Müller, N., Schwarz, M.J., Dehning, S., Douhe, A., Cerovecki, A., Goldstein-Müller, B., Spellmann, I., Hetzel, G., Maino, K., Kleindienst, N., Möller, H.J., Arolt, V., Riedel, M., 2006. The cyclooxygenase-2 inhibitor celecoxib hastherapeutic effects in major depression: results of a double-blind,randomized, placebo controlled, add-on pilot study to reboxetine. Mol.Psychiatry 11, 680–684.

Myint, A.M., Steinbusch, H.W., Goeghegan, L., Luchtman, D., Kim, Y.K.,Leonard, B.E., 2007. Effect of the COX-2 inhibitor celecoxib on behaviouraland immune changes in an olfactory bulbectomised rat model of depression.Neuroimmunomodulation 14, 65–71.

Nakamoto, N., Higuchi, H., Kanamori, H., Kurita, S., Tada, S., Takaishi, H.,Toda, K., Yamada, T., Kumagai, N., Saito, H., Hibi, T., 2006.Cyclooxygenase-2 inhibitor and interferon-beta synergistically induceapoptosis in human hepatoma cells in vitro and in vivo. Int. J. Oncol. 29,625–635.

Nakata, J., Kondo, M., Tamaoki, J., Takemiya, T., Nohara, M., Yamagata, K.,Nagai, A., 2005. Augmentation of allergic inflammation in the airways ofcyclooxygenase-2-deficient mice. Respirology 10, 149–156.

Pinardi, G., Prieto, J.C., Miranda, H.F., 2005. Analgesic synergism betweenintrathecal morphine and cyclooxygenase-2 inhibitors in mice. Pharmacol.Biochem. Behav. 82, 120–124.

Rivest, S., Lacroix, S., Vallieres, L., Nadeau, S., Zhang, J., Laflamme, N., 2000.How the blood talks to the brain parenchyma and the paraventricular nucleusof the hypothalamus during systemic inflammatory and infectious stimuli.Proc. Soc. Exp. Biol. Med. 223, 22–38.

Rummel, C., Sachot, C., Poole, S., Luheshi, G.N., 2006. Circulating interleukin-6 induces fever through a STAT3-linked activation of COX-2 in the brain.Am. J. Physiol. 291, R1316–R1326.

Sapolsky, R.M., 2004. Is impaired neurogenesis relevant to the affectivesymptoms of depression? Biol. Psychiatry 56, 137–139.

Satyanarayana, P.S., Jain, N.K., Singh, S., Kulkarni, S.K., 2004. Effect ofselective inhibition of cyclooxygenase-2 on lipopolysaccharide-inducedhyperalgesia. Inflammopharmacology 12, 57–68.

Schulkin, J., McEwen, B.S., Gold, P.W., 1994. Allostasis, amygdala, andanticipatory angst. Neurosci. Biobehav. Rev. 18, 385–396.

Tanaka, A., Hatazawa, R., Takahira, Y., Izumi, N., Filaretova, L., Takeuchi, K.,2007. Preconditioning stress prevents cold restraint stress-induced gastriclesions in rats: roles of COX-1, COX-2, and PLA2. Dig. Dis. Sci. 52,478–487.

Teismann, P., Tieu, K., Choi, D.K., Wu, D.C., Naini, A., Hunot, S., Vila, M.,Jackson-Lewis, V., Przedborski, S., 2003. Cyclooxygenase-2 is instrumentalin Parkinson's disease neurodegeneration. Proc. Natl. Acad. Sci. 100,5473–5478.

Tidey, J.W., Miczek, K.A., 1996. Social defeat stress selectively altersmesocorticolimbic dopamine release: an in vivo microdialysis study. BrainRes. 721, 140–149.

Zhang, J., Rivest, S., 2000. A functional analysis of EP4 receptor-expressingneurons in mediating the action of prostaglandin E2 within specific nuclei ofthe brain in response to circulating interleukin-1beta. J. Neurochem. 74,2134–2145.

Zhou, D., Shanks, N., Riechman, S.E., Liang, R., Kusnecov, A.W., Rabin, B.S.,1996. Interleukin 6 modulates interleukin-1- and stress-induced activation ofthe hypothalamic-pituitary-adrenal axis in male rats. Neuroendocrinology63, 227–236.