BRAIN,BEHAVIOR,

Brain, Behavior, and Immunity 18 (2004) 505–514

and IMMUNITY

www.elsevier.com/locate/ybrbi

Individual differences in behavior of inbred Lewis rats areassociated with severity of joint destruction in

adjuvant-induced arthritis

Enik€o Sajti,a,b Nico van Meeteren,b,c Annemieke Kavelaars,a Janjaap van der Net,d

Willem Hendrik Gispen,b and Cobi Heijnena,*

a Laboratory for Psychoneuroimmunology, University Medical Center Utrecht, The Netherlandsb Rudolf Magnus Institute for Neurosciences, University Medical Center Utrecht, The Netherlands

c Department of Physiotherapy, Academy of Health Sciences Utrecht, The Netherlandsd Department of Pediatric Physiotherapy, University Children Hospital, �Het Wilhelmina Kinderziekenhuis�,

University Medical Center Utrecht, The Netherlands

Received 22 October 2003; received in revised form 26 November 2003; accepted 1 December 2003

Available online 28 January 2004

Abstract

The aim of our study was to test the hypothesis that differences in behavioral characteristics are linked to severity of arthritis in

association with neuro-endocrine and immune reactivity in an inbred strain of rats. Lewis rats were selected as high-active (HA) and

low-active (LA) animals based on their exploratory activity in the open field. Subsequently, adjuvant-arthritis (AA) was induced in

both groups. We observed no differences in the severity of inflammation as determined by paw swelling and redness. However, LA

and HA animals differed in the severity of bone destruction as determined on radiographs taken on day 30 after induction of AA.

LA rats had more osteoporosis, periostal new bone formation, and bone destruction than HA rats. There were no differences

between HA and LA rats in corticosterone response after acute or chronic immune challenge. Splenocytes of LA rats had a lower

mitogen-induced IL-10 and IFNc production during AA. Histological examination revealed more intense factor VIII staining in

arthritic joints of LA animals, indicating more pronounced synovial angiogenesis. In addition, LA rats had higher plasma VEGF, an

important angiogenic factor. Expression of RANKL, a crucial factor promoting bone resorption, was also higher in joints of LA

animals. Our data demonstrate that activity in the open field, a behavioral trait, is associated with the severity of bone destruction in

AA. Lower production of bone-protective cytokines and a higher rate of angiogenesis leading to more synovial proliferation may be

responsible for the more severe joint destruction in LA animals.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Joint destruction; Behavior; Lewis rat; Cytokines; HPA-axis; Open field

1. Introduction

Rheumatoid arthritis (RA) is an autoimmune dis-

order characterized by synovial proliferation and in-

flammation, and subsequent destruction and deformity

of joints. Skeletal complications start with focal erosion

of cartilage followed by marginal and subchondral

bone loss. Extended joint destruction with ankylosis

* Corresponding author. Fax: +31-30-2505311.

E-mail address: c.heijnen@azu.nl (C. Heijnen).

0889-1591/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbi.2003.12.004

and generalized bone loss are characteristic for late

complications (Feldmann et al., 1996). These long-termskeletal complications have serious consequences as

they can lead not only to painful joint deformities but

also to progressive functional disability and increased

mortality rates (Pincus and Callahan, 1993). Joint de-

struction is characterized by pathological bone re-

sorption due to a disturbed balance in bone

remodeling. Osteoclasts, the bone-resorbing cells, take

the forehand on bone forming osteoblasts (Goldringand Gravallese, 2000). Moreover, the excessive ex-

pression of receptor activator of nuclear factor jB li-

506 E. Sajti et al. / Brain, Behavior, and Immunity 18 (2004) 505–514

gand (RANKL) by inflammatory T-cells and synovialfibroblasts has a critical function in this process.

RANKL interacts with its receptor on mononuclear

phagocyte precursors and directs their differentiation

towards osteoclasts (Kong et al., 1999a,b).

Not only RANKL, but also cytokines can modulate

osteoclastogenesis. IFNc is a cytokine well known as a

protector against bone destruction suppressing potently

osteoclast development (Takayanagi et al., 2002a,b).The synovium depends on blood supply in order to

proliferate and to invade the cartilage–bone junction.

Therefore angiogenesis, the formation of new blood

vessels, is considered to be a crucial step in pannus

formation and the subsequent destruction of cartilage

and bone. Vascular endothelial cell growth factor

(VEGF) is one of the most important angiogenic fac-

tors. It has the ability to trigger the entire sequence ofevents leading to new vessel formation (Folkman, 1995;

Nagashima et al., 1995). VEGF can be produced in the

inflamed joint by several cell types: activated synovial

monocytes/macrophages and fibroblasts as well as by

synoviocytes. It is detectable in both synovial fluid and

plasma of arthritis patients and is correlated with the

course of the disease (Harada et al., 1998; Kikuchi et al.,

1998).The occurrence and severity of late skeletal compli-

cations varies markedly among patients. We wanted to

investigate whether individual behavioral characteristics

can predict disease susceptibility or outcome. There are

a few reports available which describe a relation between

differences in behavior and the severity of inflammatory

autoimmune diseases. We have demonstrated earlier

that the behavioral reactivity of wild-type rats measuredin the resident-intruder test as the attack latency time

(ALT), was associated with EAE severity (Kavelaars

et al., 1999). Passive animals selected as rats with max-

imal ALT had less severe EAE symptoms compared to

their aggressive counterparts with a short ALT. A study

by Chover-Gonzalez investigating the severity of AA in

out-bred Wistar rats selected on the basis of their be-

havior in the learned helplessness paradigm demon-strated that helpless rats showed significantly less paw

inflammation compared to their not helpless counter-

parts (Chover-Gonzalez et al., 2000). However, these

studies used either wild-type or outbred rats, therefore a

possible selection for genetic differences in inflammatory

properties can not be ruled out.

The purpose of the present study was to examine

whether pre-existing individual differences in behaviorof Lewis rats are associated with the severity of in-

flammation and bone destruction in AA. Moreover, we

investigated whether HA and LA rats differ with re-

spect to HPA-axis reactivity, cytokine production,

plasma level of the angiogenic factor VEGF as well as

the extent of angiogenesis and synovial proliferation in

the joint.

2. Materials and methods

The Institutional Animal Use and Care Committee of

the University Medical Center Utrecht approved all

procedures in this study involving animals.

2.1. Animals

Inbred female Lewis rats were obtained from theUniversity of Limburg (Maastricht, The Netherlands).

The animals were three to four weeks old at the time of

arrival. The rats were housed individually in type 3

Macrolon cages in a temperature (21 �C) and humidity-

controlled room where a 12 h light/dark cycle (lights on

7 AM) was maintained. Food and water was available

ad libitum.

2.2. Experimental design

Three experiments with identical behavioral selection-

procedures were executed. In these experiments rats were

pre-selected upon their behavior in the open field and

assigned to two extreme groups, HA and LA animals,

respectively. Two of the experiments were performed for

the exploration of differences in the development of ar-thritis and its long term consequences between HA and

LA rats. The third experiment was designed in order to

characterize the cytokine production patterns and HPA-

axis reactivity of healthy HA and LA rats.

2.3. Open field test

Locomotor activity of 5–6-week-old rats was assessedin an open field as described earlier (Stam et al., 1997;

van Meeteren et al., 1997) during the first half of the

light phase. Animals were handled for four consecutive

days before the start of the experiments. The open field

consisted of a black round polyester arena with a di-

ameter of 130 cm and a 50-cm high border equally illu-

minated by a white TL tube. Prior to the start of the test,

rats were handled four times by the researcher. Theanimals were tested individually on four consecutive

days during the light period of the diurnal cycle with the

radio on for constant background noise. They were

gently put in the middle of the open field and allowed to

explore the arena for 4min. Ambulation was automat-

ically registered with a computer system (Noldus In-

formation Systems, Wageningen, The Netherlands).

After the test, data were analyzed by Etho Visiontracking software calculating covered distances for each

day. As a selection-criterion the sum of the distances

covered on day two, three, and four was chosen con-

forming to Denenberg (1969). Rats with scores in the

lower quartile were termed LA, that is they exhibited

low general activity. In contrast, animals with scores in

the upper quartile were selected as HA animals.

E. Sajti et al. / Brain, Behavior, and Immunity 18 (2004) 505–514 507

2.4. Induction and clinical evaluation of the arthritis

model

Four days after open field selection HA and LA

rats were pre-immunized according to the protocol of

Zhang et al. (1999) with the following modifications:

rats were pre-immunized with 100 ll (instead of 50 ll)of squalane (Sigma–Aldrich, Steinheim, Germany)

intradermally into the base of the tail instead of thefootpad.

After two weeks, arthritis was induced by an intra-

dermal injection of 100 ll of Freund�s complete adjuvant

containing 5mg of heat-killed Mycobacterium tubercu-

losis (strain H37Ra; Difco, Detroit, MI) in 1ml Freund�sincomplete adjuvant (Difco) in the base of the tail

(Pearson, 1956). Rats were observed and examined daily

in a blinded fashion for clinical signs of arthritis usingstandard methods. Each paw was scored from 0 to 4 for

the degree of erythema, swelling, and joint deformity.

The highest achievable score is 16 per animal (Trentham

et al., 1977).

All time points were considered in relation to the AA

induction day, designated as day 1. Arthritis in one of

the joints was developed in 100% of the treated animals

by day 11.

2.5. Radiological studies

Onday 30 after immunization with Freund�s Complete

Adjuvant (CFA), rats were sacrificed by decapitation,

and the hind paws were examined radiographically. Joint

radiographs were taken at 10mA, 50 kV, and 0,25 s

of exposure to Kodak Insight occlusal film. The pawswere radiographed at a 25 cm focus to film distance. Ra-

diographs were evaluated in a blinded manner, using the

grading scale described by Ackerman et al. (1979).

2.6. Histology

Whole ankle joints of the hind paws were fixed in

formaline for at least 2 days before decalcification withEDTA for a period of three weeks. After decalcification,

the joints were imbedded in paraffin, serially sectioned

onto microscope slides, and then deparaffinized. Tissue

sections (7 lm) were stained with haematoxylin–eosine.

Immunohistochemical staining was performed for

RANKL and factor VIII. For RANKL, tissue sections

were treated with 3% H2O2 for 20min at room tem-

perature. Sections were incubated for 20min with10mM citrate (pH 6.0) at 100 �C and incubated again

with the primary antibody directed against RANKL

(rabbit polyclonal antibody raised against the epitope

corresponding to amino acids 46–317 of RANKL of

human origin [FL-317], Santa Cruz Biotechnology,

Santa Cruz, CA). Rabbit IgG antibody (Dako X0936;

Dako, Carpinteria, CA) was used as an isotype control.

After rinsing, sections were incubated for 30min withbiotinylated goat anti-rabbit horseradish peroxidase

(Dako), developed with 3; 30-diaminobenzidine (Sigma,

St. Louis, MO) and counterstained with haematoxylin.

For vascular endothelial specific staining, sections were

stained with anti-human factor VIII antibody.

2.7. HPA axis activation

Nine-week-old HA and LA rats were i.p. injected at 9

AM with 1 lg/kg, 2.5 lg/ml LPS (E. coli, Sigma, St.

Louis, MO). The control groups were injected with

equal volumes of saline. Rats were decapitated 120min

after the injection.

2.8. Corticosterone assay

After decapitation trunk blood was collected in

EDTA-containing tubes on ice, immediately centrifuged

(3000 rpm, 10min, 4 �C) and stored ()20 �C) until assay.Plasma corticosterone levels were determined by radio-

immuno assay as described previously (Nijsen et al.,

2000). The sensitivity was 0.4 ng/ml; the intra- and in-

terassay variations were 4 and 7%, respectively. Plasma

corticosterone concentrations are expressed as lg/dl.

2.9. Peritoneal macrophages isolation and stimulation

After decapitation, 20ml of ice-cold RPMI-1640

(Gibco, Grand Island, NY) was injected into the peri-

toneum, and peritoneal macrophages were harvested

after 5min of gentle massage of the abdomen. Cells were

cultured in 24-well plates (5� 105 cell/well) in the pres-ence of varying concentrations of LPS. Culture super-

natants were collected after 24 h of incubation at 37 �Cand stored at )80 �C until assay.

2.10. Splenocyte stimulation

After decapitation, spleens were removed, mashed

gently through filter chambers (NPBI, Emmer-Compa-scum, The Netherlands). Cells were resuspended in

culture medium (RPMI-1640,) supplemented with 5%

fetal calf serum (Gibco, Grand Island, NY), 2mM LL-

glutamine, 100U/ml penicillin, 100 lg/ml streptomycin,

100 lg/ml gentamycin, and 50 lM 2-mercaptoethanol.

Splenocytes were cultured in quadruplicate in 200 llround-bottom microtiter wells (Costar, Cambridge,

MA) at 105 cells/well in the presence of 1 lg/ml Conca-navalin A (Calbiochem, La Jolla, CA) and different

concentrations of dexamethasone. Cultures were incu-

bated for 96 h at 37 �C in a humidified atmosphere, 5%

CO2. For the final 18 h cultures were pulsed with 1 lCi/well [3H]thymidine (Amersham, Bucks, UK). Thymidine

uptake was measured using a liquid scintillation bcounter.

508 E. Sajti et al. / Brain, Behavior, and Immunity 18 (2004) 505–514

2.11. TNFa, IL-10, and IFNc production

Supernatants of Concanavalin A stimulated spleno-

cytes were collected after 48 h of culture. IL-10 and

IFNc content was determined by ELISA (U-Cytech,

Utrecht, The Netherlands) according to the manufac-

turer�s procedure.Supernatants of LPS stimulated peritoneal macro-

phages were collected after 24 h of culture. IL-10 andTNFa was determined by ELISA (U-Cytech, Utrecht,

The Netherlands) according to the manufacturer�sprocedure.

2.12. Statistics

Clinical course of AA, cytokine production by peri-

toneal macrophages and dexamethasone inhibition ofsplenocyte proliferation were evaluated with GLM re-

peated measures. Differences in X-ray scores, plasma

VEGF, and splenocyte cytokines were tested with Stu-

dent�s T test.

Plasma corticosterone levels after LPS injection were

analyzed with two-way ANOVA. Differences in plasma

corticosterone levels in arthritic rats were analyzed with

Student�s T test.p < :05 was considered statistically significant. Sta-

tistical analysis was performed using SPSS, version 10.

3. Results

3.1. Selection of HA and LA animals

At six weeks of age rats were subjected to an open

field test to study general locomotor activity as de-

scribed in Section 2. The total distance covered by each

rat was calculated for each test day. As a selection cri-

terion we have chosen for the sum of the distances

Fig. 1. Selection of HA and LA rats. HA rats with scores in the upper

quartile and LA rats with scores in the lower quartile. The Y-axis rep-

resents the sum of the distances covered on test day two, three, and four.

covered on day two, three, and four. The activity scoresfrom test day two onward are stable for each individual

animal (Denenberg, 1969). Rats with scores in the upper

quartile were assigned to the HA group whereas rats

with scores in the lower quartile to the LA group. Fig. 1

shows the scores of a representative selection procedure.

The 30 highest and lowest active animals of the tested

120 rats were used for further experiments. The rest of

the animals, the middle group, were used in other ex-periments.

3.2. Clinical course of adjuvant arthritis in HA and LA

rats

As shown in Fig. 2A we did not observe any differ-

ence in the severity of AA, as assessed by the clinical

score based on inflammatory signs like joint swelling,redness, and limitation in joint motion. In particular,

there was no difference in kinetics or intensity in onset,

peak or the late phase of the disease. Furthermore, the

groups had a similar weight loss during the entire course

of arthritis (Fig. 2B).

Fig. 2. (A) Clinical course of AA. After preimmunization with squa-

lane, AA was induced by intradermal injection of Complete Freund�sAdjuvant (CFA) containing 5mg/ml Mycobacterium tuberculosis into

the base of the tail. Rats were examined daily in a blinded protocol for

clinical signs of disease. HA rats (m), LA rats (j). (B) Mean weight

gain as a percentage of the body weight on day 11. HA rats (m), LA

rats (j). Data represent means and SEM. N ¼ 16 rats per group.

GLM, repeated measures, p ¼ ns.

E. Sajti et al. / Brain, Behavior, and Immunity 18 (2004) 505–514 509

3.3. Long-term bone destruction in HA and LA rats

Despite a similar clinical course of arthritis, LA rats

suffered from more pronounced bone destruction than

HA animals as seen on radiographs taken on day 30 of

the disease when almost no active inflammatory signs

were visible anymore. The differences in the total X-ray

scores, assessing osteoporosis, periostal new bone for-

mation, erosions, and swelling are depicted in Fig. 3A.LA animals had a more pronounced decrease in bone-

density, destruction of bony structures, and ossifications

not contiguous with the normal bone-line. The differ-

ences in bone destruction between HA and LA animals

are also evident on the two radiographs representing the

ankle of a rat with a representative score from each

group (Figs. 3C and D). The presence of marked bone

resorption in combination with adjacent new bone for-mation suggested strongly the presence of a disordered

pattern of bone remodeling in the LA animals.

3.4. Histology

In haematoxylin–eosin stained sections of ankle joints

of LA rats the normal tissue architecture was completely

lost. A dramatic joint-space loss was visible with thepresence of a massive proliferative and erosive pannus.

The cartilage lining the bones was severely eroded. In

contrast, sections of ankle joints of HA animals revealed

significantly less severe lesions that were mainly re-

stricted to the lateral parts of the joints. The integrity of

the cartilage was preserved in the HA animals, especially

in the centers of the joint surfaces. Furthermore, the HA

rats exhibited markedly less cortical and trabecular boneloss (Fig. 4A vs. D).

3.5. RANKL and vascularization of the arthritic joint

RANKL has a critical role in bone remodeling pro-

moting bone resorption. Specific immuno-histochemical

Fig. 3. Bone destruction in the ankles of HA and LA rats. Radiographs taken

described by Ackerman et al. assessing osteoporosis, periostal new bone form

N ¼ 16 rats per group. tð24Þ ¼ 3:2; p < :01, **p < :01. Radiographs of the h

were taken on day 30 after induction of AA.

staining for RANKL showed an abundant presence ofRANKL in ankle joints of LA animals in contrast to the

joints of HA animals (Fig. 4B vs. E).

Angiogenesis is known to play an important role in

the proliferation of the bone-destroying pannus. To in-

vestigate the level of vascularization in the joint, we

stained slides for the endothelial marker factor VIII. As

is shown in Fig. 4F, a more intensive staining for factor

VIII was observed in the ankles of LA rats, suggesting amore pronounced vascularization of the invasive pannus

compared to the ankles of HA rats (Fig. 4C).

One of the crucial angiogenic factors produced in

arthritis is VEGF. We measured VEGF in the plasma of

HA and LA animals on day 30 after induction of AA. In

Fig. 5 we demonstrate that HA animals (with a better

bone integrity) had significantly lower VEGF levels as

compared to the LA rats.

3.6. Cytokine profiles of HA and LA rats

To investigate whether cytokine patterns were differ-

ent in HA and LA animals we measured the capacity of

splenocytes to produce IL-10 and IFNc and the capacity

of peritoneal macrophages to produce TNF-a and IL-

10. At baseline conditions there was no difference inmitogen-induced splenocyte in vitro IL-10 production

between HA and LA animals. However, after AA, IL-10

production by splenocytes from HA rats was signifi-

cantly higher than from LA rats (Fig. 6A). The capacity

to produce IFNc was higher in HA animals both before

the onset and after AA (Fig. 6B).

To investigate whether peritoneal macrophages of the

two groups of animals differed in their capacity to pro-duce cytokines, peritoneal macrophages of HA and LA

animals were in vitro stimulated with different concen-

trations of LPS. In Figs. 7A and B it is shown that LPS

induces TNF-a and IL-10 production dose dependently,

but no differences between the HA and LA animals were

observed.

on day 30 after induction of AA were evaluated using the grading scale

ation, erosions and swelling (A). Data represent the mean and SEM.

indlimb of a control rat (B), HA rat (C), and LA rat (D). Radiographs

Fig. 4. Paraffin imbedded sections of ankle joints of HA and LA rats on day 30 after induction of AA. (A) and (D) Haematoxylin–eosin staining

(magnification 5�). Note the complete joint-space loss with the presence of a massive proliferative and erosive pannus in the LA animals (B). In HA

animals lesions were restricted to the lateral parts of the joints. The integrity of the cartilage was preserved, especially in the centers of the joint

surfaces (A). (B) and (E) Immunohistochemistry for RANKL (magnification 20�) LA animals (E) show high expression of RANKL in the invasive

pannus (arrow heads). In the HA (B) animals RANKL expression in the cartilage (arrow heads). (C) and (F) immunohistochemistry for factor VIII

(magnification 20�). A more intensive staining in LA animals (F), indicating the presence of more blood vessels (arrow heads) in the pannus as

compared to the HA rats (C).

510 E. Sajti et al. / Brain, Behavior, and Immunity 18 (2004) 505–514

3.7. Plasma corticosterone levels in HA and LA rats

The HPA-axis is known to play an important role

in interfering with immune processes by secreting

corticosterone. We tested the reactivity of the HPA-

axis in HA and LA animals upon an acute immune

challenge as well as after the induction of the chronic

inflammatory stress of AA. As an acute immune

Fig. 5. Plasma VEGF measured on day 30 after the induction of AA.

Data represent means and SEM. N ¼ 6 rats per group. tð10Þ ¼ 2:3;

p < :05, *p < :05.

stimulus we used a low dose of LPS. Healthy animals

were injected i.p. with 1 lg/kg LPS and decapitatedafter 120min, at the peak of the corticosterone re-

sponse. As shown in Fig. 8, LPS induced an increase

in plasma corticosterone concentration in both groups

compared to the saline injections (two-way ANOVA,

significant treatment effect, p < :001) but there were no

significant differences in the magnitude of the response

between the HA and LA rats (two-way ANOVA, in-

teraction effect not significant). Moreover, the plasmalevels of corticosterone of HA and LA animals 30 days

after induction of the disease were similar as well

(Fig. 8).

3.8. Glucocorticoid receptor sensitivity in HA and LA rats

To test if an altered communication between corti-

costerone and immune cells could contribute to theobserved differences in bone destruction in HA and LA

animals, we investigated the glucocorticoid receptor

sensitivity of splenocytes by co-incubating the cells with

different concentrations of dexamethasone in a mitogen-

induced proliferation assay. Dexamethasone is known

to inhibit the proliferation of cells dose-dependently.

However, no differences were found in the slopes of the

dexamethasone inhibition curves of proliferating cells ofthe two groups of rats (Fig. 9).

Fig. 7. Cytokine production by peritoneal macrophages of HA rats (m)

and LA rats (j). Isolated peritoneal macrophages were stimulated

with different concentrations of LPS in vitro. After 24 h of culture Il-10

(A) and TNFa (B) were measured in the supernatants by ELISA.

GLM, repeated measures, p ¼ ns.

Fig. 6. (A) IL-10 production of Concanavalin A stimulated splenocytes

before the induction of AA and on day 30 of AA. tð8Þ ¼ 4:8; p < :01.

(B) IFNc production of Concanavalin A stimulated splenocytes before

the induction of AA and on day 30 after induction of AA.

tð20Þ ¼ 2:2; p < :05 and tð8Þ ¼ 3:2; p < :05 for basal conditions and

for arthritic groups, respectively. Data represent means and SEM.

N ¼ 11 rats per group for the basal conditions and N ¼ 5 for the ar-

thritis groups. *p < :05; **p < :01.

Fig. 8. HPA-axis reactivity. Healthy HA and LA rats were injected i.p.

with 1lg/kg LPS or saline and decapitated after 120min. Two-way

ANOVA, interaction effect, p ¼ ns, treatment effect, p < :001. Basal

corticosterone in the plasma of HA and LA animals on day 30 after

induction of AA was measured as well. Student�s T test, p ¼ ns.

E. Sajti et al. / Brain, Behavior, and Immunity 18 (2004) 505–514 511

4. Discussion

One important obstacle in the treatment of RA is the

limited ability to select and to recognize those patients

who will develop severe long-term complications. In the

current study we show that a pre-existing behavioral

characteristic is associated with the severity of bone

destruction in an animal model of arthritis.

Individual differences in behavioral profiles are drivenby genetic and environmental factors. In contrast to

most behavioral studies, we used an inbred strain of

rats. This approach provided the possibility to reduce

the contribution of genetic background to the relation

between behavior and disease outcome. It should be

noted, however, that we cannot exclude that genetic

differences between the animals contribute to our find-

ings. We demonstrated that rats selected upon a be-havioral characteristic in the open field, prior to the

induction of arthritis, show marked differences in bone

destruction in the late stage of the disease.

The behavior of a rat in the open field embraces

several aspects. Denenberg clearly demonstrated that

the activity measured on the first test day differs from

the activity measured on the second, third, and fourth

days. Subsequent factor analysis led him to suggest that

a different behavioral dimension is measured by theactivity on day one as compared to the activity of the

animal measured on consecutive test days (Denenberg,

1969). On the first day the novelty aspect predominates,

therefore the activity mirrors a fear response that

Fig. 9. Dexamethasone sensitivity of splenocytes of HA rats (m) and

LA rats (j). Splenocytes were incubated with different concentrations

of dexamethasone. After three days of culture proliferation was mea-

sured by [3H]thymidine incorporation. GLM, repeated measures,

p ¼ ns.

512 E. Sajti et al. / Brain, Behavior, and Immunity 18 (2004) 505–514

describes the emotional reactivity of the animal and canbe regarded as a state parameter. The activity of the first

day is positively correlated with defecation, another

frequently used parameter for emotional reactivity in the

open field. In contrast, activity on consecutive days is

negatively correlated with both activity on day one and

defecation. It represents an independent and stable be-

havioral dimension that could be interpreted as a trait

characteristic.A study by Chover-Gonzalez et al. (1999) examining

the associations between behavioral dimensions de-

scribing open field emotional reactivity and AA disease

activity reports no correlation between either activity or

number of deposited fecal pellets during the first expo-

sure to the open field and AA susceptibility or severity.

In our study as well, trait was not associated with the

inflammatory component of arthritis in a way that HAand LA animals did not differ with respect to the in-

flammatory signs of AA. However, our data show that

bone destruction—as reflected by osteoporosis, erosions

of bone, and cartilage loss—was profound in LA ani-

mals, whereas HA animals had only mild bone damage.

Furthermore, in contrast to HA animals, LA rats had

severe pathological bone proliferation and ankylosis

leading to substantial joint deformities. Moreover, werecently described that using the same experimental set-

up, this trait characteristic was also correlated to tumor

growth, tumor metastasis and tumor angiogenesis (Sajti

et al., 2004). We propose therefore that trait character-

istics may represent stable behavioral dimensions that

may have an organizing influence on physiological

processes and thereby tune disease outcome.

The observed uncoupling of inflammation and jointdestruction in our data is in line with earlier reports on

disease characteristics in humans. Rheumatoid arthritis

patients having the same inflammatory score can presentwith a destructive or non-destructive phenotype. In a

clinical study, Bresnihan et al. (1998) showed that

treatment with IL-1 receptor antagonist had beneficial

effects on bone destruction. The inflammatory process,

however, remained unaffected. Similar observations

have been made in rodents. IL-4 gene therapy in

experimental arthritis in mice has cartilage and bone-

protective effects despite ongoing inflammation (Lub-berts et al., 1999, 2000). Another important finding

demonstrating complete abolishment of bone loss with

essentially no effect on inflammation is osteoproteregin

(OPG) treatment. Systemic administration of OPG, a

decoy receptor for RANKL, in arthritic rats was shown

to result in a marked reduction in cortical and trabec-

ular bone loss and a dramatic decrease in osteoclast

numbers without significant effects on joint inflamma-tion (Kong et al., 1999b). OPG treatment did not reduce

joint inflammation quantitatively, implying that the in-

flammatory process associated with arthritis does not

depend on the RANK/RANKL pathway.

In our histological preparations, we observed a

marked difference in the expression of RANKL, an es-

sential factor in osteoclatogenesis and bone erosion, in

the two groups of rats. LA rats showed an abundantexpression of RANKL in the erosive pannus suggesting

that a difference in expression of RANKL could be a

potential mechanism explaining the differences in bone

damage between the two groups. The expression of

RANKL is under control of different cytokines. IL-10

and IFNc have been suggested to be bone-protective

regulatory mediators (Moore et al., 2001). IFNc sup-

presses osteoclastogenesis by interfering with theRANKL/RANK pathway thereby protecting bone for-

mation (Lubberts et al., 1999; Takayanagi et al., 2000).

However, IFNc is known to have a dual role in the in-

flammation accompanying arthritis, enhancing in early

stages whereas suppressing in later stages (Boissier et al.,

1995). We measured the production of these two cyto-

kines in splenocytes of our HA and LA rats. LA ani-

mals, with a pronounced joint damage, had lower levelsof IFNc both before and after the induction of arthritis.

Regarding IL-10, only the HA animals showed an in-

crease in the production of this protective cytokine upon

the induction of the disease. Although measured in a

distant immune compartment, these results suggest that

HA rats have a more favorable cytokine milieu for re-

tention of bone integrity thereby preventing long-term

skeletal complications. Furthermore, there is evidencethat cytokine production by splenocytes is closely re-

lated to disease activity in AA (Cobelens et al., 2002).

Another important mechanism, which could explain

the differences in bone erosion between the two groups,

is angiogenesis. The significantly higher plasma VEGF

concentration in LA animals strongly suggests that a

more pronounced angiogenesis contributes to the

E. Sajti et al. / Brain, Behavior, and Immunity 18 (2004) 505–514 513

development of pronounced skeletal complications inthese rats. Furthermore, specific staining for factor VIII,

an endothelial marker, revealed more blood vessels in

the proliferating synovium of LA rats.

To dissect possible endocrine correlates we measured

the reactivity of the HPA-axis in our two groups of rats.

The results showed no significant differences between the

corticosterone responses of HA and LA animals, neither

upon a chronic nor acute immune stimulus. Thesefindings are in contrast with the hypothesis stating that

susceptibility to autoimmune disease occurs in the

presence of a hyporeactive HPA-axis (Sternberg et al.,

1989a,b). More recent studies, however, have shown

that in other rat strains, corticosterone is not the dom-

inating endocrine mediator. Harbuz et al. (2003) clearly

conclude from their studies that susceptibility for and

severity of autoimmune diseases are more complexphenomena and depend on more than one parameter.

Our present results are in substantial agreement with

other behavioral studies suggesting a link between be-

havioral characteristics of the individual and subsequent

disease severity regardless HPA-axis reactivity. An ear-

lier study by our group (Kavelaars et al., 1999) dem-

onstrated that the latency of a rat to attack an intruder

was inversely correlated with the severity of experi-mental autoimmune encephalomyelitis. A study by

Chover-Gonzalez et al. (2000) using the learned help-

lessness paradigm to divide single populations of rats

into subpopulations showed that learned helpless rats

develop paw swelling in AA sooner and with increased

severity compared to the not helpless rats. Strikingly, in

both studies a different corticosterone response to acute

stress was not predictive for disease severity.The impact of the level of glucocorticoids on the in-

flammatory process will ultimately depend on the ca-

pacity of the specific receptors on peripheral tissues to

respond to circulating glucocorticoids. Therefore we

also tested the sensitivity of splenocytes to the synthetic

glucocorticoid analogue dexamethasone. However, no

differences were found between the groups in the sensi-

tivity of splenocytes to respond to dexamethasone,suggesting no differences in the communication between

the HPA-axis and the immune system between the two

groups of animals.

Considering species differences, one should be cau-

tious in attempting to extrapolate these results to hu-

mans. However, studies on the course of RA have

provided a basis for an impact of psychological factors

on disease outcome. There is evidence that neuroticism,a relatively stable personality trait, strongly influences

self-rated disease symptoms, and wellbeing in RA

(Persson and Sahlberg, 2002). Furthermore, neuroticism

at the moment of diagnosis of RA is significantly related

to psychological distress at 3- and 5-year follow-up

(Evers et al., 2002). Whether neuroticism is also related

to joint impairment has to be further investigated.

Joint impairment has a big impact on functionalability; therefore the control of bone erosive processes is

one of the most challenging objectives in the treatment

of RA. The considerable heterogeneity (clinical, patho-

logical, and immunological) of the disease makes it im-

portant to tailor the treatment on the individual patient.

Reliable prediction of those at risk for joint destruction

will almost certainly translate into long-term prevention

of disability as patients are aggressively treated early intheir disease course.

Taken these data together, we would like to speculate

that stable behavioral traits might become of clinical use

to identify patients at high risk to develop severe joint

destruction in rheumatoid arthritis allowing early and

more effective treatment of the disease.

Acknowledgments

The authors are greatly indebted to Jan Brakee for his

help with the animal experiments and to Jitske Zijlstra

for excellent technical assistance.

References

Ackerman, N.R., Rooks, W.H., Shott, L., Genant, H., Maloney, P.,

West, E., 1979. Effects of naproxen on connective tissue changes in

the adjuvant arthritic rat. Arthritis Rheum. 22, 1365–1374.

Boissier, M.C., Chiocchia, G., Bessis, N., Hajnal, J., Garotta, G.,

Nicoletti, F., Fournier, C., 1995. Biphasic effect of interferon-

gamma in murine collagen-induced arthritis. Eur. J. Immunol. 25,

1184–1190.

Bresnihan, B., Alvaro-Gracia, J.M., Cobby, M., Doherty, M., Doml-

jan, Z., Emery, P., Nuki, G., Pavelka, K., Rau, R., Rozman, B.,

Watt, I., Williams, B., Aitchison, R., McCabe, D., Musikic, P.,

1998. Treatment of rheumatoid arthritis with recombinant human

interleukin-1 receptor antagonist. Arthritis Rheum. 41, 2196–2204.

Chover-Gonzalez, A.J., Harbuz, M.S., Tejedor-Real, P., Gibert-

Rahola, J., Larsen, P.J., Jessop, D.S., 1999. Effects of stress on

susceptibility and severity of inflammation in adjuvant-induced

arthritis. Ann. N. Y. Acad. Sci. 876, 276–286.

Chover-Gonzalez, A.J., Jessop, D.S., Tejedor-Real, P., Gibert-Rahola,

J., Harbuz, M.S., 2000. Onset and severity of inflammation in rats

exposed to the learned helplessness paradigm. Rheumatology

(Oxford) 39, 764–771.

Cobelens, P.M., Kavelaars, A., van der Zee, R., van Eden, W.,

Heijnen, C.J., 2002. Dynamics of mycobacterial HSP65-induced T-

cell cytokine expression during oral tolerance induction in adjuvant

arthritis. Rheumatology (Oxford) 41, 775–779.

Denenberg, V.H., 1969. Open-field behavior in the rat: what does it

mean? Ann. N. Y. Acad. Sci. 159, 852–859.

Evers, A.W., Kraaimaat, F.W., Geenen, R., Jacobs, J.W., Bijlsma,

J.W., 2002. Longterm predictors of anxiety and depressed mood in

early rheumatoid arthritis: a 3 and 5 year followup. J. Rheumatol.

29, 2327–2336.

Feldmann, M., Brennan, F.M., Maini, R.N., 1996. Rheumatoid

arthritis. Cell 85, 307–310.

Folkman, J., 1995. Angiogenesis in cancer, vascular, rheumatoid and

other disease. Nat. Med. 1, 27–31.

514 E. Sajti et al. / Brain, Behavior, and Immunity 18 (2004) 505–514

Goldring, S.R., Gravallese, E.M., 2000. Mechanisms of bone loss in

inflammatory arthritis: diagnosis and therapeutic implications.

Arthritis Res. 2, 33–37.

Harada, M., Mitsuyama, K., Yoshida, H., Sakisaka, S., Taniguchi, E.,

Kawaguchi, T., Ariyoshi, M., Saiki, T., Sakamoto, M., Nagata, K.,

Sata, M., Matsuo, K., Tanikawa, K., 1998. Vascular endothelial

growth factor in patients with rheumatoid arthritis. Scand. J.

Rheumatol. 27, 377–380.

Harbuz, M.S., Chover-Gonzalez, A.J., Jessop, D.S., 2003. Hypothal-

amo–pituitary–adrenal axis and chronic immune activation. Ann.

N. Y. Acad. Sci. 992, 99–106.

Kavelaars, A., Heijnen, C.J., Tennekes, R., Bruggink, J.E., Koolhaas,

J.M., 1999. Individual behavioral characteristics of wild-type rats

predict susceptibility to experimental autoimmune encephalomy-

elitis. Brain Behav. Immun. 13, 279–286.

Kikuchi, K., Kubo, M., Kadono, T., Yazawa, N., Ihn, H., Tamaki,

K., 1998. Serum concentrations of vascular endothelial

growth factor in collagen diseases. Br. J. Dermatol. 139, 1049–

1051.

Kong, Y.Y., Feige, U., Sarosi, I., Bolon, B., Tafuri, A., Morony, S.,

Capparelli, C., Li, J., Elliott, R., McCabe, S., Wong, T., Campa-

gnuolo, G., Moran, E., Bogoch, E.R., Van, G., Nguyen, L.T.,

Ohashi, P.S., Lacey, D.L., Fish, E., Boyle, W.J., Penninger, J.M.,

1999a. Activated T cells regulate bone loss and joint destruction in

adjuvant arthritis through osteoprotegerin ligand. Nature 402,

304–309.

Kong, Y.Y., Yoshida, H., Sarosi, I., Tan, H.L., Timms, E., Capparelli,

C., Morony, S., Oliveira-dos-Santos, A.J., Van, G., Itie, A., Khoo,

W., Wakeham, A., Dunstan, C.R., Lacey, D.L., Mak, T.W., Boyle,

W.J., Penninger, J.M., 1999b. OPGL is a key regulator of

osteoclastogenesis, lymphocyte development and lymph-node or-

ganogenesis. Nature 397, 315–323.

Lubberts, E., Joosten, L.A., Chabaud, M., Van Den, B.L., Oppers, B.,

Coenen-De Roo, C.J., Richards, C.D., Miossec, P., Van Den Berg,

W.B., 2000. IL-4 gene therapy for collagen arthritis suppresses

synovial IL-17 and osteoprotegerin ligand and prevents bone

erosion. J. Clin. Invest. 105, 1697–1710.

Lubberts, E., Joosten, L.A., Van Den, B.L., Helsen, M.M., Bakker,

A.C., van Meurs, J.B., Graham, F.L., Richards, C.D., Van Den

Berg, W.B., 1999. Adenoviral vector-mediated overexpression of

IL-4 in the knee joint of mice with collagen-induced arthritis

prevents cartilage destruction. J. Immunol. 163, 4546–4556.

Moore, K.W., de Waal, M.R., Coffman, R.L., O�Garra, A., 2001.

Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immu-

nol. 19, 683–765.

Nagashima, M., Yoshino, S., Ishiwata, T., Asano, G., 1995. Role of

vascular endothelial growth factor in angiogenesis of rheumatoid

arthritis. J. Rheumatol. 22, 1624–1630.

Nijsen, M.J., Croiset, G., Stam, R., Bruijnzeel, A., Diamant, M.,

de Wied, D., Wiegant, V.M., 2000. The role of the CRH type 1

receptor in autonomic responses to corticotropin- releasing

hormone in the rat. Neuropsychopharmacology 22, 388–

399.

Pearson, C.M., 1956. Development of arthritis, periarthritis and

periotitis in rats given adjuvants. Proc. Soc. Exp. Biol. Med. 91, 95–

101.

Persson, L.O., Sahlberg, D., 2002. The influence of negative illness

cognitions and neuroticism on subjective symptoms and mood in

rheumatoid arthritis. Ann. Rheum. Dis. 61, 1000–1006.

Pincus, T., Callahan, L.F., 1993. The �side effects� of rheumatoid

arthritis: joint destruction, disability and early mortality. Br. J.

Rheumatol. 32 (Suppl. 1), 28–37.

Sajti, E., Kavelaars, A., van Meeteren, N., Teunis, M., Gispen, W.H.,

Heijnen, C.J., 2004. Tumor angiogenesis and metastasis formation

are associated with individual differences in behavior of inbred

Lewis rats. Brain Behav. Immun. in press.

Stam, R., Croiset, G., Akkermans, L.M., Wiegant, V.M., 1997.

Behavioural and intestinal responses to novelty in rats selected for

diverging reactivity in the open field test. Behav. Brain Res. 88,

231–238.

Sternberg, E.M., Hill, J.M., Chrousos, G.P., Kamilaris, T., Listwak,

S.J., Gold, P.W., Wilder, R.L., 1989b. Inflammatory mediator-

induced hypothalamic–pituitary–adrenal axis activation is defective

in streptococcal cell wall arthritis-susceptible Lewis rats. Proc.

Natl. Acad. Sci. USA 86, 2374–2378.

Sternberg, E.M., Young III, W.S., Bernardini, R., Calogero, A.E.,

Chrousos, G.P., Gold, P.W., Wilder, R.L., 1989a. A central

nervous system defect in biosynthesis of corticotropin-releasing

hormone is associated with susceptibility to streptococcal cell wall-

induced arthritis in Lewis rats. Proc. Natl. Acad. Sci. USA 86,

4771–4775.

Takayanagi, H., Kim, S., Matsuo, K., Suzuki, H., Suzuki, T., Sato, K.,

Yokochi, T., Oda, H., Nakamura, K., Ida, N., Wagner, E.F.,

Taniguchi, T., 2002a. RANKL maintains bone homeostasis

through c-Fos-dependent induction of interferon-beta. Nature

416, 744–749.

Takayanagi, H., Kim, S., Taniguchi, T., 2002b. Signaling crosstalk

between RANKL and interferons in osteoclast differentiation.

Arthritis Res. 4 (Suppl. 3), S227–S232.

Takayanagi, H., Ogasawara, K., Hida, S., Chiba, T., Murata, S., Sato,

K., Takaoka, A., Yokochi, T., Oda, H., Tanaka, K., Nakamura,

K., Taniguchi, T., 2000. T-cell-mediated regulation of osteoclasto-

genesis by signalling cross-talk between RANKL and IFN-gamma.

Nature 408, 600–605.

Trentham, D.E., Townes, A.S., Kang, A.H., 1977. Autoimmunity to

type II collagen an experimental model of arthritis. J. Exp. Med.

146, 857–868.

van Meeteren, N.L., Brakkee, J.H., Helders, P.J., Croiset, G., Gispen,

W.H., Wiegant, V.M., 1997. Recovery of function after sciatic

nerve crush lesion in rats selected for diverging locomotor activity

in the open field. Neurosci. Lett. 238, 131–134.

Zhang, L., Mia, M.Y., Zheng, C.L., Hossain, M.A., Yamasaki, F.,

Tokunaga, O., Kohashi, O., 1999. The preventive effects of

incomplete Freund�s adjuvant and other vehicles on the develop-

ment of adjuvant-induced arthritis in Lewis rats. Immunology 98,

267–272.