Ž .Brain Research 816 1999 563–571

Research report

Brain-IL-1b induces local inflammation but systemic anti-inflammatoryresponse through stimulation of both hypothalamic–pituitary–adrenal axis

and sympathetic nervous system

Christian Woiciechowsky a,), Britta Schoning a, Nadja Daberkow a, Katrin Asche a,¨Gisela Stoltenburg b, Wolfgang R. Lanksch a, Hans-Dieter Volk c

a Department of Neurosurgery, Charite-Campus Virchow-Klinikum, Humboldt-UniÕersitat zu Berlin, Augustenburger Platz 1, D-13444 Berlin, Germany´ ¨b Institute of Neuropathology, UniÕersitatsklinikum Benjamin Franklin, Freie UniÕersitat Berlin, D-12200 Berlin, Germany¨ ¨

c Institute of Medical Immunology, Charite-Campus Mitte, Humboldt-UniÕersitat zu Berlin, Augustenburger Platz 1, D-10098 Berlin, Germany´ ¨

Accepted 10 November 1998

Abstract

It is well established that systemic inflammation induces a counter-regulatory anti-inflammatory response particularly resulting indeactivation of monocytesrmacrophages. However, recently we demonstrated a systemic anti-inflammatory response without preceding

Ž .signs of systemic inflammation in patients with brain injuryrsurgery and release of cytokines into the cerebrospinal fluid CSF . In orderto analyze the mechanisms and pathways of systemic immunodepression resulting from sterile cerebral inflammation we established an

Ž . Ž . Ž .animal model using continuous intra-cerebroventricular i.c.v. or intra-hypothalamic i.h. infusion of rat recombinant rr tumor necrosisŽ . Ž . Ž .factor TNF -a and interleukin IL -1b for 48 h. Controls received intra-venous i.v. cytokine administration. Interestingly, i.c.v. and i.h.

Ž .infusion of IL-1b but not TNF-a produced distinct signs of central nervous system CNS inflammation. Correspondingly, i.c.v. infusionof IL-1b particularly diminished the TNF-a but increased the IL-10 concentration in whole blood cultures after endotoxin stimulation.

Ž .All parameters normalized within 48 h after termination of the infusion. Blocking the hypothalamic–pituitary–adrenal HPA axis byŽ .hypophysectomy HPX led to complete recovery of the diminished TNF-a concentration and temporarily inhibited the IL-10 increase.

Ž .Blocking the sympathetic nervous system SNS transmission by application of the b -adrenoreceptor antagonist propranolol not only2

inhibited the increase but further downregulated the endotoxin induced IL-10 concentration in the media of whole blood cell cultures,whereas the TNF-a decrease was only partially prevented. Interestingly, HPX and propranolol also diminished the cell invasion into theCSF. In summary, activation of both the HPA axis and the SNS plays an important role in systemic anti-inflammatory response resultingfrom cytokines in brain and cerebral inflammation. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Monocytermacrophage; Neuroimmunology; Cytokine; Interleukin-1b; Tumor necrosis factor-a; Interleukin-10

1. Introduction

Local infection or sterile trauma induce a local inflam-matory response. The release of pro-inflammatory cy-

Ž .tokines e.g. TNF-a , IL-1b activates an inflammatorycascade improving wound healing and anti-microbial de-fense. Monocytesrmacrophages are important players inthis scenario. However, overwhelming response can resultin systemic inflammation and septic shock. In order tocontrol the potentially harmful inflammatory response, theimmune system can release several anti-inflammatory me-

) Corresponding author. Fax: q49-30-45060977; E-mail:christian.woiciechowsky@charite.de

Ž .diators like IL-10, IL-1 receptor antagonist ra and solubleTNF-receptors. TNF-a , IL-1b and prostaglandins bythemselves are powerful inducers of the compensatoryanti-inflammatory response. However, there is evidencethat in addition to the auto-regulatory pathways of theimmune cells the delicate balance between pro- and anti-inflammatory response is controlled by the CNS.

This is most evident in the release of different media-tors by pituitary and adrenal glands that follows over-spillof IL-1b, TNF-a and IL-6 into the circulationw x4,10,18,26,32,37 . Moreover, pro-inflammatory cytokinesŽ .e.g., IL-1b can enhance sympathetic nerve activityw x15,20,34 . Remarkably, in vitro studies demonstrated thatcatecholamines are able to upregulate the endotoxin in-

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 98 01238-4

( )C. Woiciechowsky et al.rBrain Research 816 1999 563–571564

duced release of the anti-inflammatory cytokine IL-10 inperipheral blood mononuclear cells whereas the TNF-a

w xproduction was down-regulated 7,22,27,35 . Conse-quently, an activation of CNS pathways by mediators ofthe immune system may induce a brain-mediated anti-in-flammatory response in order to control systemic inflam-

w xmation 16,21 .However, the question is, what happens if the immuno-

inhibitory CNS pathways are activated without systemicinflammation? Recently, we demonstrated that postopera-tive release of pro-inflammatory cytokines into the CSFwithout any signs of systemic inflammation was associatedwith immunodepression and an increased risk of infections

w xin neurosurgical patients 1,2,39,40 . Immunodepressionwas reflected by the down-regulation of monocytic HLA-DR expression and ex vivo TNF-a production. In parallel,elevated plasma IL-10 levels were seen. Similar findings

w xwere observed in patients suffering from brain injury 39 .That means that cytokines in the brain can trigger asystemic anti-inflammatory condition without precedingsystemic inflammation. Persistence of this phenomenonincreases the risk for infectious complications.

In order to further complete the knowledge about localinflammation in the CNS and the consequences for thesystemic immune response, an animal model was estab-

Žlished using continuous i.c.v. or i.h. hypothalamus is the.hypothesized site of action for brain-IL-1b infusion of rat

Ž .recombinant rr IL-1b and rrTNF-a , both most importantpro-inflammatory cytokines, for two days. The aim of thestudy was to characterize local and systemic immuneconsequences resulting from cytokines in the brain. There-fore, we measured cell numbers in the CSF and monitoredchanges in the functional state of inflammatory immune

Ž .cells particularly monocytes by measuring the concentra-tion of TNF-a and IL-10 in the supernatants of wholeblood cell cultures as well as spleen cell cultures afterendotoxin stimulation. In order to evaluate the importanceof the HPA axis and the SNS for the immune effectsadditional experiments were performed with hypophysec-tomized rats and blocking b -adrenoreceptors with propra-2

nolol. To analyze the recovery of the immunologicalchanges, investigations were extended to 48 h after termi-nation of the IL-1b infusion in one group.

2. Material and methods

2.1. Animals

Approval for the study was achieved from the AnimalProtection Board of the Senat of Berlin. A total of 120

Žmale Sprague–Dawley rats Harlan Winkelmann, Borchen,.Germany weighing 250–350 g at the time of surgery were

used. The animals were divided into 12 groups of 10 ratsŽeach. Three different solutions: vehicle 4% HSA dis-

. Žsolved in physiological saline , DRK-Blutspendedienst,

. ŽSpringe, Germany , rrIL-1b R&D systems, Minneapolis,. Ž .USA and rrTNF-a Laboserv, Staufenberg, Germany

were administered in three different locations: i.c.v., i.h.and i.v. For the continuous application of the cytokines,

Žosmotic minipumps 96-h duration, 100 ml, 1 mlrh; Model.1003D Alza, Palo Alto, CA, USA were used. The concen-

tration of cytokines in pumps was 1 mg dissolved in 100ml vehicle solution. The delivery rate was 10 ng cytokinein one hour. The length of the infusion was 48 h. In orderto guarantee an immediate infusion of the cytokines afterthe implantation, minipumps were pre-incubated for 16 hat 378C prior to surgery according to the manufacturer’sinstructions. In order to investigate the mechanisms howi.c.v. infusion of IL-1b can induce systemic immune ef-fects additional experiments were performed with hy-

Žpophysectomized rats HPX was performed by the animal.supplier 1 week prior to operative procedures and rats

treated with the b -adrenoreceptor antagonist propranolol2ŽŽ . Ž .s - y -propranolol hydrochloride, Sigma–Aldrich,

.Deisenhofen, Germany . Propranolol was injected intra-Ž .peritoneally 10 mgrkg before, 12, 24 and 36 h after

initiation of cytokine infusion. The dose and injectioninterval were calculated according to previous descriptions

w xof effective SNS-blocking in rats 13,31 . In order to studythe recovery of the immunological changes the experimentwas extended to 48 h after termination of the IL-1b

Ž .infusion 96 h in summary in one group of animals.

2.2. Implantation of minipumps for cytokine infusion

The implantation of ALZET-pumps into the brain wasperformed as follows: rats were anesthetized using a gas

Ž .mixture of isoflurane 1.0% vrv and N OrO2 2Ž .50.0%r25.0%, vrv and an L-shaped cannula of theALZET brain infusion kit was placed into the left ventricle

Table 1Intra-cerebral application of IL-1b but not TNF-a induces local inflam-mation and body weight loss

Infusion side Solution Cell counts= Body weight510 rml in % of 0 h

value

i.c.v. vehicle 0.2"0.1 99.2"0.9TNF-a 3.3"0.1 99.0"0.7IL-1b 312.0"90.4 82.9"1.1IL-1bqpropranolol 124.4"34.9)) 89.2"1.6))

IL-1bqHPX 93.0"13.5)) 87.0"0.7))

i.h. vehicle 0.0 100.0"0.6TNF-a 5.0"0.6 96.5"0.7IL-1b 43.6"3.7 87.2"1.7

Cell counts were determined in the Neubauer counting chamber afteraspiration of the CSF through a needle puncture of the cisterna magna 48h after initiation of cytokine infusion. The data are expressed as mean"

SEM.)) P -0.01 versus i.c.v. infusion of rrIL-1b only, Mann–WhitneyU-test.

( )C. Woiciechowsky et al.rBrain Research 816 1999 563–571 565

or the left anterior hypothalamus. The coordinates were:0.8 mm posterior and 1.2 mm lateral to bregma and 3.8mm below the dura for the ventricle and 1.8 mm posterior,0.7 mm lateral to bregma and 9.0 mm below the dura forthe anterior hypothalamus according to Paxinos and Wat-

w xson 25 . The cannula was fixed with two stainless steelscrews and mounted to the skull with dental cement. Thescrews acted as an anchor to secure both the externalportion of the cannula and the entire implantation site. Theminipumps were implanted subcutaneously in the nape ofthe neck and attached to the cannula via a tubing. For thei.v. infusion a PE-10 tube was placed aseptically into the

left femoral vein to the level of the V. cava inferior andconnected to the pump.

2.3. Experimental procedures

During the days of cytokine infusion all animals wereobserved for motor behavior and feeding. Forty-eight or 96h after initiation of infusion, animals were weighed andthen euthanized. Afterwards, spleens and brains were ex-cised. The brains were frozen in liquid nitrogen and storedat y808C until used for histological examination. In orderto confirm the correct localization of the cannula and to

Fig. 1. Changes in the endotoxin induced TNF-a and IL-10 concentrations in the media of whole blood cell cultures during continuous infusion of IL-1b,Ž . Ž . Ž .TNF-a or vehicle solution into the ventricular system i.c.v. the hypothalamus i.h. or blood i.v. . Blood samples were obtained at different time points

and cultured with 100 ngrml endotoxin. TNF-a concentration was determined after 4 h and IL-10 concentration after 24 h of stimulation in supernatants6 Ž .by ELISA. The concentrations were calculated in pgr10 WBC and the data are expressed in percentage of the pretreatment 0 h value of the same

animal"S.E.M. ))) p-0.001, )) p-0.01, ) p-0.05, vs. vehicle application at the same time point, Mann–Whitney U-test.

( )C. Woiciechowsky et al.rBrain Research 816 1999 563–571566

demonstrate signs of meningoencephalitis cryostate sec-Ž .tions 10 mm of each brain at the level of the cannula

were mounted onto a glass slide, dried and stained withhematoxylin and eosin, analyzed at the light microscopic

w xlevel and compared with the anatomic atlas 25 . Thespleens were immediately used for cell culture studies.

2.4. Blood samples

ŽHeparin-anticoagulated venous blood samples 500 ml.each from the ophthalmic venous plexus were collected

from the rats 1 day before surgery, 4, and 48 h aftersurgery and additionally in one group 48 h after termina-tion of cytokine infusion using the method described by

w xReilly 30 .

2.5. Counting of white blood cells

At each time point the number of white blood cellsŽ .WBC was determined using the Neubauer countingchamber in order to calculate the cytokine concentrationper number of WBC in culture. In our opinion, the cy-tokine concentration per cell more sufficiently express thefunctional state of immune cells than the cytokine concen-tration per milliliter plasma.

2.6. Cytokine leÕels in plasma

Plasma was yielded from heparin-anticoagulated venousblood samples at each time point and immediately frozenat y808C until the measurement. Cytokine levels in plasmawere determined using the following commercial available

ŽELISAs: ultra sensitive rat TNF-a and rat IL-10 Cyto-.screen USe, Laboserv, Giessen, Germany .

2.7. CSF samples

After euthanization the CSF of all animals was obtainedthrough a needle puncture of the cisterna magna. The CSFsamples were objected to cell count and cell differentia-tion.

2.8. Cytokine concentration in supernatants of whole bloodcell cultures after endotoxin stimulation

Heparin-anticoagulated whole blood samples were di-luted 1:5 with RPMI 1640 medium supplemented with 2nM L-Glutamin, and 10 Urml StreptomycinrAmpicillinŽ .Biochrom, Berlin, Germany and incubated for 4 and 24 hŽ .378C, 5% CO , respectively, with 100 ngrml endotoxin2

Žfrom Escherichia coli 0127:B8 Sigma, Deisenhofen, Ger-.many . Cell-free supernatants were harvested and stored at

y808C until the measurement of cytokine concentrationsŽ .by rat specific ELISA. TNF-a Cytoscreene concentra-

tions were determined in supernatants of 4 h cultures

Ž .whereas IL-10 Cytoscreene levels were determined insupernatants of 24 h cultures. Cytokine concentrationswere corrected by WBC counts and shown in percent ofthe pretreatment level of the same animal.

2.9. Cytokine concentration in supernatants of spleen cellcultures after endotoxin stimulation

Spleens were removed from animals receiving i.c.v.infusion and dispersed into cell suspension in RPMI-1640supplemented with 2 nM L-Glutamin, 10 Urml and 10

Ž .Urml StreptomycinrAmpicillin and 10% FCS Biochrom .Ž .Visible cells )90% were counted using trypanblue stain-

ing. For the cell culture studies a concentration of 5=106

cells was used. For assessing the endotoxin induced cy-tokine secretion, spleen cells were incubated for 4 and 24 hŽ .378C, 5% CO with 100 ngrml endotoxin from E. coli2

Ž .0127:B8 Sigma . Cell-free supernatants were harvestedand stored at y808C until the measurement of cytokine

Žconcentrations by rat specific ELISA. TNF-a Cyto-.screene concentrations were determined in supernatants

Fig. 2. Changes in the endotoxin induced TNF-a and IL-10 concentra-tions in the media of whole blood cell cultures during continuous i.c.v.infusion of vehicle solution, IL-1b or IL-1b and additional HPX or

Žtreatment with the b -adrenoreceptor antagonist propranolol 10 mgrkg2.every 12 h . Blood samples were obtained at different time points and

cultured with 100 ngrml endotoxin. TNF-a concentration was deter-mined after 4 h and IL-10 concentration after 24 h of stimulation insupernatants by ELISA. The concentrations were calculated in pgr106

Ž .WBC and the data are expressed in percentage of the pretreatment 0 hvalue of the same animal"S.E.M. )) p-0.01, ) p-0.05 vs. vehicleapplication at the same time point, Mann–Whitney U-test. qqq p-0.001,qq p-0.01, vs. IL-1b application alone at the same time point, Mann–Whitney U-test.

( )C. Woiciechowsky et al.rBrain Research 816 1999 563–571 567

Ž .of 4 h cultures whereas IL-10 Cytoscreene levels weredetermined in supernatants of 24 h cultures.

2.10. Statistical analysis

Data are presented as mean"S.E.M. Statistical analy-ses included the Mann–Whitney U-test for the comparisonof two study groups and the Wilcoxon test for pairedsamples. A p lower than 5% was considered significant.

3. Results

3.1. Histological Õerification of the cannula position

In all animals with implanted brain cannulas the correctposition of the cannula was verified histologically. Onlydata from animals with correct cannula placement and

Žwithout injury to the surrounding brain at least 9 from 10.animals in each group were evaluated.

3.2. Alterations in body weight and behaÕior of the ani-mals during cytokine infusion

The i.c.v. and i.h. infusion of IL-1b at the dose of 10ngrh produced signs of physical discomfort in all animalsincluding piloerection and decreased physical activity dur-ing the infusion time of 48 h. The infused dose of 10 ngrhwas calculated according to previous clinical and animalstudies demonstrating high levels of pro-inflammatory cy-tokines after brain injury or meningitis and adapted tostudies showing effective brain-IL-1 concentrations for

w xalterations of blood lymphocytes 14,15,17,19,23,24,33 .Treatment of rats with TNF-a or vehicle infusion as wellas i.v. infusion of IL-1b did not perceptibly distress theanimals. Correspondingly, body weight was dramatically

Ždecreased by i.c.v. and i.h. administration of IL-1b Table.1 . HPX and treatment with the b -adrenoreceptor antago-2

nist propranolol did not protect animals from physicalŽ .discomfort and loss of body weight Table 1 . However,

there was a significant lower loss of body weight inhypophysectomized rats and after treatment with propra-

Ž .nolol Table 1 . No animals with a dysfunctional pumpshowed any reduction in body weight or increase in CNSinflammation.

3.3. IL-1b but not TNF-a induced meningoencephalitis

The continuous i.c.v. and i.h. infusion of IL-1b at adose of 10 ngrh resulted in a CNS inflammation. Table 1shows the cell numbers in the CSF 48 h after initiation ofinfusion. The differentiation of the cells revealed predomi-

Ž .nantly neutrophils not shown . The i.c.v. infusion ofIL-1b induced the most extensive invasion of cells into theCSF causing a meningo-encephalitis as could be demon-strated in histological sections. However, hypophysec-

tomized rats and rats treated with propranolol showedsignificantly lower cell infiltrations compared with animals

Ž .receiving i.c.v. infusion of IL-1b only Table 1 . In con-trast, the i.c.v. and i.h. administration of TNF-a inducedonly marginal signs of meningo-encephalitis.

3.4. Intra-cerebral IL-1b application switches the inflam-matory capacity of blood immune cells

The continuous i.c.v. and i.h. infusion of IL-1b signifi-cantly diminished the endotoxin induced TNF-a concen-tration in the media of whole blood cultures during theentire observation period beginning 4 h after initiation of

Ž .infusion Fig. 1 . In contrast, rats receiving i.v. infusion ofIL-1b showed only a temporarily diminished TNF-a con-centration in the supernatants of whole blood cell culturesŽ . Ž .after 4 h Fig. 1 .

In contrast to the diminished TNF-a , the concentrationof the anti-inflammatory cytokine IL-10 measured in 24 hwhole blood cultures, was significantly increased afteri.c.v. infusion of IL-1b with peak-levels after 4 h ofinfusion. Although the IL-10 concentration subsequentlydecreased, it still remained elevated at the end of the

Ž .infusion period after 48 h compared with vehicle infusedŽ .rats Fig. 1 . Furthermore, a non-significant elevation of

the endotoxin induced IL-10 concentration was seen inanimals receiving i.v. infusion of TNF-a . Interestingly, wealso measured higher TNF-a concentrations in media ofwhole blood cell cultures 2 h after initiation of i.v. TNF-ainfusion compared with the other groups where TNF-a

Ž .was diminished data not shown . Since TNF-a can upreg-ulate his own production and the production of IL-10 in

w xvitro 27 we speculate that the infused TNF-a may also

Fig. 3. Endotoxin induced TNF-a and IL-10 concentrations in the mediaof spleen cells from animals receiving i.c.v. infusion of IL-1b, TNF-a orvehicle solution. The spleens were excised 48 h after initiation of the

Ž 6 .infusion and dispersed into cell suspension. Spleen cells 5=10 wereŽ .cultured with endotoxin 100 ngrml . TNF-a concentration was deter-

mined after 4 h and IL-10 concentration after 24 h of stimulation insupernatants by ELISA. Data are shown as mean cytokine concentrationin pgr106 spleen cells"S.E.M. ) p-0.05 vs. vehicle and TNF-agroup, Mann–Whitney U-test.

( )C. Woiciechowsky et al.rBrain Research 816 1999 563–571568

Fig. 4. Changes in the endotoxin induced TNF-a and IL-10 concentrations in the media during and after termination of continuous i.c.v. infusion of IL-1b

for 48 h. Blood samples were obtained at different time points. Blood samples were obtained at different time points and cultured with 100 ngrmlendotoxin. TNF-a concentration was determined after 4 h and IL-10 concentration after 24 h of stimulation in supernatants by ELISA. The concentrationswere corrected by WBC counts and the data are expressed as pgr106 WBC"S.E.M. aap-0.01, ap-0.05 vs. 0 h value, Wilcoxon test for pairedsamples in follow up.

be responsible for the elevated IL-10 concentration 4 hŽ .after initiation of infusion Fig. 1 .

Neither HPX nor propranolol were able to abolish theIL-1b induced early decrease of the TNF-a concentration

Ž . Ž .in the media 4 h after surgery Fig. 2 . However, propra-nolol partially prevented this down-regulation of TNF-aŽ .Fig. 2 . Furthermore, HPX but not propranolol abolishedthe long lasting decrease of the TNF-a concentration in

Ž .whole blood cultures Fig. 2 . Additionally, the brain-IL-1b

Ž .induced IL-10 peak after 4 h was prevented by HPX andŽ .propranolol Fig. 2 . However, blocking b -adrenorecep-2

tors with propranolol even significantly decreased IL-10during the 48 h of IL-1b infusion compared with the other

Ž .groups Fig. 2 .

3.5. Intra-cerebral application of IL-1b diminished TNF-aconcentration in the supernatants of spleen cell cultures

Corresponding to the functional changes of blood im-mune cells, the endotoxin induced TNF-a concentration inthe media was also decreased in spleen cell cultures 48 hafter IL-1b infusion, whereas the IL-10 concentration was

Ž .only slightly different from vehicle controls Fig. 3 .

3.6. The brain-IL-1b induced switch of blood immune cellsinto an anti-inflammatory state reÕersed completely 48 hafter termination of cytokine infusion

Forty-eight hours after termination of the cytokine infu-sion the TNF-a and IL-10 concentrations in the medianormalized, demonstrating that the observed systemic im-mune changes are directly affected by the cerebral cy-

Ž .tokine application Fig. 4 .

3.7. Cytokine leÕels in plasma

No detectable plasma levels of TNF-a and IL-10 weredemonstrable in any group at any time point. This excludes

plasma concentrations higher than 0.7 pgrml for TNF-aŽ .and 80 pgrml for IL-10 sensitivity of the assays .

4. Discussion

In response to an inciting event such as infection,hemorrhage, or trauma the body unleashes an inflamma-tory reaction that is capable of amplifying itself indefi-nitely. However, little is known how the body mightdown-regulate the inflammatory cascade. Evidence is ac-cumulating that in response to the original inciting eventŽ .the inflammatory response the body also mounts an

w xanti-inflammatory response 29 . Several studies demon-strated the induction of anti-inflammatory factors by pro-

Žinflammatory mediators e.g. TNF-a and prostaglandins. w xinduce IL-10 as negative feedback regulation 8,27,36 .

However, the control of the inflammatory response isnot limited to the immune cells by themselves. The CNSmay be involved in the regulation of the pro- and anti-in-flammatory reactions in order to modulate excessive, dele-terious effects of cytokines after they have produced theirinitial, beneficial effects. So, a systemic anti-inflammatoryresponse with over-spill of pro-inflammatory cytokinesinto the circulation can activate the HPA axis and the SNSand induces a compensatory anti-inflammatory responsew x5,15,16,21,32,35 . IL-1b, IL-6 and TNF-a are the mostimportant cytokines in this process of brain-mediateddown-regulation of the inflammatory cascade. If thesemediators balance each other and the initial insult isovercome, homeostasis will be restored. If balance cannotbe established a dominant systemic inflammatory or anti-inflammatory response syndrome will ensue.

Recently, we demonstrated an association between thepostoperative release of pro-inflammatory cytokines intothe CSF and systemic immunodepression in neurosurgicalpatients with an increased risk of infection without signs of

w xpreceding systemic inflammatory response 1,2,39 . There-

( )C. Woiciechowsky et al.rBrain Research 816 1999 563–571 569

fore, we speculate that a local inflammation in the CNSwith over-spill of cytokines into the CSF may cause abrain-mediated anti-inflammatory response independentfrom a systemic inflammation.

Here, we clearly demonstrate, that IL-1b but not TNF-aat least at the dose infused causes a distinct inflammationwhen administered into the CNS. The signs of inflamma-tion were more generalized in rats receiving i.c.v. infusioncompared with animals receiving i.h. infusion. This wasvisible by higher cell numbers in the CSF and a histologi-cally verified meningo-encephalitis. The i.h. infusion ofIL-1b only caused a local encephalitis with minor signs ofbasal meningitis on the site of infusion. The inflammatoryresponse of i.c.v. and i.h. infusion of TNF-a was onlymarginal with low cell numbers in the CSF and withouthistological signs of meningo-encephalitis. Quagliarello et

w xal. 28 also observed that TNF-a is less effective thanIL-1b in induction of meningitis and injury of the bloodbrain barrier. The local inflammatory response to cerebralIL-1b infusion was diminished by HPX and propranolol.Since we noticed a decrease of white blood cell countsafter HPX and propranolol compared with IL-1b infusion

Ž .alone data not shown , we speculate that this phenomenonalso may be responsible for the lower cell counts in theCSF. This is in context with descriptions that glucocorti-coids induce leucocytosis, and HPX prevents this effectw x38 .

Corresponding to the CNS inflammation, we were ableto show systemic immunological effects. Beginning 4 hafter initiation of i.c.v. infusion of IL-1b, a decreasedendotoxin induced TNF-a and an increased IL-10 concen-tration in the media of whole blood cell cultures were seen.Although the IL-10 concentration in the supernatants de-creased in the course of 48 h the differences still remainedsignificant compared with the vehicle group. Furthermore,

Žanimals receiving i.h. hypothalamus is the hypothesized.site of action of central IL-1b infusion of IL-1b showed

similar but less severe effects compared with the i.c.v.group, mainly the IL-10 peak disappeared. In the i.v. groupthe immunological changes were shorter lasting. This cor-responds well to the described fact that IL-1b is usuallymore potent in the CNS than in the periphery. Interest-ingly, TNF-a could not be detected in plasma at any timepoint suggesting a brain-mediated anti-inflammatory re-sponse without an adequate systemic inflammation.

What are the mechanisms by which brain-IL-1b in-duces monocytic deactivation? In previous clinical studies,we demonstrated that postoperative immunodepression inneurosurgical patients was associated with sympatheticactivation as well as stimulation of the HPA axisw x1,2,39,41 . The release of glucocorticoids and catechol-amines may influence the function of monocytesrmacro-phages. In fact, glucocorticoids and catecholamines sup-press the production of pro-inflammatory cytokines andthe latter also induce IL-10 release in peripheral blood

w xmononuclear cells 11,16,22,27,38 . Consequently, we

speculated that the systemic immunological effects withdecreased endotoxin induced TNF-a and increased IL-10concentration in the media of whole blood cultures re-sulted from stimulation of the SNS as well as the HPAaxis by brain-IL-1b.

In order to evaluate this hypothesis, additional experi-ments with animals receiving i.c.v. infusion of IL-1b wereprovided by turning off the HPA axis through HPX andblocking sympathetic b -adrenoreceptors with propranolol2w x9,12 .

HPX and treatment with propranolol diminished theinvasion of immune cells into the CSF. Furthermore, theloss of body weight was less extreme in both groupscompared with IL-1b infusion only. HPX also completelyabolished the long-lasting brain-IL-1b induced decrease ofthe TNF-a concentration in the media and prevented theearly IL-10 peak. On the other hand, propranolol suffi-ciently abolished the IL-10 peak after 4 h and caused afurther decrease with lowest values 48 h after initiation ofthe treatment.

These data suggest the predominant role of the HPAaxis for the prolonged decreased endotoxin-induced TNF-a

Žconcentration, whereas the early suppressive effects after.4 h were more influenced by the SNS. This confirms in

vivo and in vitro results, where it was shown that catechol-amines can increase the endotoxin induced IL-10 produc-

w xtion very fast 15,22,27,36,39 .However, administration of IL-1b into CNS also in-

duced migration of immune cells into CSF and brainparenchyma. Subsequent release of various mediators bythese immune cells could also activate cerebral pathwaysand produce systemic effects. Interestingly, the enhancedIL-10 concentration was seen after i.c.v. but not after i.h.infusion, although the hypothalamus is the supposed site of

w xaction of brain-IL-1b 3,6,15 . Therefore, we believe thatthe more severe CNS inflammation after i.c.v. infusionmay affect additional neuro-immune pathways which con-tribute to the systemic immunodepression.

Moreover, termination of the IL-1b infusion resulted innormalization of all peripheral variables measured within48 h, demonstrating that all effects depend on the infusionof IL-1b into the CSF.

5. Conclusion

Our results demonstrate that i.c.v. and i.h. infusion ofIL-1b at the dose of 10 ngrh for 48 h cause a severe CNSinflammation whereas TNF-a infusion at the same dose isless effective. Correspondingly, an acute systemic immuno-depression with decreased endotoxin induced concentra-

Ž .tion of pro-inflammatory cytokines TNF-a and increasedŽ .concentration of anti-inflammatory cytokines IL-10 in

the supernatants of whole blood cell cultures was ob-served. These data suggest that pro-inflammatory cy-tokines in the brain and cerebral inflammation may induce

( )C. Woiciechowsky et al.rBrain Research 816 1999 563–571570

a so called ‘central’ anti-inflammatory response syndromewithout an adequate systemic inflammation by activation

Ž .of immuno-inhibitory CNS pathways HPA axis, SNS .This may generate systemic immunodepression in patientswith cerebral injury as could be demonstrated in our

w xclinical studies 1,2,39 .

Acknowledgements

This work was supported by a grant from the DeutscheForschungsgemeinschaft, SFB 507rC5.

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