Journal of Neuroimmunology 219 (2010) 75–80

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Journal of Neuroimmunology

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Chemical destruction of brain noradrenergic neurons affects spleniccytokine production

Harald Engler a,b,⁎, Raphael Doenlen a, Carsten Riether a, Andrea Engler a, Hugo O. Besedovsky c,Adriana del Rey c, Gustavo Pacheco-López a, Manfred Schedlowski a,b

a Institute of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, University of Duisburg-Essen, 45122 Essen, Germanyb Laboratory of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, ETH Zurich, 8092 Zurich, Switzerlandc Department of Immunophysiology, Institute of Physiology and Pathophysiology, Philipps University Marburg, 35037 Marburg, Germany

⁎ Corresponding author. Institute of Medical Psychologogy, University Hospital Essen, Hufelandstrasse 55, D-45201 723 4506; fax: +49 201 723 5948.

E-mail address: harald.engler@uk-essen.de (H. Engle

0165-5728/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.jneuroim.2009.12.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 October 2009Received in revised form 1 December 2009Accepted 1 December 2009

Keywords:NoradrenalineBrainstemLocus coeruleusDSP-4SpleenCytokine

The neurotransmitter noradrenaline (NA) plays a pivotal role in immune regulation. Here we used theselective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to investigate the impact ofcentral NA depletion on cytokine production by splenic monocytes/macrophages and T cells. Intraperitonealadministration of DSP-4 in adult rats induced a substantial reduction of noradrenergic neurons in the locuscoeruleus and the A5 cell group. The degeneration of brainstem noradrenergic neurons was accompanied bya significant decrease in the production of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α bylipopolysaccharide-stimulated splenocytes. In addition, upon T cell receptor stimulation with anti-CD3,isolated splenocytes of DSP-4 treated animals produced significantly less interferon (IFN)-γ but not IL-2 andIL-4. The proportion of monocytes/macrophages and T cells in the spleen remained unaffected by theneurotoxin treatment, however, the percentage of natural killer cells decreased significantly. The findingssuggest that a certain level of central noradrenergic tone is required for normal functioning of peripheralimmune cells.

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1. Introduction

The sympathetic nervous system (SNS) and its principal neuro-transmitter, noradrenaline (NA), play a pivotal role in the neuralregulation of the immune system (Besedovsky and del Rey, 1996;Elenkov et al., 2000; Sanders and Straub, 2002; Sternberg, 2006).Primary and secondary lymphoid organs are richly innervated bysympathetic nerve fibers and noradrenergic terminals are found in theclose vicinity of lymphocytes and macrophages, forming synapse-likecontacts (Felten et al., 1985). Moreover, adrenergic receptors, mainlyof the β2-subtype, have been identified on most of the cells thatparticipate in innate and adaptive immune responses, providing thebiochemical basis for noradrenergic influences on immune cells (Kinand Sanders, 2006). Further evidence for the involvement of the SNSin immune regulation derived from pharmacological studies showingthat systemic administration of exogenous NA alters leukocytedistribution and cytokine production in humans (Schedlowski et al.,1993, 1996; Torres et al., 2005). In addition, the mobilization ofimmune cells during the acute stress response was found to bemediated by the SNS (Benschop et al., 1996; Engler et al., 2004).

However, not only increased peripheral NA levels but also lack insympathetic output following chemical destruction of peripheralnoradrenergic nerve terminals with the neurotoxin 6-hydroxydopa-mine (6-OHDA) has been shown to be associated with immunealterations in lymphoid tissues (Bellinger et al., 2005; Besedovskyet al., 1979; Madden et al., 1989). This suggests that a certain level ofsympathetic tone is required for normal immune functioning in theperiphery.

The vast majority of studies investigating the role of NA in immuneregulation have focused on the peripheral SNS, whereas very little isknown about the central noradrenergic system. In the brain, thelargest cluster of noradrenergic cell bodies is located within the locuscoeruleus (LC/A6). Projections from this brainstem nucleus areextremely widespread, innervating the entire cortex, subcorticalregions (e.g., hippocampus, amygdala, thalamus, hypothalamus, andcerebellum), other brainstem nuclei, and the spinal cord (Jones andMoore, 1977; Sara, 2009). Bilateral electrolytic lesions of the LC wereshown to affect humoral and cellular immune responses in ratsindicating a potential involvement of this nucleus in neuro-immunecommunication (Jovanova-Nesic et al., 1993; Nikolic et al., 1993).However, a major drawback of this technique is the lack of specificityfor a certain neurotransmitter, damaging all neurons and fibers ofpassage in the targeted area. Further evidence for the role of the brainnoradrenergic system in peripheral immune regulation derived fromanimal studies in which the catecholaminergic neurotoxin 6-OHDA

76 H. Engler et al. / Journal of Neuroimmunology 219 (2010) 75–80

was injected into the lateral ventricle or the cisterna magna. Centraladministration of 6-OHDA caused a substantial NA depletion invarious brain regions and alterations in peripheral immune para-meters including suppressed antibody production, impaired cytokineproduction and decreased cell proliferation (Cross et al., 1986; Crossand Roszman, 1988; Pacheco-López et al., 2003). However, since theneurotoxic actions of 6-OHDA are not restricted to noradrenergicneurons but also affect dopaminergic neurons, it is possible that theobserved immune changes resulted from the lack of central dopamine(DA) and not from noradrenergic depletion. This is particularlyimportant in view of recent findings from our laboratory showing thatcentral DA depletion indeed can lead to suppression of peripheralcytokine production (Engler et al., 2009). Therefore, additionalstudies are needed to further elucidate the specific role of the centralnoradrenergic system in peripheral immune regulation.

The present study aimed at investigating in adult rats whetherthe destruction of central noradrenergic neurons with the selectiveneurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)would affect cytokine production by monocytes/macrophages and Tcells from the spleen. The spleen was chosen since its innervation ispredominately sympathetic and regions that are rich in macrophagesand T cells are densely innervated by noradrenergic nerve fibers (Feltenand Olschowka, 1987; Nance and Sanders, 2007). In addition,transneuronal retrograde tracing studies have shown that the pre-preganglionic neurons projecting to the spleen are located in thenoradrenergic cell groups A5 and A6 (Buijs et al., 2008; Cano et al.,2001).

2. Materials and methods

2.1. Animals

Adult male Fischer 344 rats (280–300 g) were purchased fromHarlan Europe (Horst, The Netherlands) andwere individually housedin standard plastic cages with metal wire lids. Animals weremaintained on a reversed 12:12-h light/dark cycle (lights off at0700h) and had ad libitum access to water and standard diet. Ratswere allowed to acclimate to the new surroundings for 2 weeks beforeinitiation of any experimental procedure. Procedures were approvedby the Cantonal Veterinary Office of Zurich and are in accordance withthe Swiss Federal Act on Animal Protection and the Swiss AnimalProtection Ordinance.

2.2. Noradrenaline depletion

The neurotoxin N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine(DSP-4) hydrochloride (Sigma-Aldrich, Buchs, Switzerland) was usedfor the destruction of noradrenergic neurons in the brain (Jonssonet al., 1981; Dudley et al., 1990). DSP-4 is a site-directed alkylatingagent with high affinity for the NA transporter. Following peripheraladministration, it readily crosses the blood-brain barrier and producesa retrograde degeneration of noradrenergic neurons originating fromthe LC (Fritschy and Grzanna, 1991). The selectivity of DSP-4 forNA neurons depends on the species and the strain in which it isadministered. In some rodent strains, DSP-4 treatment has beenshown to affect brain serotonin levels as well, although to a muchlesser extent than NA (Fornai et al., 1996). However, previous studieshave demonstrated that DSP-4 treatment of Fischer 344 rats affectsonly noradrenergic neurons, leaving serotonergic and dopaminergicneurons intact (Chrobak et al., 1985; Martin and Elgin, 1988). Forinjection, DSP-4 was dissolved in sterile normal saline (B. BraunMelsungen, Melsungen, Germany) and was administered intraper-itoneally at a dose of 50 mg/kg. Because of its instability in solution,the drug was dissolved just prior to use. Control animals receivedinjections of 0.5 ml of normal saline.

2.3. Tissue collection

Samples were collected 8 weeks after DSP-4 or saline injection.Animals were deeply anesthetized by inhalation of isoflurane (Attane,Mirnrad Inc., NY, USA). The spleen was aseptically removed and onepart of the organ was snap-frozen in liquid nitrogen and stored at−80 °C for later determination of NA levels. The other part wastransferred to a sterile tube containing Hanks' balanced salt solution(HBSS, Invitrogen, Basel, Switzerland) and was used for immunolog-ical analyses. After removal of the spleen, the animals weretranscardially perfused with 0.01 M phosphate-buffered saline (PBS)followed by 0.1 M PBS containing 4% paraformaldehyde (PFA). Afterperfusion, the brain was removed and postfixed in 0.1 M PBSwith PFAfor 24 h at 4 °C.

2.4. Splenic cytokine production

The functional capacity of the splenocytes from DSP-4 and saline-injected animals was assessed using a standard ex vivo stimulationassay. Single cell suspensions of the spleen were obtained bymechanically disrupting the tissue in cold HBSS (Invitrogen). Redblood cells were removed using PharM Lyse (BD Pharmingen,Allschwil, Switzerland). Splenocytes were washed with cold cellculture medium (RPMI 1640 supplemented with GlutaMAX I, 25 mMHEPES, 10% fetal bovine serum, 50 μg/ml gentamicin; Invitrogen) andfiltered through a 70-µm nylon cell strainer. Cell concentrations weredetermined with an automatic animal cell counter (Vet abc, scilanimal care company GmbH, Viernheim, Germany) and adjusted to afinal concentration of 2.5×106 cells/ml in cell culture medium.Splenocytes (5×105 cells) were stimulated in 96-well flat-bottommicrotiter plates with either 10 μg/ml lipopolysaccharide (LPS;Escherichia coli serotype 0111:B4, Sigma-Aldrich) for 24 h or 1 µg/mlsoluble anti-rat CD3 monoclonal antibody (NA/LE, clone: G4.18; BDPharmingen) for 72 h. After incubation (37 °C, 5% CO2, humidifiedatmosphere), culture supernatants were collected and stored at−80 °C until analysis. Basal cytokine production was determined inunstimulated samples.

2.5. Cytokine determination

Cytokine concentrations in culture supernatants were quantifiedusing multiplexed bead-based assays (Bio-Plex Cytokine Assays, Bio-Rad Laboratories AG, Reinach, Switzerland). Samples were preparedaccording to the manufacturer's instructions and were analyzed on adual-laser LSR II flow cytometer using FACSDiva software (BDImmunocytometry Systems, Allschwil, Switzerland). Absolute cyto-kine levels were calculated based on the mean fluorescence intensityof cytokine standard dilutions.

2.6. Leukocyte phenotyping

Splenocyte suspensions were incubated at 4 °C for 45 minwith thefollowing fluorochrome-conjugated monoclonal antibodies: anti-ratCD3 (clone 1F4, BD Pharmingen), anti-rat CD4 (clone OX-35, BDPharmingen), anti-rat CD8a (clone OX-8, BD Pharmingen), anti-ratCD45 (clone OX-1, BD Pharmingen), anti-rat CD45RA (clone OX-33,BD Pharmingen), anti-rat CD161 (clone 10/78, AbD Serotec, Düssel-dorf, Germany), anti-rat CD172a (clone ED9, AbD Serotec). Antibodylabeling was performed by a standard lyse-wash procedure usingFACS lysing solution (BD Immunocytometry Systems) and supple-mented PBS (Dulbecco's PBS, 2% fetal bovine serum, 0.1% NaN3). Tenthousand cells per sample were analyzed on a LSR II flow cytometerusing FACS Diva software (BD Immunocytometry Systems). Lympho-cytes, monocytes/macrophages and neutrophil granulocytes wereidentified by forward and side scatter characteristics and differencesin CD45, CD4 and CD172a expression. Lymphocyte subpopulations

77H. Engler et al. / Journal of Neuroimmunology 219 (2010) 75–80

were identified by lineage specific markers: CD3+/CD4+ (T helpercells), CD3+/CD8a+ (cytotoxic T cells), CD3−/CD161+ (natural killercells), CD3−/CD45RA+ (B cells).

2.7. Splenic noradrenaline content

Splenic NA levels were determined by HPLC as previouslydescribed elsewhere (del Rey et al., 2006). Briefly, tissue sampleswere homogenized in 0.4 M perchloric acid, centrifuged, and aliquotsof the supernatant were injected into an HPLC system withelectrochemical detection. Peaks were quantified by peak heightevaluation using Chromeleon software (Version 6.01, Dionex, Sunny-vale, CA, USA).

2.8. Immunohistochemistry

Serial 40-µm coronal sections were cut through the LC and the A5using a vibratome (Leica VT1000S, Leica Microsystems, Nussloch,Germany). Free floating sections were incubated for 30 min in PBScontaining 0.5% H2O2 to block endogenous peroxidase. After rinsing inPBS, sections were incubated at room temperature for 1 h in PBS with0.3% Triton X-100 (PBS-T) containing 5% normal goat serum (NGS).Sections were then incubated at 4 °C for 24 h with rabbit polyclonalanti-tyrosine hydroxylase (TH) IgG (1:800, Santa Cruz Biotechnology,Santa Cruz, CA, USA) diluted in PBS-T containing 2% NGS. Subse-quently, sections were rinsed and incubated for 1 h with anti-rabbitIgG (1:500, Vector Laboratories, Burlingame, CA, USA) diluted in PBS-Tcontaining 2% NGS, followed by 1% avidin–biotin complex (Vector-stain Elite ABC kit, Vector Laboratories). Finally, sections were washedin 0.1 M Tris–HCl (pH 7.4) and the immunoreaction was visualizedwith 3,3′-diaminobenzidine tetrahydrochloride (1.25%) and 0.08%H2O2 in Tris–HCl.

2.9. Stereology

Stereological analyses were performed with a computer-assistedimage analysis system consisting of a Leica DM5500B microscope(Leica Microsystems, Heerbrugg, Switzerland) equipped with amotorized stage (Märzhäuser, Wetzlar, Germany) and a MicrofireCCD camera (Optronics, Goleta, CA, USA), and Mercator Pro softwarewith Mosaic module (Explora Nova, La Rochelle, France). The opticalfractionator method was used to estimate the total numbers of TH-positive cells in the LC and the A5 in an unbiased way (Howard andReed, 2005). The first section was randomly selected and the sectionsampling fraction (ssf) was 1/2. The optical fractionator was used atregular predetermined dx and dy distances within the LC(150×150 μm) and the A5 (175×175 μm). The area associated witheach frame (asf) was 2500 μm2. The height sampling fraction (hsf)was corresponding to 60% of the section thickness. The total numberof neurons (N) was calculated according to the following formula:N=1/hsf×1/asf×1/ssf×∑Q, where Q equals the number of cellscounted within the dissector. All analyses were carried out by aperson who was blind for the group assignment.

2.10. Statistical analysis

The Shapiro–Wilk test was used to determine whether the datameet the assumption of normality. Group means were compared bytwo-tailed Student's t-test. Treatment effects over time wereevaluated using repeated measures analysis of variance (ANOVA).Results are expressed as mean±SEM. The level of significance was setat pb0.05. Statistics were calculated using SPSS for Windows (Version15.0.1, SPSS, Chicago, IL, USA).

3. Results

3.1. Body mass development

The neurotoxin DSP-4 significantly affected body mass develop-ment (time: F(2,120)=584.82, pb0.001; group: F(1,30)=42.22,pb0.001; time×group: F(2,120)=65.65, pb0.001). Animals treatedwith DSP-4 lost about 8% of their initial body mass within the firstweek after injection. Thereafter, the animals quickly regained weightand body mass had returned to baseline levels at 2 weeks after theneurotoxin administration. Four weeks after injection, body masses ofDSP-4 and saline-treated rats did not significantly differ and bothgroups showed a comparable gain in body mass until sacrifice at8 weeks.

3.2. Brain histology and splenic NA content

Treatment with the neurotoxin DSP-4 resulted in the loss ofnoradrenergic neurons in the LC and the area A5 (Fig. 1). Eight weeksafter neurotoxin administration, the total numbers of TH-immunore-active cells in these brain regions were significantly reduced by 40%and 35%, respectively, compared to saline-injected controls (LC:t=3.96, p=0.001; A5: t=2.28, p=0.039). Splenic NA levels werenot significantly different between DSP-4 and saline-treated animals(t=1.71, p=0.109). No significant group differences in spleen masswere found (control: 802±27 mg, DSP-4: 815±8 mg; t=0.46;p=0.646).

3.3. Splenic cytokine production

Neurotoxin treatmentmarkedly affected the cytokine production byisolated splenocytes (Fig. 2). Eight weeks after DSP-4 administration,the LPS-stimulated secretion of TNF-α, IL-1β, and IL-6 was significantlyreduced by 50%, 31%, and 33%, respectively, compared to saline-injectedanimals (TNF-α: t=5.58, pb0.001; IL-1β: t=5.45, pb0.001; IL-6:t=4.08, p=0.001). In contrast, the production of IL-10 tended to beincreased, but the groupdifferences did not reach statistical significance(t=1.94, p=0.073). Upon T cell receptor stimulation with anti-CD3,the production of IFN-γwas significantly reduced in splenocyte culturesof DSP-4 treated animals compared to saline controls (t=2.53,p=0.024), whereas the secretion of IL-2 and IL-4 remained unaffectedby the neurotoxin treatment (IL-2: t=0.18, p=0.861; IL-4: t=0.32,p=0.753). The production of pro-inflammatory cytokines showed asignificant positive associationwith the number of TH-positive neuronsin the LC (IL-1β: r=0.62, p=0.01; IL-6: r=0.67, p=0.005; TNF-α:r=0.69, p=0.003) whereas IL-10 production showed a significantnegative correlation (r=−0.51, p=0.041).

3.4. Leukocyte subpopulations in the spleen

Flow cytometry analysis showed that DSP-4 treatment had onlysmall effects on leukocyte subpopulations in the spleen (Table 1).Neurotoxin-treated animals showed a significant decrease in thepercentage of natural killer (NK) cells compared to saline-injectedcontrols (t=3.88, p=0.002). Percentages of total T cells (t=0.75,p=0.467), CD4+ T cells (t=0.21, p=0.837), CD8+ T cells (t=1.53,p=0.148), B cells (t=0.69, p=0.499), and monocytes/macrophages(t=1.50, p=0.156) did not significantly differ between the twotreatment groups.

4. Discussion

The neurotoxin DSP-4 is a site-directed alkylating agent with highaffinity for the NA transporter (Jonsson et al., 1981; Dudley et al.,1990). Following systemic injection, it penetrates the blood-brainbarrier, accumulates within noradrenergic nerve terminals, and

Fig. 1. Effect of DSP-4 treatment on brainstem noradrenergic nuclei and splenic noradrenaline content. (A) Photomicrographs of brain slices illustrating the degeneration ofnoradrenergic neurons in the LC and the A5 cell group. Brains were collected 8 weeks after the injection and TH immunohistochemistry was performed. Scale bar=100 µm. (B) Totalnumbers of TH-positive neurons in LC and A5 estimated by stereology, and splenic noradrenaline content shown as ng/g wet tissue weight. Data are expressed as mean and S.E.M.;Student's t-test, *pb0.05, ***pb0.001; n=8 per group.

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produces a retrograde degeneration of noradrenergic neuronsoriginating from the LC. Here we show that intraperitoneal admin-istration of 50 mg/kg DSP-4 in adult Fischer 344 rats induced apronounced loss of TH-positive neurons in the LC and the area A5.Two month after neurotoxin injection, the number of noradrenergicneurons in these brain regions was reduced by 35–40%, which is inline with previous reports using the same dose and route ofadministration (Fritschy and Grzanna, 1992). Importantly, the effectsof DSP-4 are not limited to noradrenergic neurons in the brainstem.Due to thewidespread projections arising from the LC, the destructionof noradrenergic neurons in this nucleus leads to an extensive NAdepletion in the brain and the spinal cord (Chrobak et al., 1985;Jonsson et al., 1981; Martin and Elgin, 1988; Ögren et al., 1980;Srinivasan and Schmidt, 2004).

The degeneration of noradrenergic neurons in the LC and the A5cell group was accompanied by alterations in the function ofperipheral immune cells. Cultured splenocytes of DSP-4-treatedanimals produced significantly lower amounts of IL-1β, IL-6, andTNF-α upon stimulation with bacterial LPS than cells from saline-

injected controls. In addition, the production of IL-10 tended to beincreased following DSP-4 treatment. This was not due to alterationsin the number of cytokine producing cells because the proportion ofsplenic monocytes/macrophages remained unaffected by the neuro-toxin treatment. Hence, a sustained impairment of the centralnoradrenergic system seems to cause a functional shift in spleniccytokine balance by suppressing the production of pro-inflammatorycytokines and, at least partially, promoting the secretion of anti-inflammatory mediators.

The loss of noradrenergic neurons in the brainstem was not onlyassociated with alterations in the production of monocyte/macro-phage-derived cytokines. Splenic immune cells from DSP-4 treatedanimals also produced significantly less IFN-γ upon T cell receptorstimulationwith anti-CD3 antibody. In contrast, the production of IL-2and IL-4 remained unaffected. Interleukin-2 is mainly produced by Tcells and stimulates the production of IFN-γ by both T and NK cells(Handa et al., 1983). DSP-4 treatment caused a progressive decreasein NK cells but did not affect the number of splenic T cells. Thus, thereduction in IFN-γ production might have been the consequence of

Fig. 2. Effect of DSP-4 treatment on ex vivo cytokine production by splenocytes. The neurotoxin was administered intraperitoneally and spleens were collected 8 weeks after theinjection. Splenocytes were stimulated with (A) 10 μg/ml lipopolysaccharide or (B) 1 μg/ml anti-CD3 mAb, and cytokine levels were determined in culture supernatants. Data areexpressed as mean and S.E.M.; Student's t-test, *pb0.05, **pb0.01, ***pb0.001; n=8 per group.

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the changes in the cellular composition of the spleen. However, IFN-γlevels in the culture supernatants were not correlated with splenic NKcell numbers. Therefore, it is more likely that central NA depletioninduced a selective suppression of IFN-γ production in T cells withoutaffecting the synthesis of IL-2. Although both IFN-γ and IL-2 areprototypical T helper cell type 1 (Th1) cytokines, their expression in Tcells was shown to be independently regulated (Penix et al., 1993).

Earlier reports have shown that DSP-4 induces not only a long-lasting depletion of NA in the brain but causes also an acute reductionof NA levels in peripheral tissues including the spleen (Archer et al.,1982; Fety et al., 1986; Jaim-Etcheverry and Zieher, 1980; Shirokawaet al., 2000). The decrease in splenic NA content following DSP-4treatment (20–40%) was less pronounced compared to the massiveNA depletion (80–90%) after peripheral chemical sympathectomywith 6-OHDA (Bellinger et al., 2005; Besedovsky et al., 1979; Maddenet al., 1989). Nevertheless, peripheral sympathectomy has beenshown to affect splenic cytokine production (Callahan and Moynihan,2002; Madden et al., 2000). For example, Madden et al. (2000) founda suppression of IFN-γ production, but not IL-2 production, byconcanavalin-A stimulated splenocytes from sympathectomized F344rats. This effect was fully reversible after the recovery of splenic NAlevels. To exclude the possibility that a reduction of splenic NA levelswas responsible for the DSP-4 induced alterations in splenic cytokine

Table 1Effect of DSP-4 treatment on leukocyte subpopulations in the spleen.

Cell type Control DSP-4

Total T cells 29.9±0.9 30.7±0.6CD4+ T cells 19.6±0.8 19.7±0.4CD8+ T cells 10.4±0.3 11.0±0.2B cells 45.7±0.9 46.5±0.7NK cells 6.4±0.1 5.5±0.2**Monocytes/macrophages 4.7±0.3 4.1±0.2

Values represent percentage of cells. Data are expressed as mean±S.E.M.Student's t-test, **pb0.01; n=8 per group.

production, we compared the NA content of the spleens from saline-and neurotoxin-injected animals. Importantly, there was no differ-ence in splenic NA concentration between the two treatment groupsat 8 weeks after neurotoxin administration, suggesting that theobserved effects on cytokine production were indeed the conse-quence of central NA depletion. This is supported by the results of thecorrelation analyses that revealed strong relationships between thenumber of TH-positive cells in the LC and the amounts of cytokinesproduced by the isolated splenocytes.

In summary, this study provides novel data on the impact of centralNAdepletion onperipheral immune function and suggests that a certainlevel of central noradrenergic tone is required for normal immunefunctioning in the periphery. However, it remains open whetherthe observed immune effects are a direct consequence of reducednoradrenergic output from the brainstemor an indirect result of alteredneurotransmission in brain regions to which the LC and A5 project.Further studies are needed toelucidate theafferentmechanisms that areinvolved in this process. Importantly, loss of noradrenergic neurons inthe LC and A5 cell group are common features of many neurodegen-erative diseases and psychiatric disorders (Arima and Akashi, 1990;Benarroch et al., 2008; Chan-Palay and Asan, 1989; German et al., 1992;Mann, 1983; Marien et al., 2004; Matthews et al., 2002; Ressler andNemeroff, 1999; Zarow et al., 2003). In addition, although to a lesserextent, the number of noradrenergic LC neurons declines during normalaging (Lohr and Jeste, 1988;Mann, 1983). In thepast, studies on the age-and disease-associated degeneration of the central noradrenergicsystem have primarily focused on behavioral and cognitive deficits.Based on the results of the present study it can be speculated that thealtered cytokine production by peripheral immune cells of elderlypeople and patients suffering from neurodegenerative diseases orpsychiatric disorders might be as well related to a decline in centralnoradrenergic tone (De Luigi et al., 2002; Hasegawa et al., 2000; Pandaet al., 2009; Richartz et al., 2005). The biological and clinical relevance ofthese findings need to be further evaluated in future experiments, e.g.,by using viral and bacterial infection models.

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Acknowledgements

The authors thank Anja Wettstein and Thomas Wyss for excellenttechnical assistance. The study was partly supported by the SwissFederal Institute of Technology (ETH) Zurich and the GermanResearch Foundation (DFG; GK1045/2).

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