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Chemical destruction of brain noradrenergic neurons affects splenic cytokine production
Journal of Neuroimmunology 219 (2010) 75–80
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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
Chemical destruction of brain noradrenergic neurons affects splenic cytokine 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 b c
Institute of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, University of Duisburg-Essen, 45122 Essen, Germany Laboratory of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, ETH Zurich, 8092 Zurich, Switzerland Department of Immunophysiology, Institute of Physiology and Pathophysiology, Philipps University Marburg, 35037 Marburg, Germany
a r t i c l e
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Article history: Received 7 October 2009 Received in revised form 1 December 2009 Accepted 1 December 2009 Keywords: Noradrenaline Brainstem Locus coeruleus DSP-4 Spleen Cytokine
a b s t r a c t The neurotransmitter noradrenaline (NA) plays a pivotal role in immune regulation. Here we used the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to investigate the impact of central NA depletion on cytokine production by splenic monocytes/macrophages and T cells. Intraperitoneal administration of DSP-4 in adult rats induced a substantial reduction of noradrenergic neurons in the locus coeruleus and the A5 cell group. The degeneration of brainstem noradrenergic neurons was accompanied by a significant decrease in the production of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α by lipopolysaccharide-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 and IL-4. The proportion of monocytes/macrophages and T cells in the spleen remained unaffected by the neurotoxin treatment, however, the percentage of natural killer cells decreased significantly. The findings suggest that a certain level of central noradrenergic tone is required for normal functioning of peripheral immune cells. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The sympathetic nervous system (SNS) and its principal neurotransmitter, noradrenaline (NA), play a pivotal role in the neural regulation 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 by sympathetic nerve fibers and noradrenergic terminals are found in the close vicinity of lymphocytes and macrophages, forming synapse-like contacts (Felten et al., 1985). Moreover, adrenergic receptors, mainly of the β2-subtype, have been identified on most of the cells that participate in innate and adaptive immune responses, providing the biochemical basis for noradrenergic influences on immune cells (Kin and Sanders, 2006). Further evidence for the involvement of the SNS in immune regulation derived from pharmacological studies showing that systemic administration of exogenous NA alters leukocyte distribution and cytokine production in humans (Schedlowski et al., 1993, 1996; Torres et al., 2005). In addition, the mobilization of immune cells during the acute stress response was found to be mediated by the SNS (Benschop et al., 1996; Engler et al., 2004). ⁎ Corresponding author. Institute of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, Hufelandstrasse 55, D-45122 Essen, Germany. Tel.: +49 201 723 4506; fax: +49 201 723 5948. E-mail address: [email protected] (H. Engler). 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.12.001
However, not only increased peripheral NA levels but also lack in sympathetic output following chemical destruction of peripheral noradrenergic nerve terminals with the neurotoxin 6-hydroxydopamine (6-OHDA) has been shown to be associated with immune alterations in lymphoid tissues (Bellinger et al., 2005; Besedovsky et al., 1979; Madden et al., 1989). This suggests that a certain level of sympathetic tone is required for normal immune functioning in the periphery. The vast majority of studies investigating the role of NA in immune regulation have focused on the peripheral SNS, whereas very little is known about the central noradrenergic system. In the brain, the largest cluster of noradrenergic cell bodies is located within the locus coeruleus (LC/A6). Projections from this brainstem nucleus are extremely widespread, innervating the entire cortex, subcortical regions (e.g., hippocampus, amygdala, thalamus, hypothalamus, and cerebellum), other brainstem nuclei, and the spinal cord (Jones and Moore, 1977; Sara, 2009). Bilateral electrolytic lesions of the LC were shown to affect humoral and cellular immune responses in rats indicating a potential involvement of this nucleus in neuro-immune communication (Jovanova-Nesic et al., 1993; Nikolic et al., 1993). However, a major drawback of this technique is the lack of specificity for a certain neurotransmitter, damaging all neurons and fibers of passage in the targeted area. Further evidence for the role of the brain noradrenergic system in peripheral immune regulation derived from animal studies in which the catecholaminergic neurotoxin 6-OHDA
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was injected into the lateral ventricle or the cisterna magna. Central administration of 6-OHDA caused a substantial NA depletion in various brain regions and alterations in peripheral immune parameters including suppressed antibody production, impaired cytokine production and decreased cell proliferation (Cross et al., 1986; Cross and Roszman, 1988; Pacheco-López et al., 2003). However, since the neurotoxic actions of 6-OHDA are not restricted to noradrenergic neurons but also affect dopaminergic neurons, it is possible that the observed immune changes resulted from the lack of central dopamine (DA) and not from noradrenergic depletion. This is particularly important in view of recent findings from our laboratory showing that central DA depletion indeed can lead to suppression of peripheral cytokine production (Engler et al., 2009). Therefore, additional studies are needed to further elucidate the specific role of the central noradrenergic system in peripheral immune regulation. The present study aimed at investigating in adult rats whether the destruction of central noradrenergic neurons with the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) would affect cytokine production by monocytes/macrophages and T cells from the spleen. The spleen was chosen since its innervation is predominately sympathetic and regions that are rich in macrophages and T cells are densely innervated by noradrenergic nerve fibers (Felten and Olschowka, 1987; Nance and Sanders, 2007). In addition, transneuronal retrograde tracing studies have shown that the prepreganglionic neurons projecting to the spleen are located in the noradrenergic 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 from Harlan Europe (Horst, The Netherlands) and were individually housed in standard plastic cages with metal wire lids. Animals were maintained on a reversed 12:12-h light/dark cycle (lights off at 0700h) and had ad libitum access to water and standard diet. Rats were allowed to acclimate to the new surroundings for 2 weeks before initiation of any experimental procedure. Procedures were approved by the Cantonal Veterinary Office of Zurich and are in accordance with the Swiss Federal Act on Animal Protection and the Swiss Animal Protection Ordinance. 2.2. Noradrenaline depletion The neurotoxin N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine (DSP-4) hydrochloride (Sigma-Aldrich, Buchs, Switzerland) was used for the destruction of noradrenergic neurons in the brain (Jonsson et al., 1981; Dudley et al., 1990). DSP-4 is a site-directed alkylating agent with high affinity for the NA transporter. Following peripheral administration, it readily crosses the blood-brain barrier and produces a retrograde degeneration of noradrenergic neurons originating from the LC (Fritschy and Grzanna, 1991). The selectivity of DSP-4 for NA neurons depends on the species and the strain in which it is administered. In some rodent strains, DSP-4 treatment has been shown to affect brain serotonin levels as well, although to a much lesser extent than NA (Fornai et al., 1996). However, previous studies have demonstrated that DSP-4 treatment of Fischer 344 rats affects only noradrenergic neurons, leaving serotonergic and dopaminergic neurons intact (Chrobak et al., 1985; Martin and Elgin, 1988). For injection, DSP-4 was dissolved in sterile normal saline (B. Braun Melsungen, Melsungen, Germany) and was administered intraperitoneally at a dose of 50 mg/kg. Because of its instability in solution, the drug was dissolved just prior to use. Control animals received injections 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 one part of the organ was snap-frozen in liquid nitrogen and stored at −80 °C for later determination of NA levels. The other part was transferred to a sterile tube containing Hanks' balanced salt solution (HBSS, Invitrogen, Basel, Switzerland) and was used for immunological analyses. After removal of the spleen, the animals were transcardially perfused with 0.01 M phosphate-buffered saline (PBS) followed by 0.1 M PBS containing 4% paraformaldehyde (PFA). After perfusion, the brain was removed and postfixed in 0.1 M PBS with PFA for 24 h at 4 °C. 2.4. Splenic cytokine production The functional capacity of the splenocytes from DSP-4 and salineinjected animals was assessed using a standard ex vivo stimulation assay. Single cell suspensions of the spleen were obtained by mechanically disrupting the tissue in cold HBSS (Invitrogen). Red blood cells were removed using PharM Lyse (BD Pharmingen, Allschwil, Switzerland). Splenocytes were washed with cold cell culture medium (RPMI 1640 supplemented with GlutaMAX I, 25 mM HEPES, 10% fetal bovine serum, 50 μg/ml gentamicin; Invitrogen) and filtered through a 70-µm nylon cell strainer. Cell concentrations were determined with an automatic animal cell counter (Vet abc, scil animal care company GmbH, Viernheim, Germany) and adjusted to a final concentration of 2.5 × 106 cells/ml in cell culture medium. Splenocytes (5 × 105 cells) were stimulated in 96-well flat-bottom microtiter plates with either 10 μg/ml lipopolysaccharide (LPS; Escherichia coli serotype 0111:B4, Sigma-Aldrich) for 24 h or 1 µg/ml soluble anti-rat CD3 monoclonal antibody (NA/LE, clone: G4.18; BD Pharmingen) for 72 h. After incubation (37 °C, 5% CO2, humidified atmosphere), culture supernatants were collected and stored at −80 °C until analysis. Basal cytokine production was determined in unstimulated samples. 2.5. Cytokine determination Cytokine concentrations in culture supernatants were quantified using multiplexed bead-based assays (Bio-Plex Cytokine Assays, BioRad Laboratories AG, Reinach, Switzerland). Samples were prepared according to the manufacturer's instructions and were analyzed on a dual-laser LSR II flow cytometer using FACSDiva software (BD Immunocytometry Systems, Allschwil, Switzerland). Absolute cytokine levels were calculated based on the mean fluorescence intensity of cytokine standard dilutions. 2.6. Leukocyte phenotyping Splenocyte suspensions were incubated at 4 °C for 45 min with the following fluorochrome-conjugated monoclonal antibodies: anti-rat CD3 (clone 1F4, BD Pharmingen), anti-rat CD4 (clone OX-35, BD Pharmingen), anti-rat CD8a (clone OX-8, BD Pharmingen), anti-rat CD45 (clone OX-1, BD Pharmingen), anti-rat CD45RA (clone OX-33, BD Pharmingen), anti-rat CD161 (clone 10/78, AbD Serotec, Düsseldorf, Germany), anti-rat CD172a (clone ED9, AbD Serotec). Antibody labeling was performed by a standard lyse-wash procedure using FACS lysing solution (BD Immunocytometry Systems) and supplemented PBS (Dulbecco's PBS, 2% fetal bovine serum, 0.1% NaN3). Ten thousand cells per sample were analyzed on a LSR II flow cytometer using FACS Diva software (BD Immunocytometry Systems). Lymphocytes, monocytes/macrophages and neutrophil granulocytes were identified by forward and side scatter characteristics and differences in CD45, CD4 and CD172a expression. Lymphocyte subpopulations
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were identified by lineage specific markers: CD3+/CD4+ (T helper cells), CD3+/CD8a+ (cytotoxic T cells), CD3−/CD161+ (natural killer cells), CD3−/CD45RA+ (B cells).
2.7. Splenic noradrenaline content Splenic NA levels were determined by HPLC as previously described elsewhere (del Rey et al., 2006). Briefly, tissue samples were homogenized in 0.4 M perchloric acid, centrifuged, and aliquots of the supernatant were injected into an HPLC system with electrochemical detection. Peaks were quantified by peak height evaluation using Chromeleon software (Version 6.01, Dionex, Sunnyvale, CA, USA).
2.8. Immunohistochemistry Serial 40-µm coronal sections were cut through the LC and the A5 using a vibratome (Leica VT1000S, Leica Microsystems, Nussloch, Germany). Free floating sections were incubated for 30 min in PBS containing 0.5% H2O2 to block endogenous peroxidase. After rinsing in PBS, sections were incubated at room temperature for 1 h in PBS with 0.3% Triton X-100 (PBS-T) containing 5% normal goat serum (NGS). Sections were then incubated at 4 °C for 24 h with rabbit polyclonal anti-tyrosine hydroxylase (TH) IgG (1:800, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in PBS-T containing 2% NGS. Subsequently, sections were rinsed and incubated for 1 h with anti-rabbit IgG (1:500, Vector Laboratories, Burlingame, CA, USA) diluted in PBS-T containing 2% NGS, followed by 1% avidin–biotin complex (Vectorstain Elite ABC kit, Vector Laboratories). Finally, sections were washed in 0.1 M Tris–HCl (pH 7.4) and the immunoreaction was visualized with 3,3′-diaminobenzidine tetrahydrochloride (1.25%) and 0.08% H2O2 in Tris–HCl.
2.9. Stereology Stereological analyses were performed with a computer-assisted image analysis system consisting of a Leica DM5500B microscope (Leica Microsystems, Heerbrugg, Switzerland) equipped with a motorized stage (Märzhäuser, Wetzlar, Germany) and a Microfire CCD camera (Optronics, Goleta, CA, USA), and Mercator Pro software with Mosaic module (Explora Nova, La Rochelle, France). The optical fractionator method was used to estimate the total numbers of THpositive cells in the LC and the A5 in an unbiased way (Howard and Reed, 2005). The first section was randomly selected and the section sampling fraction (ssf) was 1/2. The optical fractionator was used at regular predetermined dx and dy distances within the LC (150 × 150 μm) and the A5 (175 × 175 μm). The area associated with each frame (asf) was 2500 μm2. The height sampling fraction (hsf) was corresponding to 60% of the section thickness. The total number of neurons (N) was calculated according to the following formula: N = 1 / hsf × 1 / asf × 1 / ssf × ∑Q, where Q equals the number of cells counted within the dissector. All analyses were carried out by a person who was blind for the group assignment.
2.10. Statistical analysis The Shapiro–Wilk test was used to determine whether the data meet the assumption of normality. Group means were compared by two-tailed Student's t-test. Treatment effects over time were evaluated using repeated measures analysis of variance (ANOVA). Results are expressed as mean ± SEM. The level of significance was set at p b 0.05. Statistics were calculated using SPSS for Windows (Version 15.0.1, SPSS, Chicago, IL, USA).
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3. Results 3.1. Body mass development The neurotoxin DSP-4 significantly affected body mass development (time: F(2,120) = 584.82, p b 0.001; group: F(1,30) = 42.22, p b 0.001; time × group: F(2,120) = 65.65, p b 0.001). Animals treated with DSP-4 lost about 8% of their initial body mass within the first week after injection. Thereafter, the animals quickly regained weight and body mass had returned to baseline levels at 2 weeks after the neurotoxin administration. Four weeks after injection, body masses of DSP-4 and saline-treated rats did not significantly differ and both groups showed a comparable gain in body mass until sacrifice at 8 weeks. 3.2. Brain histology and splenic NA content Treatment with the neurotoxin DSP-4 resulted in the loss of noradrenergic neurons in the LC and the area A5 (Fig. 1). Eight weeks after neurotoxin administration, the total numbers of TH-immunoreactive 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 were not significantly different between DSP-4 and saline-treated animals (t = 1.71, p = 0.109). No significant group differences in spleen mass were found (control: 802 ± 27 mg, DSP-4: 815 ± 8 mg; t = 0.46; p = 0.646). 3.3. Splenic cytokine production Neurotoxin treatment markedly affected the cytokine production by isolated splenocytes (Fig. 2). Eight weeks after DSP-4 administration, the LPS-stimulated secretion of TNF-α, IL-1β, and IL-6 was significantly reduced by 50%, 31%, and 33%, respectively, compared to saline-injected animals (TNF-α: t = 5.58, p b 0.001; IL-1β: t = 5.45, p b 0.001; IL-6: t = 4.08, p = 0.001). In contrast, the production of IL-10 tended to be increased, but the group differences did not reach statistical significance (t = 1.94, p = 0.073). Upon T cell receptor stimulation with anti-CD3, the production of IFN-γ was significantly reduced in splenocyte cultures of DSP-4 treated animals compared to saline controls (t = 2.53, p = 0.024), whereas the secretion of IL-2 and IL-4 remained unaffected by 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 a significant positive association with the number of TH-positive neurons in 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 significant negative correlation (r = −0.51, p = 0.041). 3.4. Leukocyte subpopulations in the spleen Flow cytometry analysis showed that DSP-4 treatment had only small effects on leukocyte subpopulations in the spleen (Table 1). Neurotoxin-treated animals showed a significant decrease in the percentage of natural killer (NK) cells compared to saline-injected controls (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 two treatment groups. 4. Discussion The neurotoxin DSP-4 is a site-directed alkylating agent with high affinity for the NA transporter (Jonsson et al., 1981; Dudley et al., 1990). Following systemic injection, it penetrates the blood-brain barrier, accumulates within noradrenergic nerve terminals, and
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Fig. 1. Effect of DSP-4 treatment on brainstem noradrenergic nuclei and splenic noradrenaline content. (A) Photomicrographs of brain slices illustrating the degeneration of noradrenergic 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) Total numbers 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, *p b 0.05, ***p b 0.001; n = 8 per group.
produces a retrograde degeneration of noradrenergic neurons originating from the LC. Here we show that intraperitoneal administration of 50 mg/kg DSP-4 in adult Fischer 344 rats induced a pronounced loss of TH-positive neurons in the LC and the area A5. Two month after neurotoxin injection, the number of noradrenergic neurons in these brain regions was reduced by 35–40%, which is in line with previous reports using the same dose and route of administration (Fritschy and Grzanna, 1992). Importantly, the effects of DSP-4 are not limited to noradrenergic neurons in the brainstem. Due to the widespread projections arising from the LC, the destruction of noradrenergic neurons in this nucleus leads to an extensive NA depletion 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 A5 cell group was accompanied by alterations in the function of peripheral immune cells. Cultured splenocytes of DSP-4-treated animals produced significantly lower amounts of IL-1β, IL-6, and TNF-α upon stimulation with bacterial LPS than cells from saline-
injected controls. In addition, the production of IL-10 tended to be increased following DSP-4 treatment. This was not due to alterations in the number of cytokine producing cells because the proportion of splenic monocytes/macrophages remained unaffected by the neurotoxin treatment. Hence, a sustained impairment of the central noradrenergic system seems to cause a functional shift in splenic cytokine balance by suppressing the production of pro-inflammatory cytokines and, at least partially, promoting the secretion of antiinflammatory mediators. The loss of noradrenergic neurons in the brainstem was not only associated with alterations in the production of monocyte/macrophage-derived cytokines. Splenic immune cells from DSP-4 treated animals also produced significantly less IFN-γ upon T cell receptor stimulation with anti-CD3 antibody. In contrast, the production of IL-2 and IL-4 remained unaffected. Interleukin-2 is mainly produced by T cells and stimulates the production of IFN-γ by both T and NK cells (Handa et al., 1983). DSP-4 treatment caused a progressive decrease in NK cells but did not affect the number of splenic T cells. Thus, the reduction in IFN-γ production might have been the consequence of
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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 the injection. 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 are expressed as mean and S.E.M.; Student's t-test, *p b 0.05, **p b 0.01, ***p b 0.001; n = 8 per group.
the changes in the cellular composition of the spleen. However, IFN-γ levels in the culture supernatants were not correlated with splenic NK cell numbers. Therefore, it is more likely that central NA depletion induced a selective suppression of IFN-γ production in T cells without affecting the synthesis of IL-2. Although both IFN-γ and IL-2 are prototypical T helper cell type 1 (Th1) cytokines, their expression in T cells was shown to be independently regulated (Penix et al., 1993). Earlier reports have shown that DSP-4 induces not only a longlasting depletion of NA in the brain but causes also an acute reduction of NA levels in peripheral tissues including the spleen (Archer et al., 1982; Fety et al., 1986; Jaim-Etcheverry and Zieher, 1980; Shirokawa et al., 2000). The decrease in splenic NA content following DSP-4 treatment (20–40%) was less pronounced compared to the massive NA depletion (80–90%) after peripheral chemical sympathectomy with 6-OHDA (Bellinger et al., 2005; Besedovsky et al., 1979; Madden et al., 1989). Nevertheless, peripheral sympathectomy has been shown to affect splenic cytokine production (Callahan and Moynihan, 2002; Madden et al., 2000). For example, Madden et al. (2000) found a suppression of IFN-γ production, but not IL-2 production, by concanavalin-A stimulated splenocytes from sympathectomized F344 rats. This effect was fully reversible after the recovery of splenic NA levels. To exclude the possibility that a reduction of splenic NA levels was responsible for the DSP-4 induced alterations in splenic cytokine Table 1 Effect of DSP-4 treatment on leukocyte subpopulations in the spleen. Cell type
Control
DSP-4
Total T cells CD4+ T cells CD8+ T cells B cells NK cells Monocytes/macrophages
29.9 ± 0.9 19.6 ± 0.8 10.4 ± 0.3 45.7 ± 0.9 6.4 ± 0.1 4.7 ± 0.3
30.7 ± 0.6 19.7 ± 0.4 11.0 ± 0.2 46.5 ± 0.7 5.5 ± 0.2** 4.1 ± 0.2
Values represent percentage of cells. Data are expressed as mean ± S.E.M. Student's t-test, **p b 0.01; n = 8 per group.
production, we compared the NA content of the spleens from salineand neurotoxin-injected animals. Importantly, there was no difference in splenic NA concentration between the two treatment groups at 8 weeks after neurotoxin administration, suggesting that the observed effects on cytokine production were indeed the consequence of central NA depletion. This is supported by the results of the correlation analyses that revealed strong relationships between the number of TH-positive cells in the LC and the amounts of cytokines produced by the isolated splenocytes. In summary, this study provides novel data on the impact of central NA depletion on peripheral immune function and suggests that a certain level of central noradrenergic tone is required for normal immune functioning in the periphery. However, it remains open whether the observed immune effects are a direct consequence of reduced noradrenergic output from the brainstem or an indirect result of altered neurotransmission in brain regions to which the LC and A5 project. Further studies are needed to elucidate the afferent mechanisms that are involved in this process. Importantly, loss of noradrenergic neurons in the LC and A5 cell group are common features of many neurodegenerative 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 and Nemeroff, 1999; Zarow et al., 2003). In addition, although to a lesser extent, the number of noradrenergic LC neurons declines during normal aging (Lohr and Jeste, 1988; Mann, 1983). In the past, studies on the ageand disease-associated degeneration of the central noradrenergic system have primarily focused on behavioral and cognitive deficits. Based on the results of the present study it can be speculated that the altered cytokine production by peripheral immune cells of elderly people and patients suffering from neurodegenerative diseases or psychiatric disorders might be as well related to a decline in central noradrenergic tone (De Luigi et al., 2002; Hasegawa et al., 2000; Panda et al., 2009; Richartz et al., 2005). The biological and clinical relevance of these 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 excellent technical assistance. The study was partly supported by the Swiss Federal Institute of Technology (ETH) Zurich and the German Research Foundation (DFG; GK1045/2).
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Contents lists available at ScienceDirect
Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
Chemical destruction of brain noradrenergic neurons affects splenic cytokine 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 b c
Institute of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, University of Duisburg-Essen, 45122 Essen, Germany Laboratory of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, ETH Zurich, 8092 Zurich, Switzerland Department of Immunophysiology, Institute of Physiology and Pathophysiology, Philipps University Marburg, 35037 Marburg, Germany
a r t i c l e
i n f o
Article history: Received 7 October 2009 Received in revised form 1 December 2009 Accepted 1 December 2009 Keywords: Noradrenaline Brainstem Locus coeruleus DSP-4 Spleen Cytokine
a b s t r a c t The neurotransmitter noradrenaline (NA) plays a pivotal role in immune regulation. Here we used the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to investigate the impact of central NA depletion on cytokine production by splenic monocytes/macrophages and T cells. Intraperitoneal administration of DSP-4 in adult rats induced a substantial reduction of noradrenergic neurons in the locus coeruleus and the A5 cell group. The degeneration of brainstem noradrenergic neurons was accompanied by a significant decrease in the production of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α by lipopolysaccharide-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 and IL-4. The proportion of monocytes/macrophages and T cells in the spleen remained unaffected by the neurotoxin treatment, however, the percentage of natural killer cells decreased significantly. The findings suggest that a certain level of central noradrenergic tone is required for normal functioning of peripheral immune cells. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The sympathetic nervous system (SNS) and its principal neurotransmitter, noradrenaline (NA), play a pivotal role in the neural regulation 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 by sympathetic nerve fibers and noradrenergic terminals are found in the close vicinity of lymphocytes and macrophages, forming synapse-like contacts (Felten et al., 1985). Moreover, adrenergic receptors, mainly of the β2-subtype, have been identified on most of the cells that participate in innate and adaptive immune responses, providing the biochemical basis for noradrenergic influences on immune cells (Kin and Sanders, 2006). Further evidence for the involvement of the SNS in immune regulation derived from pharmacological studies showing that systemic administration of exogenous NA alters leukocyte distribution and cytokine production in humans (Schedlowski et al., 1993, 1996; Torres et al., 2005). In addition, the mobilization of immune cells during the acute stress response was found to be mediated by the SNS (Benschop et al., 1996; Engler et al., 2004). ⁎ Corresponding author. Institute of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, Hufelandstrasse 55, D-45122 Essen, Germany. Tel.: +49 201 723 4506; fax: +49 201 723 5948. E-mail address: [email protected] (H. Engler). 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.12.001
However, not only increased peripheral NA levels but also lack in sympathetic output following chemical destruction of peripheral noradrenergic nerve terminals with the neurotoxin 6-hydroxydopamine (6-OHDA) has been shown to be associated with immune alterations in lymphoid tissues (Bellinger et al., 2005; Besedovsky et al., 1979; Madden et al., 1989). This suggests that a certain level of sympathetic tone is required for normal immune functioning in the periphery. The vast majority of studies investigating the role of NA in immune regulation have focused on the peripheral SNS, whereas very little is known about the central noradrenergic system. In the brain, the largest cluster of noradrenergic cell bodies is located within the locus coeruleus (LC/A6). Projections from this brainstem nucleus are extremely widespread, innervating the entire cortex, subcortical regions (e.g., hippocampus, amygdala, thalamus, hypothalamus, and cerebellum), other brainstem nuclei, and the spinal cord (Jones and Moore, 1977; Sara, 2009). Bilateral electrolytic lesions of the LC were shown to affect humoral and cellular immune responses in rats indicating a potential involvement of this nucleus in neuro-immune communication (Jovanova-Nesic et al., 1993; Nikolic et al., 1993). However, a major drawback of this technique is the lack of specificity for a certain neurotransmitter, damaging all neurons and fibers of passage in the targeted area. Further evidence for the role of the brain noradrenergic system in peripheral immune regulation derived from animal studies in which the catecholaminergic neurotoxin 6-OHDA
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was injected into the lateral ventricle or the cisterna magna. Central administration of 6-OHDA caused a substantial NA depletion in various brain regions and alterations in peripheral immune parameters including suppressed antibody production, impaired cytokine production and decreased cell proliferation (Cross et al., 1986; Cross and Roszman, 1988; Pacheco-López et al., 2003). However, since the neurotoxic actions of 6-OHDA are not restricted to noradrenergic neurons but also affect dopaminergic neurons, it is possible that the observed immune changes resulted from the lack of central dopamine (DA) and not from noradrenergic depletion. This is particularly important in view of recent findings from our laboratory showing that central DA depletion indeed can lead to suppression of peripheral cytokine production (Engler et al., 2009). Therefore, additional studies are needed to further elucidate the specific role of the central noradrenergic system in peripheral immune regulation. The present study aimed at investigating in adult rats whether the destruction of central noradrenergic neurons with the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) would affect cytokine production by monocytes/macrophages and T cells from the spleen. The spleen was chosen since its innervation is predominately sympathetic and regions that are rich in macrophages and T cells are densely innervated by noradrenergic nerve fibers (Felten and Olschowka, 1987; Nance and Sanders, 2007). In addition, transneuronal retrograde tracing studies have shown that the prepreganglionic neurons projecting to the spleen are located in the noradrenergic 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 from Harlan Europe (Horst, The Netherlands) and were individually housed in standard plastic cages with metal wire lids. Animals were maintained on a reversed 12:12-h light/dark cycle (lights off at 0700h) and had ad libitum access to water and standard diet. Rats were allowed to acclimate to the new surroundings for 2 weeks before initiation of any experimental procedure. Procedures were approved by the Cantonal Veterinary Office of Zurich and are in accordance with the Swiss Federal Act on Animal Protection and the Swiss Animal Protection Ordinance. 2.2. Noradrenaline depletion The neurotoxin N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine (DSP-4) hydrochloride (Sigma-Aldrich, Buchs, Switzerland) was used for the destruction of noradrenergic neurons in the brain (Jonsson et al., 1981; Dudley et al., 1990). DSP-4 is a site-directed alkylating agent with high affinity for the NA transporter. Following peripheral administration, it readily crosses the blood-brain barrier and produces a retrograde degeneration of noradrenergic neurons originating from the LC (Fritschy and Grzanna, 1991). The selectivity of DSP-4 for NA neurons depends on the species and the strain in which it is administered. In some rodent strains, DSP-4 treatment has been shown to affect brain serotonin levels as well, although to a much lesser extent than NA (Fornai et al., 1996). However, previous studies have demonstrated that DSP-4 treatment of Fischer 344 rats affects only noradrenergic neurons, leaving serotonergic and dopaminergic neurons intact (Chrobak et al., 1985; Martin and Elgin, 1988). For injection, DSP-4 was dissolved in sterile normal saline (B. Braun Melsungen, Melsungen, Germany) and was administered intraperitoneally at a dose of 50 mg/kg. Because of its instability in solution, the drug was dissolved just prior to use. Control animals received injections 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 one part of the organ was snap-frozen in liquid nitrogen and stored at −80 °C for later determination of NA levels. The other part was transferred to a sterile tube containing Hanks' balanced salt solution (HBSS, Invitrogen, Basel, Switzerland) and was used for immunological analyses. After removal of the spleen, the animals were transcardially perfused with 0.01 M phosphate-buffered saline (PBS) followed by 0.1 M PBS containing 4% paraformaldehyde (PFA). After perfusion, the brain was removed and postfixed in 0.1 M PBS with PFA for 24 h at 4 °C. 2.4. Splenic cytokine production The functional capacity of the splenocytes from DSP-4 and salineinjected animals was assessed using a standard ex vivo stimulation assay. Single cell suspensions of the spleen were obtained by mechanically disrupting the tissue in cold HBSS (Invitrogen). Red blood cells were removed using PharM Lyse (BD Pharmingen, Allschwil, Switzerland). Splenocytes were washed with cold cell culture medium (RPMI 1640 supplemented with GlutaMAX I, 25 mM HEPES, 10% fetal bovine serum, 50 μg/ml gentamicin; Invitrogen) and filtered through a 70-µm nylon cell strainer. Cell concentrations were determined with an automatic animal cell counter (Vet abc, scil animal care company GmbH, Viernheim, Germany) and adjusted to a final concentration of 2.5 × 106 cells/ml in cell culture medium. Splenocytes (5 × 105 cells) were stimulated in 96-well flat-bottom microtiter plates with either 10 μg/ml lipopolysaccharide (LPS; Escherichia coli serotype 0111:B4, Sigma-Aldrich) for 24 h or 1 µg/ml soluble anti-rat CD3 monoclonal antibody (NA/LE, clone: G4.18; BD Pharmingen) for 72 h. After incubation (37 °C, 5% CO2, humidified atmosphere), culture supernatants were collected and stored at −80 °C until analysis. Basal cytokine production was determined in unstimulated samples. 2.5. Cytokine determination Cytokine concentrations in culture supernatants were quantified using multiplexed bead-based assays (Bio-Plex Cytokine Assays, BioRad Laboratories AG, Reinach, Switzerland). Samples were prepared according to the manufacturer's instructions and were analyzed on a dual-laser LSR II flow cytometer using FACSDiva software (BD Immunocytometry Systems, Allschwil, Switzerland). Absolute cytokine levels were calculated based on the mean fluorescence intensity of cytokine standard dilutions. 2.6. Leukocyte phenotyping Splenocyte suspensions were incubated at 4 °C for 45 min with the following fluorochrome-conjugated monoclonal antibodies: anti-rat CD3 (clone 1F4, BD Pharmingen), anti-rat CD4 (clone OX-35, BD Pharmingen), anti-rat CD8a (clone OX-8, BD Pharmingen), anti-rat CD45 (clone OX-1, BD Pharmingen), anti-rat CD45RA (clone OX-33, BD Pharmingen), anti-rat CD161 (clone 10/78, AbD Serotec, Düsseldorf, Germany), anti-rat CD172a (clone ED9, AbD Serotec). Antibody labeling was performed by a standard lyse-wash procedure using FACS lysing solution (BD Immunocytometry Systems) and supplemented PBS (Dulbecco's PBS, 2% fetal bovine serum, 0.1% NaN3). Ten thousand cells per sample were analyzed on a LSR II flow cytometer using FACS Diva software (BD Immunocytometry Systems). Lymphocytes, monocytes/macrophages and neutrophil granulocytes were identified by forward and side scatter characteristics and differences in CD45, CD4 and CD172a expression. Lymphocyte subpopulations
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were identified by lineage specific markers: CD3+/CD4+ (T helper cells), CD3+/CD8a+ (cytotoxic T cells), CD3−/CD161+ (natural killer cells), CD3−/CD45RA+ (B cells).
2.7. Splenic noradrenaline content Splenic NA levels were determined by HPLC as previously described elsewhere (del Rey et al., 2006). Briefly, tissue samples were homogenized in 0.4 M perchloric acid, centrifuged, and aliquots of the supernatant were injected into an HPLC system with electrochemical detection. Peaks were quantified by peak height evaluation using Chromeleon software (Version 6.01, Dionex, Sunnyvale, CA, USA).
2.8. Immunohistochemistry Serial 40-µm coronal sections were cut through the LC and the A5 using a vibratome (Leica VT1000S, Leica Microsystems, Nussloch, Germany). Free floating sections were incubated for 30 min in PBS containing 0.5% H2O2 to block endogenous peroxidase. After rinsing in PBS, sections were incubated at room temperature for 1 h in PBS with 0.3% Triton X-100 (PBS-T) containing 5% normal goat serum (NGS). Sections were then incubated at 4 °C for 24 h with rabbit polyclonal anti-tyrosine hydroxylase (TH) IgG (1:800, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in PBS-T containing 2% NGS. Subsequently, sections were rinsed and incubated for 1 h with anti-rabbit IgG (1:500, Vector Laboratories, Burlingame, CA, USA) diluted in PBS-T containing 2% NGS, followed by 1% avidin–biotin complex (Vectorstain Elite ABC kit, Vector Laboratories). Finally, sections were washed in 0.1 M Tris–HCl (pH 7.4) and the immunoreaction was visualized with 3,3′-diaminobenzidine tetrahydrochloride (1.25%) and 0.08% H2O2 in Tris–HCl.
2.9. Stereology Stereological analyses were performed with a computer-assisted image analysis system consisting of a Leica DM5500B microscope (Leica Microsystems, Heerbrugg, Switzerland) equipped with a motorized stage (Märzhäuser, Wetzlar, Germany) and a Microfire CCD camera (Optronics, Goleta, CA, USA), and Mercator Pro software with Mosaic module (Explora Nova, La Rochelle, France). The optical fractionator method was used to estimate the total numbers of THpositive cells in the LC and the A5 in an unbiased way (Howard and Reed, 2005). The first section was randomly selected and the section sampling fraction (ssf) was 1/2. The optical fractionator was used at regular predetermined dx and dy distances within the LC (150 × 150 μm) and the A5 (175 × 175 μm). The area associated with each frame (asf) was 2500 μm2. The height sampling fraction (hsf) was corresponding to 60% of the section thickness. The total number of neurons (N) was calculated according to the following formula: N = 1 / hsf × 1 / asf × 1 / ssf × ∑Q, where Q equals the number of cells counted within the dissector. All analyses were carried out by a person who was blind for the group assignment.
2.10. Statistical analysis The Shapiro–Wilk test was used to determine whether the data meet the assumption of normality. Group means were compared by two-tailed Student's t-test. Treatment effects over time were evaluated using repeated measures analysis of variance (ANOVA). Results are expressed as mean ± SEM. The level of significance was set at p b 0.05. Statistics were calculated using SPSS for Windows (Version 15.0.1, SPSS, Chicago, IL, USA).
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3. Results 3.1. Body mass development The neurotoxin DSP-4 significantly affected body mass development (time: F(2,120) = 584.82, p b 0.001; group: F(1,30) = 42.22, p b 0.001; time × group: F(2,120) = 65.65, p b 0.001). Animals treated with DSP-4 lost about 8% of their initial body mass within the first week after injection. Thereafter, the animals quickly regained weight and body mass had returned to baseline levels at 2 weeks after the neurotoxin administration. Four weeks after injection, body masses of DSP-4 and saline-treated rats did not significantly differ and both groups showed a comparable gain in body mass until sacrifice at 8 weeks. 3.2. Brain histology and splenic NA content Treatment with the neurotoxin DSP-4 resulted in the loss of noradrenergic neurons in the LC and the area A5 (Fig. 1). Eight weeks after neurotoxin administration, the total numbers of TH-immunoreactive 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 were not significantly different between DSP-4 and saline-treated animals (t = 1.71, p = 0.109). No significant group differences in spleen mass were found (control: 802 ± 27 mg, DSP-4: 815 ± 8 mg; t = 0.46; p = 0.646). 3.3. Splenic cytokine production Neurotoxin treatment markedly affected the cytokine production by isolated splenocytes (Fig. 2). Eight weeks after DSP-4 administration, the LPS-stimulated secretion of TNF-α, IL-1β, and IL-6 was significantly reduced by 50%, 31%, and 33%, respectively, compared to saline-injected animals (TNF-α: t = 5.58, p b 0.001; IL-1β: t = 5.45, p b 0.001; IL-6: t = 4.08, p = 0.001). In contrast, the production of IL-10 tended to be increased, but the group differences did not reach statistical significance (t = 1.94, p = 0.073). Upon T cell receptor stimulation with anti-CD3, the production of IFN-γ was significantly reduced in splenocyte cultures of DSP-4 treated animals compared to saline controls (t = 2.53, p = 0.024), whereas the secretion of IL-2 and IL-4 remained unaffected by 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 a significant positive association with the number of TH-positive neurons in 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 significant negative correlation (r = −0.51, p = 0.041). 3.4. Leukocyte subpopulations in the spleen Flow cytometry analysis showed that DSP-4 treatment had only small effects on leukocyte subpopulations in the spleen (Table 1). Neurotoxin-treated animals showed a significant decrease in the percentage of natural killer (NK) cells compared to saline-injected controls (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 two treatment groups. 4. Discussion The neurotoxin DSP-4 is a site-directed alkylating agent with high affinity for the NA transporter (Jonsson et al., 1981; Dudley et al., 1990). Following systemic injection, it penetrates the blood-brain barrier, accumulates within noradrenergic nerve terminals, and
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Fig. 1. Effect of DSP-4 treatment on brainstem noradrenergic nuclei and splenic noradrenaline content. (A) Photomicrographs of brain slices illustrating the degeneration of noradrenergic 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) Total numbers 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, *p b 0.05, ***p b 0.001; n = 8 per group.
produces a retrograde degeneration of noradrenergic neurons originating from the LC. Here we show that intraperitoneal administration of 50 mg/kg DSP-4 in adult Fischer 344 rats induced a pronounced loss of TH-positive neurons in the LC and the area A5. Two month after neurotoxin injection, the number of noradrenergic neurons in these brain regions was reduced by 35–40%, which is in line with previous reports using the same dose and route of administration (Fritschy and Grzanna, 1992). Importantly, the effects of DSP-4 are not limited to noradrenergic neurons in the brainstem. Due to the widespread projections arising from the LC, the destruction of noradrenergic neurons in this nucleus leads to an extensive NA depletion 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 A5 cell group was accompanied by alterations in the function of peripheral immune cells. Cultured splenocytes of DSP-4-treated animals produced significantly lower amounts of IL-1β, IL-6, and TNF-α upon stimulation with bacterial LPS than cells from saline-
injected controls. In addition, the production of IL-10 tended to be increased following DSP-4 treatment. This was not due to alterations in the number of cytokine producing cells because the proportion of splenic monocytes/macrophages remained unaffected by the neurotoxin treatment. Hence, a sustained impairment of the central noradrenergic system seems to cause a functional shift in splenic cytokine balance by suppressing the production of pro-inflammatory cytokines and, at least partially, promoting the secretion of antiinflammatory mediators. The loss of noradrenergic neurons in the brainstem was not only associated with alterations in the production of monocyte/macrophage-derived cytokines. Splenic immune cells from DSP-4 treated animals also produced significantly less IFN-γ upon T cell receptor stimulation with anti-CD3 antibody. In contrast, the production of IL-2 and IL-4 remained unaffected. Interleukin-2 is mainly produced by T cells and stimulates the production of IFN-γ by both T and NK cells (Handa et al., 1983). DSP-4 treatment caused a progressive decrease in NK cells but did not affect the number of splenic T cells. Thus, the reduction in IFN-γ production might have been the consequence of
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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 the injection. 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 are expressed as mean and S.E.M.; Student's t-test, *p b 0.05, **p b 0.01, ***p b 0.001; n = 8 per group.
the changes in the cellular composition of the spleen. However, IFN-γ levels in the culture supernatants were not correlated with splenic NK cell numbers. Therefore, it is more likely that central NA depletion induced a selective suppression of IFN-γ production in T cells without affecting the synthesis of IL-2. Although both IFN-γ and IL-2 are prototypical T helper cell type 1 (Th1) cytokines, their expression in T cells was shown to be independently regulated (Penix et al., 1993). Earlier reports have shown that DSP-4 induces not only a longlasting depletion of NA in the brain but causes also an acute reduction of NA levels in peripheral tissues including the spleen (Archer et al., 1982; Fety et al., 1986; Jaim-Etcheverry and Zieher, 1980; Shirokawa et al., 2000). The decrease in splenic NA content following DSP-4 treatment (20–40%) was less pronounced compared to the massive NA depletion (80–90%) after peripheral chemical sympathectomy with 6-OHDA (Bellinger et al., 2005; Besedovsky et al., 1979; Madden et al., 1989). Nevertheless, peripheral sympathectomy has been shown to affect splenic cytokine production (Callahan and Moynihan, 2002; Madden et al., 2000). For example, Madden et al. (2000) found a suppression of IFN-γ production, but not IL-2 production, by concanavalin-A stimulated splenocytes from sympathectomized F344 rats. This effect was fully reversible after the recovery of splenic NA levels. To exclude the possibility that a reduction of splenic NA levels was responsible for the DSP-4 induced alterations in splenic cytokine Table 1 Effect of DSP-4 treatment on leukocyte subpopulations in the spleen. Cell type
Control
DSP-4
Total T cells CD4+ T cells CD8+ T cells B cells NK cells Monocytes/macrophages
29.9 ± 0.9 19.6 ± 0.8 10.4 ± 0.3 45.7 ± 0.9 6.4 ± 0.1 4.7 ± 0.3
30.7 ± 0.6 19.7 ± 0.4 11.0 ± 0.2 46.5 ± 0.7 5.5 ± 0.2** 4.1 ± 0.2
Values represent percentage of cells. Data are expressed as mean ± S.E.M. Student's t-test, **p b 0.01; n = 8 per group.
production, we compared the NA content of the spleens from salineand neurotoxin-injected animals. Importantly, there was no difference in splenic NA concentration between the two treatment groups at 8 weeks after neurotoxin administration, suggesting that the observed effects on cytokine production were indeed the consequence of central NA depletion. This is supported by the results of the correlation analyses that revealed strong relationships between the number of TH-positive cells in the LC and the amounts of cytokines produced by the isolated splenocytes. In summary, this study provides novel data on the impact of central NA depletion on peripheral immune function and suggests that a certain level of central noradrenergic tone is required for normal immune functioning in the periphery. However, it remains open whether the observed immune effects are a direct consequence of reduced noradrenergic output from the brainstem or an indirect result of altered neurotransmission in brain regions to which the LC and A5 project. Further studies are needed to elucidate the afferent mechanisms that are involved in this process. Importantly, loss of noradrenergic neurons in the LC and A5 cell group are common features of many neurodegenerative 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 and Nemeroff, 1999; Zarow et al., 2003). In addition, although to a lesser extent, the number of noradrenergic LC neurons declines during normal aging (Lohr and Jeste, 1988; Mann, 1983). In the past, studies on the ageand disease-associated degeneration of the central noradrenergic system have primarily focused on behavioral and cognitive deficits. Based on the results of the present study it can be speculated that the altered cytokine production by peripheral immune cells of elderly people and patients suffering from neurodegenerative diseases or psychiatric disorders might be as well related to a decline in central noradrenergic tone (De Luigi et al., 2002; Hasegawa et al., 2000; Panda et al., 2009; Richartz et al., 2005). The biological and clinical relevance of these 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 excellent technical assistance. The study was partly supported by the Swiss Federal Institute of Technology (ETH) Zurich and the German Research Foundation (DFG; GK1045/2).
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