Address correspondence and reprint requests to Manfred Schedlowski, PhD, Department of Medical Psychology, Medical Faculty, University of Duisburg-Essen, Hufelandstraße 55, Essen, Germany. E-mail: firstname.lastname@example.org
Experimental and clinical evidence has demonstrated extensive communication between the CNS and the immune system. To analyse the role of central catecholamines in modulating peripheral immune functions, we injected the neurotoxin 6-hydroxydopamine (6-OHDA) i.c.v. in rats. This treatment significantly reduced brain catecholamine content 2, 4 and 7 days after injection, and in the periphery splenic catecholamine levels were reduced 4 days after treatment. Central catecholamine depletion induced an inhibition of splenic and blood lymphocyte proliferation and splenic cytokine production and expression (interleukin-2 and interferon-γ) 7 days after injection. In addition, central treatment with 6-OHDA reduced the percentage of spleen and peripheral blood natural killer (CD161 +) cells, and T-cytotoxic (CD8 +) cells in peripheral blood. The reduction in splenocyte proliferation was not associated with a glucocorticoid alteration but was completely abolished by prior peripheral sympathectomy. These data demonstrate a crucial role of central and peripheral catecholamines in modulating immune function.
However, the effect of central catecholamine depletion on peripheral cellular immune functions is somewhat unclear. Initially in vitro experiments reported that central catecholamine depletion does not affect the responsiveness of T lymphocytes in the spleen (Cross and Roszman 1988). In contrast, in other in vitro studies there was an impairment or an enhancement of splenic T-lymphocyte proliferation after injection of 6-OHDA into the striatum and nucleus accumbens (Deleplanque et al. 1992, 1994; Neveu et al. 1992). Specific depletion of the central and peripheral dopaminergic system by i.p. injection of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) attenuated the splenic T- and B-cell proliferation to mitogens (Bieganowska et al. 1993; Tsao et al. 1997). On the other hand, central catecholamines have a consistent role in cellular immune functions when tested in vivo. For example, mice injected intrastriatally with 6-OHDA have an impaired response to an intracellular bacterium (Filipov et al. 2002). In rats with a hyperactive dopaminergic system, reduced tumour growth, experimental metastasis formation, and angiogenesis have been reported (Teunis et al. 2002). A recent experiment by Alaniz and colleagues clearly illustrated the differences between in vitro and in vivo experiments: knock-out mice lacking dopamine beta-hydroxylase (i.e. they cannot produce noradrenaline or adrenaline, but produce dopamine instead) had normal numbers of blood leucocytes and normal T- and B-cell development. However, when challenged in vivo by infection with intracellular pathogens, these knock-out mice were more susceptible to infection (Alaniz et al. 1999).
The time at which immune assays are performed after central catecholamine depletion appears to be crucial to the results observed. Although no effects were reported on cellular immune variables 2 days after 6-OHDA treatment into the cisterna magna (Cross and Roszman 1988), mitogen-induced T-lymphocyte proliferation was suppressed 2 weeks after lesioning central dopaminergic structures in mice (Deleplanque et al. 1992). However, when immunological variables were analysed 4–6 weeks after central dopaminergic lesioning, a significant enhancement in T-lymphocytes proliferation was observed (Neveu et al. 1992).
In order to further clarify the role of efferent pathways in this neuro–immune interaction, we designed a set of experiments to analyse cellular immune functions in the periphery at various times after central catecholamine depletion with the neurotoxin 6-OHDA.
Materials and methods
Naive male Dark Agouti (DA) rats (Harlan Laboratories, Borchen, Germany), weighing between 220 and 250 g were used. All rats were allowed to habituate to the animal laboratory conditions for 3 weeks before starting the experiments. Animals were individually housed in standard plastic laboratory cages with a wire mesh lid. Cages were kept in an air-conditioned, sound-proof holding room at an ambient temperature of 24.0 ± 0.5°C. The animals had access to standard lab chow and tap water ad libitum. A 12-h light–dark cycle was maintained throughout the experiment with lights off at 07.00 hours. All animal procedures were carried out as approved by the ethics committee of the Medical Faculty, University of Essen, Germany.
To centrally deplete catecholamines, experimental animals received bilateral injections of 6-OHDA into the lateral ventricles. Control animals were injected only with the vehicle. 2, 4 and 7 days later experimental and control animals were killed by decapitation. Immediately afterwards, spleen and blood samples were obtained to measure the proliferative capacity of lymphocytes, cytokine production and leucocyte distribution. Brain and spleen samples were collected and stored (− 80°C) for later catecholamine determination and blood samples were stored for corticosterone analysis. An additional experimental group was peripherally sympathectomized with 6-OHDA i.p. injections, and 3 days later these animals were subsequently treated to deplete central catecholamines as well.
Central catecholamine depletion
Central catecholamines were depleted by stereotaxically injecting 100 µg freshly prepared 6-OHDA (Sigma, Taufkirchen, Germany) dissolved in 10 µL ascorbate solution (2 mg/mL ascorbic acid in sterile saline solution) as a vehicle into the lateral ventricles of the animals (5 µL/ventricle). The stereotaxic coordinates used were: ± 1.4 mm lateral, − 3.4 mm ventral and – 0.8 mm anterior with respect to bregma, according to a stereotaxic atlas (Paxinos and Watson 1986).
Peripheral catecholamine depletion
6-OHDA was dissolved in sterile phosphate-buffered saline (PBS) supplemented with 0.01% ascorbic acid. The peripheral sympathectomy was completed with animals receiving a total dose of 200 mg/kg 6-OHDA i.p. over 3 days. The first day the animals received 6-OHDA at a dose of 40 mg/kg, injected in a total volume of 1 mL. On the subsequent 2 days, animals received an 80-mg/kg dose of 6-OHDA. This procedure has been shown previously in our laboratory to reduce noradrenaline and adrenaline concentrations in the spleen by 85%. This regimen does not affect splenocyte proliferation or splenocyte subset distribution (Exton et al. 2002).
Lymphocyte proliferation assay
Peripheral mononuclear cells were collected from EDTA-treated blood, isolated according to a standard Ficoll protocol and resuspended in cell culture medium (RPMI−1640 (PAA, Linz, Austria) +5% fetal bovine serum inactivated (Sigma) +1% penicillin (10 000 u/mL)-streptomycin (10 mg/mL) solution (Sigma). Splenocytes were released from tissue by injecting cell culture medium into the spleen. Blood and spleen cells were washed several times in PBS and adjusted to a concentration of 5 × 106 cells/mL. They were then cultured for 72 h in the presence of Con A (0, 1.25 and 2.5 µg/mL) at 37°C in a 5% CO2 incubator. After 48 h of mitogen stimulation the cells were pulsed with 20 µL/well [3H]thymidine (0.5 µCi) and harvested 24 h later. Radioactivity was measured using a β counter.
Cell suspensions from the spleen and blood were analysed as described previously (Exton et al. 1998). Briefly, 1 × 106 cells were added to microtitre plates and then incubated with different mouse anti-rat monoclonal antibodies (mAb) for identification: NK cells (CD161 +, TCR −; mAb 3.2.3/mAb R73); B cells (CD45 +, immunoglobulin kappa chain +; mAb Ox22/mAb Ox12); T-cyto toxic lymphocytes (CD8 +, TCR +; mAb Ox8/mAb R73); T-helper lymphocytes (CD4 +, TCR +; mAb W3/25/mAb R73). T-helper lymphocytes were further differentiated into two subsets: naive (CD45 +; mAb Ox22) and memory (CD45 −). T-helper lymphocytes were further differentiated into two subsets: naive (CD45 +; mAb Ox22) and memory (CD45 –), as well as B cells (CD45 +, Ig κ chain +; mAb Ox12). A minimum of 1 × 104 events was analysed in a FACSCalibur flow cytometer (Becton-Dickinson, Heidelberg, Germany), gating on forward and side scatter characteristics. All mAbs were purchased from Serotec (Oxford, UK).
Interleukin (IL)-2 and interferon (IFN)-γ were assayed in splenocyte supernatant cultures using commercial enzyme-linked immunosorbent assay kits for the detection of rat IL-2 (Biosource International, CA, USA) and rat IFN-γ (U-CyTech, Utrecht, The Netherlands).
RT–PCR analysis of IL-2 mRNA expression
Using RT–PCR, mRNA expression of IL-2 was analysed in splenocyte cell cultures. Rat splenocytes (5 × 106/mL) were cultured for 6 h in the presence of Con A (1.25 µg/mL), after which the cells were denatured with 700 µL 4 m guanidinium thiocyanate (Sigma, St Louis, MO, USA) following a phenol–chloroform RNA extraction (Chomczynski and Sacchi 1987). cDNA synthesis was performed in a reaction buffer containing 1 µg total RNA sample, 5 × reaction buffer, 2.5 µL oligo-dT15 (Gibco/BRL, Rockville, MD, USA), 10 mm dNTP mixture and 100 U M-MLV reverse transcriptase (Promega, Madison, WI, USA). The RT reaction was performed at 45°C for 90 min, 52°C for 30 min and 96°C for 15 min. PCR of cDNA was performed with 1 U Taq DNA polymerase (Gibco/BRL), 10 × PCR buffer without magnesium (500 mm KCl, 200 mm Tris-HCl, pH 8.4), 10 mm dNTP mixture, 50 mm MgCl2 (Promega) 10 µm primer (MWG Biotech, Ebersberg, Germany) and autoclaved distilled water. cDNA was denatured for 3 min at 95°C and then set up according to the following protocol: (1) denaturing for 30 s at 94°C; (2) annealing primers for 30 s at 61°C for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 54°C for IL-2; (3) extending the primers for 30 s at 72°C. The PCR technique employed 25 cycles of amplification for GAPDH and 30 cycles for IL-2. The primers used for the gene amplification have been described previously (Exton et al. 2002). The amplified PCR product was identified by electrophoresis on 1.5% agarose gel stained with 0.5 µg/mL ethidium bromide. GAPDH served as the internal control. The data are expressed as amount of IL-2 produced over the amount of GAPDH.
Levels of dopamine, adrenaline, noradrenaline and serotonin in the spleen and brain tissues (hypothalamus and cortex) were measured by HPLC as described previously (Exton et al. 1998).
Corticosterone levels in plasma were determined by radioimmunoassay as described previously (Exton et al. 1999).
One-way anova and Fisher post-hoc test were used to examine statistical differences between the groups. Data are expressed as mean ± SEM. p < 0.05 was considered statistically significant.
Effects of central catecholamine depletion on lymphocyte proliferation and cytokine production
Two, four, and seven days after i.c.v. 6-OHDA treatment, the proliferative capacity of T lymphocytes in the spleen was analysed. The proliferative response of T lymphocytes was not affected 2 or 4 days after central catecholamine depletion compared with that in the vehicle control group (data not shown). However, we observed a pronounced decrease in splenocyte proliferation 7 days after central catecholamine depletion compared with the vehicle control group (p < 0.05) (Fig. 1a). In the next step, we investigated whether or not this effect was limited to the spleen by testing the proliferative capacity of peripheral blood T lymphocytes at the same time point. In parallel to the reduced splenocyte responsiveness, we observed a reduction in the proliferation of peripheral blood lymphocytes 7 days after i.c.v. 6-OHDA injection compared with the vehicle control group (p < 0.05) (Fig. 1b).
To elucidate possible cellular mechanisms underlying the reduced proliferation, levels of IL-2 and IFN-γ were measured in the supernatants of the Con A-stimulated splenocytes. Compared to control groups, experimental 6-OHDA treated animals had a significantly (p < 0.05) reduced IL-2 (Fig. 2a), and IFN-γ (211 ± 43 vs. 410 ± 33pg/mL, 6-OHDA vs. vehicle, mean ± SEM) concentration. To further examine whether the suppression of splenocyte proliferation and cytokine production occurred at a transcriptional level, we measured mRNA expression of IL-2. Consistent with the effects observed at the protein and cellular level, splenocytes of 6-OHDA-treated animals showed a significant suppression of IL-2 mRNA expression (Figs 2b and c).
Splenocyte and leucocyte distribution after central catecholamine depletion
One possible explanation for the reduction in T-lymphocyte proliferative capacity 7 days after central 6-OHDA treatment might be that catecholamine depletion in the brain induces changes in the distribution of lymphocyte subpopulations in the spleen and peripheral blood. Therefore spleen and blood lymphocyte subsets were analysed by flow cytometry. Central catecholamine depletion significantly reduced the percentage of NK (CD161 +) cells (p < 0.05) in the spleen and in peripheral blood, and significantly increased T-cytotoxic (CD8 +) cells (p < 0.05) in the peripheral blood but not in the spleen, compared with levels in the vehicle control group (Table 1).
Table 1. Flow cytometric analysis of lymphocyte subpopulations from spleen and peripheral blood samples 7 days after i.c.v. treatment with 6-OHDA or vehicle Results are expressed as (mean ± S.E. * p < 0.05)
Values are mean ± SEM percentage of total gated lymphocytes compared with controls (n = 8 per group). *p < 0.05 versus vehicle (one-way anova and Fisher post-hoc test).
Effects of i.c.v. 6-OHDA treatment on central and peripheral catecholamine concentrations
Two, four, and seven days after i.c.v. injection with either 6-OHDA or vehicle, brain and spleen tissue samples were collected for catecholamine analysis by HPLC. There was a pronounced reduction in dopamine (p < 0.05), adrenaline (p < 0.05) and noradrenaline levels (p < 0.01) in the hypothalamus (Fig. 3) and in the cortex (p < 0.01) (data not shown) of animals treated with i.c.v. 6-OHDA compared with controls. In the periphery, we observed a significant but transient reduction in splenic concentrations of noradrenaline and adrenaline (p < 0.05 on day 4 after 6-OHDA treatment) (Fig. 3). In order to assess the specificity of 6-OHDA specificity for catecholamine depletion, serotonin levels were assessed in the same tissues; 6-OHDA had no effect on serotonin levels at any time point (data not shown).
Effect of central catecholamine depletion on the hypothalamic–pituitary–adrenal (HPA) axis
The reduction in lymphocyte proliferative responsiveness after central catecholamine depletion might be the result of a deregulated HPA axis, with an increased release of glucocorticoids. However, we did not observe any significant changes in plasma corticosterone concentration at 2 days (11.7 ± 1.7 vs. 12.7 ± 2.5 µg/dL, 6-OHDA vs. vehicle), 4 days (13.1 ± 1.9 vs. 15.4 ± 2.3 µg/dL) or 7 days (14.0 ± 1.0 vs. 14.5 ± 1.3) after central catecholamine depletion.
Peripheral sympathectomy before central catecholamine depletion abolishes the immune changes observed
We have previously established a regimen of peripheral sympathectomy with i.p. 6-OHDA treatment that reduces the splenic catecholamine content by 85%, and does not affect splenocyte proliferation or lymphocyte distribution (Exton et al. 2002). In order to analyse the potential role of noradrenergic mechanisms in the periphery, an experimental group of rats received a total 6-OHDA dose of 200 mg/kg i.p. over 3 days (40, 80 and 80 mg/kg). Three days later these animals were centrally depleted with i.c.v. 6-OHDA injections (100 µg). At 7 days after the i.c.v. injection we observed no differences in the proliferative capacity of splenocytes between rats that had been subjected to central and peripheral depletion of catecholamine, and those in the vehicle control group (Fig. 4), indicating that the reduction in splenocyte proliferation after central 6-OHDA treatment is mediated by peripheral noradrenergic mechanisms.
The current data further contribute to our understanding of the role of central catecholamines in regulating peripheral cellular immune functions. We showed that 7 days after central catecholamine depletion, lymphocyte proliferation in spleen and peripheral blood, splenic IL-2 and IFN-γ production, and IL-2 mRNA expression are significantly reduced. These effects do not appear to be related to 6-OHDA-induced changes in lymphocyte distribution nor to HPA axis activation with increased glucocorticoid levels. In addition, we observed a significant reduction in splenic catecholamine content 4 days after i.c.v. 6-OHDA treatment, suggesting that sympathetic mechanisms are responsible for these effects. This hypothesis was confirmed by the observation that the reduced splenocyte proliferation after i.c.v. 6-OHDA treatment was completely abolished when animals were peripherally sympathectomized before central catecholamine depletion.
The precise mechanism underlying the effect of central catecholamine depletion on peripheral immunity is unknown. However, the present data suggest a predominant role for the peripheral sympathetic nervous system in regulating immune functions. We hypothesize that central catecholamine depletion alters afferent and efferent communication between the immune system and the CNS resulting in a disruption of peripheral immune homeostasis. This view is supported by our data showing a deregulated sympathetic output in the periphery after central catecholamine depletion. Moreover, when animals were sympathectomized before central catecholamine depletion, the immunosuppressive effects were abolished, confirming a main role for peripheral catecholamines in neuro–immune communications.
The central catecholaminergic system seems to be involved in sensing and modulating peripheral immune functions. During an immune response significant alterations of catecholamine activity occur in specific brain regions (Besedovsky et al. 1983; Zalcman et al. 1991; Qiu et al. 1996; Devoino et al. 1997), indicating that this central neurotransmission system is involved in the afferent arm of immune-to-brain communication. On the other hand, central noradrenergic networks may exert direct immunomodulatory efferent actions through fibres innervating immune organs (Bellinger et al. 1989, 1992; Felten 1993). Specifically, it has been shown that the splenic sympathetic innervation is under the control of the ventromedial hypothalamic nucleus (Okamoto et al. 1996). Furthermore, these splenic efferent fibres originate from the sympathetic thoracolumbar column which is under control of the A5 and A7 noradrenergic brainstem nuclei (Cano et al. 2001). As the neuronal activity (firing rates and patterns) of brain nuclei receiving projections from catecholaminergic structures changes after 6-OHDA depletion (Ni et al. 2000, 2001), we hypothesize that centrally administrated 6-OHDA affects peripheral catecholamine output, with its immunomodulatory effects, by causing discrete alterations in central neural firing. However, further experiments are necessary to analyse the role of specific central neurotransmitter systems as well as the specific role of forebrain, brainstem and spinal cord structures controlling the proliferative capacity of T-lymphocyte and cytokine production.
In the periphery, the spleen receives sympathetic efferent fibres, with T-lymphocyte zones richly innervated by noradenergic nerve fibres (Weihe et al. 1991; Stevens and Bellinger 1997), and lymphocytes express adrenoceptors (Jetschmann et al. 1997; Elenkov et al. 2000; Sanders et al. 2001) and dopamine receptors (Le Fur et al. 1980b; Basu et al. 1993; Bergquist et al. 1994; Basu and Dasgupta 2000). In vitro and in vivo evidence demonstrates that noradrenaline regulates T- and B-lymphocyte functions and cytokine production through α- and β-adrenoceptor stimulation (Kohm and Sanders 2000; Madden 2001; Sanders and Straub 2002; Xie et al. 2002). In addition, dopamine significantly modulates the proliferative capacity of murine and human lymphocytes (Offen et al. 1995; Tsao et al. 1997; Saha et al. 2001). Interestingly, lymphocytes from patients with Parkinson's disease show lower expression of dopamine receptors (Le Fur et al. 1980a). Although no changes have been reported in cardiac concentrations of catecholamines 2 days after the central 6-OHDA treatment (Cross et al. 1986), this does not rule out the possibility that catecholaminergic activity in other peripheral tissues change after i.c.v. 6-OHDA treatment. Indeed, we found a significant decrease in splenic catecholamine concentrations 4 days after 6-OHDA injection, but not 2 or 7 days after injection, supporting our hypothesis that central 6-OHDA affects peripheral catecholamine output. In addition, 4 weeks after i.c.v. 6-OHDA injection the central and peripheral contents of noradrenaline and adrenaline are reduced in rats (Deng et al. 1993). As we observed unbalanced levels of the three catecholamines in the periphery, it is likely that these neurotransmitters induced the observed immunosuppressive effects.
Flow cytometric analysis revealed no significant changes in distribution of T-helper (CD4 +) and B cells (κ chain +) from spleen and blood in animals centrally depleted of catecholamines compared with controls. This is in agreement with a previous study in which no differences were found in splenic T- or B-cell populations 2 days after central catecholamine depletion in mice (Cross and Roszman 1988). However, 6-OHDA treatment reduced the percentage of NK cells (CD161 +) in the spleen and peripheral blood in the present study. This observation might partially explain previous reports in which NK cell activity was inhibited by 6-OHDA treatment in the cisterna magna (Cross and Roszman 1988) or in the nucleus accumbens (Deleplanque et al. 1994). We also observed a significant increase in T-cytotoxic (CD8 +) cells in the peripheral blood but not in the spleen. How central catecholamines modulate the distribution of lymphocytes is not fully understood. However, it has been shown that peripheral adrenaline and noradrenaline can affect leucocyte distribution and trafficking (Benschop et al. 1996, 1997), in particular of NK cells and CD8 + T-lymphocytes (Schedlowski et al. 1996). This suggests that the peripheral catecholamine changes observed after 6-OHDA treatment contribute to alterations in the lymphocyte subset distribution.
In summary, our data demonstrate that 7 days after central catecholamine depletion T-lymphocyte responsiveness, and cytokine (IL-2 and IFN-γ) production and mRNA expression are significantly reduced. This modification of the peripheral cellular immune response is neither due to lymphocyte subset variation nor HPA axis activation but could be blocked by prior peripheral sympathectomy. The present data confirm the role of the central catecholamines in peripheral immune homeostasis.
This work was supported by the German Academic Exchange Service (DAAD) and Facultad Odontología UNAM fellowships to Gustavo Pacheco-López, and by the German Research Foundation, grants DFG SCHE-341/9–1 and DFG SCHE-341/9–2. We acknowledge Dr S. Frede, Department of Physiology, University of Essen, for technical advice, and Dr Michael J. Harnish and Ulrieke Birner for critical reading of the manuscript.