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Dopamine, vesicular transporters and dopamine receptor expression and localization in rat thymus and spleen
Journal of Neuroimmunology 206 (2009) 5–13
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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
Dopamine, vesicular transporters and dopamine receptor expression and localization in rat thymus and spleen Fiorenzo Mignini ⁎, Daniele Tomassoni, Enea Traini, Francesco Amenta Anatomia Umana, Dipartimento di Medicina Sperimentale e Sanità Pubblica, Università di Camerino, Camerino, Italy
a r t i c l e
i n f o
Article history: Received 11 August 2008 Received in revised form 26 September 2008 Accepted 26 September 2008 Keywords: Dopamine transporter Vesicular monoamine transporter Dopamine receptors Lymphoid organ Western blot analysis Immunohistochemistry
a b s t r a c t The localization of dopamine stores and the expression and localization of vesicular monoamine transporter (VMAT) type-1 and 2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat thymus and spleen by immunohistochemical, immunochemical techniques and by RT-PCR. In the thymus dopamine immunoreactivity was developed in the cortico-medullary junction and in the medulla, but not in the thymic cortex. In the spleen, dopamine stores were found in reticular structures in the white pulp border and in the white pulp, but not in the red one. Both thymus and spleen expressed VMAT-1 and VMAT-2 immunoreactivity as well as dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity. Immunohistochemistry revealed VMAT-1, VMAT-2 and dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity primarily in the thymic cortical-medulla transitional zone and to a lesser extent in the medulla but not in the cortex. In the spleen, VMAT-1, VMAT-2 and dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity was located primarily in the white pulp border and to a lesser extent in the white pulp. These findings indicate that both thymus and spleen express a dopaminergic system characterized by the presence of dopamine, vesicular monoamine transporters and the five subtypes of dopamine receptors. The presence of these dopaminergic markers suggests that dopamine likely originating from immune cells and/or from sympathetic neuroeffector plexus is released in the lymphoid microenvironment. Based on the microanatomical localization of dopaminergic markers investigated, a role of dopamine in maturation and selection of lymphocytes and activation of immune responses is suggested. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Increasing evidence indicates the occurrence of interactions between nervous and immune systems (for a review, see Mignini et al., 2003; Meredith et al., 2005). Central nervous system influences the immune system through two major pathways. They include the neuroendocrine outflow linked to pituitary gland activity and the autonomic innervation of immune organs (Ader et al., 1995; Mignini et al., 2003). Both nervous and immune systems have relevant anatomical connections (Mignini et al., 2003; Meredith et al., 2005). Substances common to nervous and immune systems such as cytokines and neurotransmitters probably represent molecules involved in neuroimmune communications (Qiu et al., 1996). On the other hand, several nervous system disorders may have immune pathogenesis or implications (Sklar and Anisman, 1981) and stressing factors can influence the development and course of diseases (for a review see Mignini et al., 2003). In the nervous system, catecholamines have a key role in neurotransmission and are crucially involved in several physiological and
⁎ Corresponding author. Dipartimento di Medicina Sperimentale e Sanità Pubblica, Via Madonna delle Carceri, 9 62032 Camerino, Italy. Fax: +39 0737 403325. E-mail address: fi[email protected] (F. Mignini). 0165-5728/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.09.018
pathological conditions (Carlsson, 1987). The capability of noradrenergic system and of noradrenaline and adrenaline in modulating immune responses has been extensively investigated (Kohm and Sanders, 2001), whereas less information is available for dopamine. In the thymus catecholamines are located both in neural and nonneuronal compartments. Neural compartment includes sympathetic fibres in which dopamine is co-localized with noradrenaline and probably a sensory dopaminergic system (Katz et al.,1987). Non-neural system contributing to thymic catecholamine levels includes thymocytes with mainly a CD3 high phenotype and thymic epithelial cells localized predominantly on the medullary side of the corticomedullary junction and in the subcapsular cortex (Pilipović et al., 2008). Dopamine exerts several peripheral actions involving primarily the cardiovascular system and the kidney. These actions are mediated through the interaction with five subtypes of dopamine receptors belonging to the dopamine D1-like (D1 and D5) and D2-like receptor super families (D2, D3 and D4) (Sibley and Monsma, 1992; Gingrich and Caron, 1993). Dopamine receptors have been also identified in peripheral blood lymphocytes and it is thought that effects of this catecholamine on immune system are mediated by the interaction with its own receptors expressed on the surface of immune target cells (Oberbeck, 2006). From a functional point of view, high doses of dopamine in vitro can inhibit mitogen-induced proliferation and promote
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Table 1 Antibodies used and their concentrations in immunochemistry and immunohistochemistry experiments Primary antibody
Raised in
Dopamine VMAT1 VMAT2 D1 D2 D3 D4 D5 Actin (C-2)
Mouse Goat Rabbit Rabbit Rabbit Rabbit Rabbit Goat Mouse
Working dilution WB 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500
Producer
Code
Abcam plc Chemicon Chemicon Chemicon Santa Cruz Biotechnology Santa Cruz Biotechnology Chemicon Chemicon Santa Cruz Biotechnology
No. No. No. No. No. No. No. No. No.
IH 1:50 1:100 1:100 1:500 1:500 1:250 1:1000 1:250
ab8892 AB1597P AB1598P AB1765P sc-7522 sc-9114 324405 MAB 5292 sc-8432
Secondary antibody Horseradish peroxidase-conjugated antibodies (WB) Anti-mouse Donkey 1:1000 Santa Cruz Biotechnology Anti-goat Donkey 1:1000 Santa Cruz Biotechnology Anti rabbit Donkey 1:1000 Santa Cruz Biotechnology
No. sc-2314 No. sc-2020 No. sc-2313
Biotin-conjugated antibodies (IH) Anti-mouse Goat 1:200 Anti-goat Donkey 1:200 Anti-rabbit Donkey 1:200
No. AP124B No. PK6105 No. PK6101
Chemicon Vector (Vectastain ABC Kit) Vector (Vectastain ABC Kit)
WB: Western blot analysis; IH: immunohistochemistry.
apoptosis in lymphocytes (Caronti et al., 1998; Kavelaars et al., 2005; Olanow, 2007). Data from in vivo studies provided conflicting results, with evidence for both stimulatory and inhibitory effects of dopamine on lymphocyte function (Cook-Mills et al., 1995; Tsao et al., 1997; Ilani et al., 2004). Receptors are the molecular entities through which dopamine can elicit its biological actions on effector cells. Transporter systems represent another relevant marker of dopamine neurobiology. In the nervous system dopamine plasma membrane transporter and the vesicular monoamine transporters (VMAT) type-1 and type-2, remove the neurotransmitter from the extracellular space, thereby controlling extracellular lifetime of dopamine (Giros and Caron, 1993). Dopamine transporters displaying similar characteristics to those expressed by brain and peripheral tissues are also expressed by human peripheral blood lymphocytes (Amenta et al., 2001; Gordon and Barnes, 2003). A problem not clarified yet, with the exception of the demonstration of dopamine transporter (DAT) in rat thymus and spleen (Mignini et al., 2006), is if immune organs express other dopamine transporters and which anatomical relationships they may have with dopamine receptors. The localization of dopamine stores in immune organs is also still unclear. The present study has investigated in rat thymus and spleen the localization of dopamine stores by immunohistochemistry and dopamine transporter expression by assessing VMAT type-1 and type-2 transporters with immunochemical, RT-PCR and immunohistochemical techniques. These techniques were also used for characterizing and localizing microanatomically dopamine D1-like and D2-like receptor subtypes.
and killed by decapitation. Striatum, thymus, spleen and adrenal medulla of 8 rats were dissected out and used for immunochemistry or RT-PCR analysis. After removal, tissues were weighed and homogenized as detailed below. Thymus and spleen of the remaining 8 rats were removed, washed in ice-cold 0.9% saline solution, embedded in a cryoprotectant medium and frozen in isopentane cooled with liquid nitrogen. This material was used for immunohistochemistry. 2.2. Western blot analysis Samples of thymus and spleen (0.1 ± 0.02 g) were lysed in a Mixer Mill MM300 (Qiagen, Hilden, Germany) with 800 μl of lysed buffer as previously described (Mignini et al., 2006). Equal amounts of protein (40 μg) were separated by 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane by electro blotting in Towbin buffer. Transblotted membranes were incubated with VMAT-1 and VMAT-2 antibodies and dopamine D1, D2, D3, D4 and D5 receptor antibodies, as summarized in Table 1. Membranes were then incubated with corresponding horseradishperoxidase conjugated secondary antibodies (Table 1). The product of immune reaction was visualized by a chemiluminescence detection system (ECL Chemiluminescent detection system, GE Healthcare U.K.), followed by computer-driven densitometry with an IAAS 2000 image analyzer (Delta Sistemi, Rome, Italy). The specificity of immune reaction was assessed using antibodies pre-adsorbed with specific peptides used for generating them. 2.3. RNA extraction and RT-PCR analysis Total RNA was extracted from striatum, adrenal medulla, thymus and spleen using Trizol reagent according to the manufacturer's instructions as previously described (Mignini et al., 2006). mRNA samples (2 μg) were subjected to reverse transcription using the RevertAid Hminus First Strand cDNA synthesis kit (Fermentas, Life Science, M-Medical srl, Cornaredo, Italy). Oligonucleotide primers used are listed in Table 2. For PCR, 1 μl of each cDNA sample was amplified using HotStartTaq Master Mix (Qiagen), containing 10 pmol of each specific primer, for 40 cycles with thermal cycler profile denaturation at 95 °C for 30 sec, primers annealing at temperature different for each specific pair of primers (Table 2), elongation at 72 °C for 30 sec. PCR reactions were preceded by incubation at 95 °C for 15 min and a post-incubation at 72 °C for 10 min. Samples were separated and analyzed by 1.8% agarose gel electrophoresis with DNA molecular size markers as a reference (Mass Ruler, DNA Low Range, Fermentas, Cat. No. SM0383). Resulting amplicons were visualized with an ultra violet light, photographed and their size was determined by an Image Master VDS, Pharmacia Biotech. Table 2 Primers used to assess vesicular monoamine transporters or dopamine receptor subtypes mRNA expression Gene
Primer
Oligonucleotide sequence
Annealing (°C)
Size bp
VMAT-1
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5′-AGA CAG CAA CTC TTC TCT GC-3′ 5′-CTA TCC CTT GCA AGC AGT TGT-3′ 5′-ATC GCT GCA GCC TCC ATC T-3′ 5′-TGC CAC TTT CGG GAA CAC A-3′ 5′-CAT TCT GAA CCT CTG CGT GA-3′ 5′-GTT GTC ATC CTC GGT GTC CT-3′ 5′-GCA GTC GAG CTT TCA GAG CC-3′ 5′-TCT GCG GCT CAT CGT CTT AGG-3′ 5′-GGG GTC ACT GTC CTG GTC TA-3′ 5′-TGG CCC TTA TTG AAA ACT GC-3′ 5′-GAT GTG TTG GAC GCC TTT CT-3′ 5′-TCG GCA TTG AAG ATG GTG TA-3′ 5′-ACC AAG ACA CGG TCT TCC AC-3′ 5′-CAC AGT CAA GCT CCC AGA CA-3′ 5′-TGT CAC CAA CTG GGA CGA TA-3′ 5′-ACC CTC ATA GAT GGG CAC AG-3′
60
175
57
102
57
229
60 56
404 317 169
56
150
56
140
55
280
2. Materials and methods
VMAT-2
2.1. Animals and tissue treatment
D1 D2
Male Wistar rats (n = 16) of 2 months of age (weight 239 ± 20 g) were used. Animals were handled according to internationally accepted principles for care of laboratory animals (European Community Council Directive 86/609, O.J. No. L358, Dec. 18, 1986). Rats were received 4 weeks before experiments and were kept under a constant light–dark cycle (7:00 a.m. to 7:00 p.m. light period), at an ambient temperature of 22 ± 1 °C, with free access to water and laboratory chow. They were then anesthetized with pentobarbital sodium (50 mg/kg, i.p.)
D3 D4 D5 β-actin
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2.4. Immunohistochemistry Thymus and spleen were cut serially at a −20 °C microtome cryostat. Eight μm thick sections were mounted on polylysinated microscope slides and collected in 8 groups of 5 sections each. The first section of each group was stained with hematoxylin and eosin or with a 0.5% toluidine blue solution to verify microanatomical details. The second and fourth sections were used for the immunohistochemical detection of dopamine or VMAT-1, VMAT-2, dopamine D1, D2, D3, D4 and D5 receptors (test section). The third and fifth sections were used to assess the specificity of immune reaction (control sections). Endogenous peroxidase activity was blocked by H2O2 (20 min). Test sections were pre-incubated for 1 h at 4 °C in a 3% normal donkey serum dissolved in 0.1 M phosphate buffered saline (PBS) and 0.3% TritonX-100 to prevent non-specific binding of IgGs. Sections were then incubated overnight at 4 °C with primary antibodies at the concentrations indicated in Table 1. Slides were rinsed several times with PBS at room temperature, and then incubated for 1 h at room temperature in a biotinylated secondary antibody dissolved in 0.3% PBS–TritonX-100. The product of the immune reaction was then
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revealed using a biotin–streptavidin immunostaining kit with 0.05% diamino benzidine (DAB) in 0.1% H2O2 as a chromogen. Sections were then washed, dehydrated in ethanol, and mounted in a synthetic mounting medium and observed under a light microscope (Mignini et al., 2006). 3. Results 3.1. Dopamine immunohistochemistry Fig. 1 shows the localization of tissue stores of dopamine in rat thymus and spleen. In the thymus dopamine immunoreactivity was developed in the cortico-medullary junction (Fig. 1A) and in the medulla, whereas no immune reaction was observed in the thymic cortex (data not shown). At higher magnification dopamine immunoreactivity displayed a reticular localization (Fig. 1A inset). In the spleen, dopamine immunoreactivity was located primarily in the white pulp border and to a lesser extent in the red pulp (Fig. 1D). Similarly as observed in the thymus, immune reaction displayed a flattened and network-like localization (Fig. 1D inset).
Fig. 1. Dopamine immunohistochemistry in sections of rat thymus (pictures A and C) and spleen (pictures D and F). Sections were exposed to anti-dopamine antibody (pictures A and D) or to the antibody previously adsorbed with dopamine to verify the specificity of the reaction (pictures C and F). Micrographs B and E show sections close to those processed for immunohistochemistry stained with hematoxylin and eosin to verify microanatomical details. Note in thymus and spleen the localization of immune reaction in the medulla (M) or white pulp (W) but not in the cortex (C) or red pulp (R) respectively. The highest immunoreactivity was observed in the thymic cortico-medullary junction (CJM) and in the white pulp border (WPB). Insets are a higher magnification of the two areas showing the localization of dopamine immune reaction in reticular structures probably representing epithelial cells of CMJ and splenic marginal sinus cells. Calibration bars: A–C 25 μm; B–D 50 μm (for the inset 12.5 μm).
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Fig. 2. Western blot analysis of vesicular monoamine transporter (VMAT) type-1 (panel A), and VMAT type-2 (panel B) in membranes obtained from rat thymus (lanes 1), spleen (lane 2), adrenal medulla (panel A, lane 3) or striatum (panel B, lane 3). Panel C shows migration characteristics of actin used as a reference.
3.2. Western blot analysis Immunoblots with anti-VMAT-1 and anti-VMAT-2 antibodies developed a single band of immunoreactivity of approximately 55 kDa and 68 kDa respectively in thymus (Fig. 2, panels A and B lane 1) and spleen (Fig. 2, panels A and B lane 2) lysates. A similar pattern of immunoreactivity was observed for VMAT-1 in the adrenal medulla (Fig. 2, panel a, lane 3) and for VMAT-2 in striatum used as reference tissues (Fig. 2, panel B, lane 3). Immunoblots performed with anti-D1 and anti-D5 dopamine receptor antibodies revealed the development of a single band of approximately 50 kDa and 55 kDa respectively in thymus and spleen (Fig. 3, panels A and E, lanes 1 and 2) as well as in the striatum (Fig. 3, panels A and E, lane 3). Immunoblots exposed to anti-D2 dopamine receptors revealed the development of two bands of approximately 53 and 48 kDa in membrane preparations of thymus, spleen and striatum (Fig. 3, panel B). Exposure of tissue preparations to anti-D3 and anti-D4 dopamine receptors antibodies revealed the development of a single band of approximately 50 and 44 kDa respectively. These bands were similar for thymus, spleen and striatum (Fig. 3, panels C and D lanes 1–3). 3.3. RT-PCR analysis RT-PCR analysis revealed VMAT-1 mRNA expression in rat thymus, spleen and adrenal medulla (Fig. 4, panel A, lanes 1, 2 and 3 respectively) and VMAT-2 mRNA expression in rat thymus, spleen and striatum (Fig. 4, panel B, lanes 1, 2 and 3 respectively). RT-PCR revealed the presence of mRNA for D1 receptor and D5 receptor in thymus, spleen and striatum (Fig. 5, panels A and E, lanes 1, 2 and 3 respectively). Analysis of D2 receptor revealed in the thymus and spleen the predominant expression of the long isoform, whereas striatum expressed to a similar extent both long and short isoforms (Fig. 5, panel B, lanes 1, 2 and 3 respectively). RT-PCR analysis showed the expression of D3 (Fig. 5, panel C) and D4 (Fig. 5, panel D) dopamine
Fig. 3. Western blot analysis of dopamine D1 (panel A), D2 (panel B), D3 (panel C), D4 (panel D) and D5 (panel E) receptors in membranes obtained from rat thymus (lane 1), spleen (lane 2) and striatum (lane 3). For the migration characteristics of actin used as a reference see Fig. 2, panel C.
Fig. 4. RT-PCR of VMAT-1 mRNA (panel A), and VMAT-2 (panel B) in membranes obtained from rat thymus (lanes 1), spleen (lane 2), adrenal medulla (panel A, lane 3) or striatum (panel B, lane 3). Panel C shows migration characteristics of actin used as a reference. LD: DNA ladder; bp: base pair.
receptor mRNAs in the two immune organs examined as well as in the striatum (Fig. 5, panels C and D, lanes 1, 2 and 3 respectively). 3.4. Immunohistochemistry Section of rat thymus exposed to VMAT-1 or VMAT-2 antibodies developed a dark-brown immune reaction in the adventitia–media junction of arteries and arterioles located in the septa of the connective tissue (data not shown). No VMAT-1 (Fig. 6A, C) or VMAT-2 immunoreactivity was observed in the thymic cortex (Fig. 6B). An obvious immune reaction was developed in the medulla and corticomedullary junction (Fig. 6A and B). At higher magnification VMAT-1 and VMAT-2 immunoreactivity was located in structures reticular in shape (Fig. 6C and D). In the spleen, VMAT-1 and VMAT-2 were found primarily in the white pulp border and to a lesser extent in the white pulp (Fig. 7). Sections of rat thymus exposed to dopamine D1 and D5 receptor antibodies developed a faint immune reaction in the medulla (Fig. 8A and B). Sections exposed to dopamine D2-like antibodies developed a dark-brown immune reaction in the adventitia–media border of thymic arteries (data not shown), in cortico-medullary junction and in the medulla (Fig. 8C–E). Comparatively, immune reaction was more
Fig. 5. RT-PCR of dopamine D1 (panel A), D2 (panel B), D3 (panel C), D4 (panel D) and D5 (panel E) mRNA receptors in membranes obtained from rat thymus (lane 1), spleen (lane 2) and striatum (lane 3). For the migration characteristics of actin used as a reference see Fig. 4, panel C. For the significance of abbreviations see legend to Fig. 4.
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Fig. 6. Sections of rat thymus exposed to VMAT-1 (A, C, E) and VMAT-2 (B, D, F) antibodies, processed for immunohistochemistry and counterstained with methyl green. A and B represent lower magnification pictures; C and D are higher magnification fields of the cortico-medullary junction, whereas pictures E and F are sections next to those shown in A and B respectively incubated with primary antibodies adsorbed with peptides used to generate them to verify the specificity of the reaction. No VMAT-1 immunostaining was observed in thymic cortex, whereas an immune reaction was observed in the cortico-medullary junction and to a lesser extent in the medulla. A similar pattern was observed for VAMT-2 immunoreactivity, but with a lighter immunostaining compared to VMAT-1. For the significance of abbreviations see legend to Fig. 1. Calibration bars: A, B, E and F 50 μm; C and D 25 μm.
pronounced in the cortico-medullary junction than in the medulla (Fig. 8C–E). In the spleen sections exposed to dopamine D1 and D5 receptor antibodies developed a faint immunoreaction in the white pulp border (see below). Sections exposed to dopamine D2-like receptor antibodies developed a dark-brown immune reaction in the adventitia–media border of splenic arteries (data not shown). Dopamine D2, D3 and D4 receptor protein immunoreactivity was localized in the white pulp border and to a lesser extent in the white pulp (Fig. 9A–D). A similar localization was observed for both D1 (data not shown) and D5 receptors (Fig. 9E). Comparatively in the spleen, dopamine D2-like receptor immunoreactivity was more pronounced than dopamine D1-like receptor protein immunoreactivity (Fig. 9). 4. Discussion Membrane (DAT) and vesicular (VMAT –1 and –2) transport systems for monoamines (dopamine, noradrenaline and serotonin) are involved in the regulation on synaptic communication and neuronal plasticity (Levitt et al., 1997; Sherren and Pappas, 2005). Increasing evidence indicates that DAT and VMAT-2 transporters are probably the most specific markers of dopaminergic neurons (Masson et al., 1999). The
possibility that immune organs such as thymus and spleen express a dopaminergic system was first suggested by studies identifying dopamine receptors in these organs (for a review see Mignini et al., 2003). In the present work, thymic and splenic stores of dopamine were analyzed immunohistochemically using antibodies raised against dopamine itself (Yang et al., 1991). This approach was pursued in view of the difficulties reported by other studies to demonstrate dopamine localization in the thymus probably due to the low concentrations of the catecholamine (Pilipović et al., 2008). Moreover, VMAT-1 and 2, and D1-like and D2-like dopamine receptors were characterized in thymus and spleen by several techniques. The results of Western blot and RT-PCR analysis are consistent with those obtained by previous investigators characterizing these systems in brain or peripheral tissue preparations, including peripheral blood lymphocytes, thymus and spleen (Andersen, 1987; Hitri et al., 1991; Vaughan et al., 1993; Niznik et al., 1995; Masson et al., 1999; Amenta et al., 2001; Mignini et al., 2006). Dopamine immunohistochemistry, which represents an original contribution of this work, revealed the microanatomical localization of the catecholamine primarily in the thymic cortico-medullary junction and in medulla with a reticular distribution. Owing to the resolution limits of microanatomical techniques used, we were unable to identify the cellular populations positive to dopamine. From their morphology
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Fig. 7. Sections of rat spleen exposed to VMAT-1 (A, C, E) and VMAT-2 (B, D, F) antibodies, processed for immunohistochemistry and counterstained with methyl green. A and B represent lower magnification pictures; C and D are higher magnification fields of the white pulp border, whereas pictures E and F are sections next to those shown in A and B respectively incubated with primary antibodies adsorbed with peptides used to generate them to verify the specificity of the reaction. No VMAT-1 immunostaining was observed in the red pulp, a light immunoreactivity was found in the white pulp and a more pronounced immunostaining was seen in the red–white pulp marginal zone. The pattern of distribution of VMAT-2 immunoreactivity was similar, but the density of immune staining was less pronounced compared to VMAT-1. For the significance of abbreviations see legend to Fig. 1. Calibration bars: A, B, E and F 50 μm; C and D 25 μm.
these structures can correspond to processes of thymic epithelial cells or may represent lymphocytes close to these processes. Further work is in progress to clarify this point. The pattern of dopamine immunoreactivity localization is similar to that of VMAT-1 and VMAT-2, as well as of the other dopamine transporter DAT as previously described (Mignini et al., 2006). Hence, the cortico-medullary junction represents the thymic area with the highest expression of dopaminergic markers. The same is true for the white pulp border of the spleen (see below), which is another area in which a remarkable accumulation of lymphocytes is noticeable (Wang et al., 2000). Thymocytes maturation and activation are complex processes of cell multiplication and differentiation, and their regulation is only partially understood (Hayday and Pennington, 2007). Our microanatomical study has shown that in the thymic cortex the dopaminergic system is not expressed. In this portion of the thymus, epithelial cells interact with the earliest lymphocytes immigrants and immature thymocytes participate in intimate cortical lymphoepithelial complex (Pezzano et al., 2001). The observation that dopamine and the catecholamine vesicular transporters are located in the medulla, where resides most of single-positive (CD4+ and CD8+) lymphocytes suggests that the dopaminergic system may play a role in modulating
T cell maturation and in regulating activity of thymic medulla epithelial cells. VMAT-1 is primarily present in endocrine and paracrine cells of peripheral organs (Masson et al., 1999); VMAT-2 is the predominant monoamine vesicular transporter in central nervous system and has been identified also in histamine-containing cells of the stomach, adrenal medulla and blood cells. In the central nervous system, VMAT2 moves cytoplasmic dopamine into synaptic vesicles for storage and subsequent exocytotic release (Zheng et al., 2006). Reduced function and/or quantity of VMAT-2 probably compromises normal functioning of the nerve terminal (Volz et al., 2006; Fei et al., 2008). An apparent particularity of immune organs investigated is the co-expression of VMAT-1 and VMAT-2 that are considered to be mutually exclusive except than in human or rat chromaffin cells where they are coexpressed (Masson et al., 1999). These results are consistent with those obtained in peripheral blood lymphocyte (Amenta et al., 2001) and with evidence that both VMAT-1 and VMAT-2 are more widely expressed during embryonic development (Hansson et al., 1998). VMAT-2 protects dopamine from oxidation, and likely prevents excessive production of dopamine-related free radicals (Xia et al., 2001). Several in vitro studies have shown that dopamine can cause
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Fig. 8. Dopamine receptor protein immunohistochemistry in sections of rat thymus. Sections were incubated with a dopamine D1 receptor protein antibody alone (A) or plus the peptide fragment used to generate it (B) to define the specificity of immune staining, with a dopamine D2 receptor protein antibody (C), a dopamine D3 receptor protein antibody (D) or with a dopamine D4 receptor protein antibody alone (E) or plus the peptide fragment used to generate it (F). No dopamine receptor protein immunostaining was observed in the thymic cortex. A positive immune reaction was visualized in the cortico-medullary junction and to a lesser extent in the medulla. Comparatively, dopamine D2-like receptor (D2, D3 and D4) immunostaining was more pronounced than dopamine D1-like receptor immunostaining. For the significance of abbreviations see legend to Fig. 1. Calibration bar: 50 μm.
apoptosis and lysis of thymocytes (Offen et al., 1995) through a mechanism similar to that observed in the central nervous system (Jaubert et al., 2006). This effect is mediated partially by dopamine transporters and involves reactive oxygen species (ROS) formation as well as oxidative stress. The pro-apoptotic effect of dopamine in vivo remains unclear (Jaubert et al., 2006). In the spleen, dopamine and VMAT-1 and VMAT-2 immunoreactivity is located primarily in the white pulp border, which is an area containing splenic sinuses and is supplied with a rich sympathetic innervation (for a review see Mignini et al., 2003). Splenic red–white pulp marginal zone is enriched in germ-line encoded T-cells (LopesCarvalho et al., 2005). The localization of dopamine and vesicular monoamine transporter immunoreactivity in this area suggests a possible role of dopamine and of dopaminergic mechanisms in the effector phase of the immunocompetent response. From a receptor point of view, this study has shown that dopamine D1-like and D2-like receptors are expressed in the same areas of thymus and spleen where dopamine stores and transporters are apparently accumulated. These data are consistent with the hypothesis that D2-like activity may stimulate mitogenesis in a number of cell types and is implicated in cell differentiation (Swarzenski et al., 1994). The presence
of the dopamine D2 receptor in rat thymus and spleen suggests a role of this receptor subtype in the interactions between the nervous and immune system. D2 receptor exists in two alternatively spliced isoforms, which differ in the insertion of 20 amino acids in the third cytoplasmic loop, termed D2-S (short) and D2-L (long) (Dal Toso et al., 1989; Giros et al., 1989; Monsma et al., 1989; Wolfe and Morris, 1999). In this study we have observed that in the immune organs investigated the D2-L isoform predominates. In NG108-15 cells D2-L receptor isoform was mainly located in the perinuclear compartment around the Golgi apparatus, whereas the D2-S has a plasma membrane localization (Takeuchi and Fukunaga, 2003). The dopamine D2-S isoform induces calcineurin activation (Takeuchi and Fukunaga, 2003). Calcineurin activity and the duration of both calcineurin and Erk pathways activation are relevant for thymocyte lineage differentiation (Adachi and Iwata, 2002). An absolute requirement for calcineurin in positive selection by deletion of the genetic locus encoding the regulatory B1 subunit of calcineurin in thymocytes was suggested (Neilson et al., 2004). Further molecular studies are necessary to define the relevance of the D2-L isoform expression in thymus. The distribution of vesicular dopamine transporters and dopamine receptors, is consistent with our previous data on DAT localization
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Fig. 9. Dopamine receptor protein immunohistochemistry in sections of rat spleen. Sections were incubated with a dopamine D2 receptor protein antibody (A), dopamine D3 receptor protein antibody (B), a dopamine D4 receptor protein antibody alone (C) or plus the peptide fragment used to generate it (D), or with a dopamine D5 receptor protein antibody alone (E) or plus the peptide fragment used to generate it (F) to define the specificity of immune staining. A positive immunostaining more pronounced for dopamine D2-like than for D1like receptors was observed primarily in the white pulp border and to a lesser extent in the white pulp. For the significance of abbreviations see legend to Fig. 1. Calibration bar: 50 μm.
(Mignini et al., 2006) and supports the hypothesis that dopamine released in the lymphoid microenvironment may contribute to neuroimmune modulation in the early phases of thymocytes development and in the effector phases of immune responses in the spleen. Acknowledgement The present study was supported by a grant of the European Commission (Grant ICA2-CT-2000-10047). References Adachi, S., Iwata, M., 2002. Duration of calcineurin and Erk signals regulates CD4/CD8 lineage commitment of thymocytes. Cell. Immunol. 215, 45–53. Ader, D., Cohen, D., Felten, D., 1995. Psychoneuroimmunology: interactions between the nervous system and the immune system. Lancet 345, 99–103. Amenta, F., Bronzetti, E., Cantalamessa, F., El-Assouad, D., Felici, L., Ricci, A., Tayebati, S.K., 2001. Identification of dopamine plasma membrane and vesicular transporters in human peripheral blood lymphocytes. J. Neuroimmunol. 117, 133–142. Andersen, P., 1987. Biochemical and pharmacological characterization of [3H]GBR 12935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex. J. Neurochem. 48, 1887–1896. Carlsson, A., 1987. Perspectives on the discovery of central monoaminergic neurotransmission. Annu. Rev. Neurosci. 10, 19–40. Caronti, B., Calderaro, C., Passarelli, F., Palladini, G., Pontieri, F.E., 1998. Dopamine receptor mRNAs in the rat lymphocytes. Life Sci. 62, 1919–1925.
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Pilipović, I., Vidić-Danković, B., Perišić, M., Radojević, K., Čolić, M., Todorović, V., Leposavić, G., 2008. Sexual dimorphism in the catecholamine-containing thymus microenvironment: a role for gonadal hormones. J. Neuroimmunol. 195, 7–20. Qiu, Y., Peng, Y., Wang, J., 1996. Immunoregolatory role of neurotransmitter. Adv. Neuroimmunol. 6, 223–231. Sherren, N., Pappas, B.A., 2005. Selective acetylcholine and dopamine lesions in neonatal rats produce distinct patterns of cortical dendritic atrophy in adulthood. Neuroscience 136, 445–456. Sibley, D.R., Monsma Jr., F.J., 1992. Molecular biology of dopamine receptors. Trends Pharmacol. Sci. 13, 61–69. Sklar, L.S., Anisman, H., 1981. Stress and cancer. Psychol. Bull. 89, 369–406. Swarzenski, B.C., Tang, L., Oh, Y.J., O'Malley, K.L., Todd, R.D., 1994. Morphogenic potentials of D2, D3, and D4 dopamine receptors revealed in transfected neuronal cell lines. Proc. Natl. Acad. Sci. U. S. A. 18, 649–653. Takeuchi, Y., Fukunaga, K., 2003. Differential subcellular localization of two dopamine D2 receptor isoforms in transfected NG108-15 cells. J. Neurochem. 85, 1064–1074. Tsao, C.W., Lin, Y.S., Cheng, J.T., 1997. Effect of dopamine on immune cell proliferation in mice. Life Sci. 61, 361–371. Vaughan, R.A., Uhl, G.R., Kuhar, M.J., 1993. Dopamine transporter recognition by antipeptide antibodies. Cell. Mol. Neurosci. 4, 209–215. Volz, T.J., Hanson, G.R., Fleckenstein, A.E., 2006. Kinetic analysis of developmental changes in vesicular monoamine transporter-2 function. Synapse 60, 474–477. Wang, S.L., Kutsche, M., DiSciullo, G., Schachner, M., Bogen, S.A., 2000. Selective malformation of the splenic white pulp border in L1-deficient mice. J. Immunol. 165, 2465–2473. Wolfe, S.E., Morris, S.J., 1999. Dopamine D2 receptor isoforms expressed in AtT20 cells differentially couple to G proteins to acutely inhibit high voltage-activated calcium channels. J. Neurochem. 73, 2375–2382. Xia, X.G., Schmidt, N., Teismann, P., Ferger, B., Schulz, J.B., 2001. Dopamine mediates striatal malonate toxicity via dopamine transporter-dependent generation of reactive oxygen species and D2 but not D1 receptor activation. J. Neurochem. 79, 63–70. Yang, C.Y., Lukasiewicz, P., Maguire, G., Werblin, F.S., Yazulla, S., 1991. Amacrine cells in the tiger salamander retina: morphology, physiology, and neurotransmitter identification. J. Comp. Neurol. 312, 19–32. Zheng, G., Dwoskin, L.P., Crooks, P.A., 2006. Vesicular monoamine transporter 2: role as a novel target for drug development. AAPS J. 8, E682–E692.
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
Dopamine, vesicular transporters and dopamine receptor expression and localization in rat thymus and spleen Fiorenzo Mignini ⁎, Daniele Tomassoni, Enea Traini, Francesco Amenta Anatomia Umana, Dipartimento di Medicina Sperimentale e Sanità Pubblica, Università di Camerino, Camerino, Italy
a r t i c l e
i n f o
Article history: Received 11 August 2008 Received in revised form 26 September 2008 Accepted 26 September 2008 Keywords: Dopamine transporter Vesicular monoamine transporter Dopamine receptors Lymphoid organ Western blot analysis Immunohistochemistry
a b s t r a c t The localization of dopamine stores and the expression and localization of vesicular monoamine transporter (VMAT) type-1 and 2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat thymus and spleen by immunohistochemical, immunochemical techniques and by RT-PCR. In the thymus dopamine immunoreactivity was developed in the cortico-medullary junction and in the medulla, but not in the thymic cortex. In the spleen, dopamine stores were found in reticular structures in the white pulp border and in the white pulp, but not in the red one. Both thymus and spleen expressed VMAT-1 and VMAT-2 immunoreactivity as well as dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity. Immunohistochemistry revealed VMAT-1, VMAT-2 and dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity primarily in the thymic cortical-medulla transitional zone and to a lesser extent in the medulla but not in the cortex. In the spleen, VMAT-1, VMAT-2 and dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity was located primarily in the white pulp border and to a lesser extent in the white pulp. These findings indicate that both thymus and spleen express a dopaminergic system characterized by the presence of dopamine, vesicular monoamine transporters and the five subtypes of dopamine receptors. The presence of these dopaminergic markers suggests that dopamine likely originating from immune cells and/or from sympathetic neuroeffector plexus is released in the lymphoid microenvironment. Based on the microanatomical localization of dopaminergic markers investigated, a role of dopamine in maturation and selection of lymphocytes and activation of immune responses is suggested. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Increasing evidence indicates the occurrence of interactions between nervous and immune systems (for a review, see Mignini et al., 2003; Meredith et al., 2005). Central nervous system influences the immune system through two major pathways. They include the neuroendocrine outflow linked to pituitary gland activity and the autonomic innervation of immune organs (Ader et al., 1995; Mignini et al., 2003). Both nervous and immune systems have relevant anatomical connections (Mignini et al., 2003; Meredith et al., 2005). Substances common to nervous and immune systems such as cytokines and neurotransmitters probably represent molecules involved in neuroimmune communications (Qiu et al., 1996). On the other hand, several nervous system disorders may have immune pathogenesis or implications (Sklar and Anisman, 1981) and stressing factors can influence the development and course of diseases (for a review see Mignini et al., 2003). In the nervous system, catecholamines have a key role in neurotransmission and are crucially involved in several physiological and
⁎ Corresponding author. Dipartimento di Medicina Sperimentale e Sanità Pubblica, Via Madonna delle Carceri, 9 62032 Camerino, Italy. Fax: +39 0737 403325. E-mail address: fi[email protected] (F. Mignini). 0165-5728/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.09.018
pathological conditions (Carlsson, 1987). The capability of noradrenergic system and of noradrenaline and adrenaline in modulating immune responses has been extensively investigated (Kohm and Sanders, 2001), whereas less information is available for dopamine. In the thymus catecholamines are located both in neural and nonneuronal compartments. Neural compartment includes sympathetic fibres in which dopamine is co-localized with noradrenaline and probably a sensory dopaminergic system (Katz et al.,1987). Non-neural system contributing to thymic catecholamine levels includes thymocytes with mainly a CD3 high phenotype and thymic epithelial cells localized predominantly on the medullary side of the corticomedullary junction and in the subcapsular cortex (Pilipović et al., 2008). Dopamine exerts several peripheral actions involving primarily the cardiovascular system and the kidney. These actions are mediated through the interaction with five subtypes of dopamine receptors belonging to the dopamine D1-like (D1 and D5) and D2-like receptor super families (D2, D3 and D4) (Sibley and Monsma, 1992; Gingrich and Caron, 1993). Dopamine receptors have been also identified in peripheral blood lymphocytes and it is thought that effects of this catecholamine on immune system are mediated by the interaction with its own receptors expressed on the surface of immune target cells (Oberbeck, 2006). From a functional point of view, high doses of dopamine in vitro can inhibit mitogen-induced proliferation and promote
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Table 1 Antibodies used and their concentrations in immunochemistry and immunohistochemistry experiments Primary antibody
Raised in
Dopamine VMAT1 VMAT2 D1 D2 D3 D4 D5 Actin (C-2)
Mouse Goat Rabbit Rabbit Rabbit Rabbit Rabbit Goat Mouse
Working dilution WB 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500
Producer
Code
Abcam plc Chemicon Chemicon Chemicon Santa Cruz Biotechnology Santa Cruz Biotechnology Chemicon Chemicon Santa Cruz Biotechnology
No. No. No. No. No. No. No. No. No.
IH 1:50 1:100 1:100 1:500 1:500 1:250 1:1000 1:250
ab8892 AB1597P AB1598P AB1765P sc-7522 sc-9114 324405 MAB 5292 sc-8432
Secondary antibody Horseradish peroxidase-conjugated antibodies (WB) Anti-mouse Donkey 1:1000 Santa Cruz Biotechnology Anti-goat Donkey 1:1000 Santa Cruz Biotechnology Anti rabbit Donkey 1:1000 Santa Cruz Biotechnology
No. sc-2314 No. sc-2020 No. sc-2313
Biotin-conjugated antibodies (IH) Anti-mouse Goat 1:200 Anti-goat Donkey 1:200 Anti-rabbit Donkey 1:200
No. AP124B No. PK6105 No. PK6101
Chemicon Vector (Vectastain ABC Kit) Vector (Vectastain ABC Kit)
WB: Western blot analysis; IH: immunohistochemistry.
apoptosis in lymphocytes (Caronti et al., 1998; Kavelaars et al., 2005; Olanow, 2007). Data from in vivo studies provided conflicting results, with evidence for both stimulatory and inhibitory effects of dopamine on lymphocyte function (Cook-Mills et al., 1995; Tsao et al., 1997; Ilani et al., 2004). Receptors are the molecular entities through which dopamine can elicit its biological actions on effector cells. Transporter systems represent another relevant marker of dopamine neurobiology. In the nervous system dopamine plasma membrane transporter and the vesicular monoamine transporters (VMAT) type-1 and type-2, remove the neurotransmitter from the extracellular space, thereby controlling extracellular lifetime of dopamine (Giros and Caron, 1993). Dopamine transporters displaying similar characteristics to those expressed by brain and peripheral tissues are also expressed by human peripheral blood lymphocytes (Amenta et al., 2001; Gordon and Barnes, 2003). A problem not clarified yet, with the exception of the demonstration of dopamine transporter (DAT) in rat thymus and spleen (Mignini et al., 2006), is if immune organs express other dopamine transporters and which anatomical relationships they may have with dopamine receptors. The localization of dopamine stores in immune organs is also still unclear. The present study has investigated in rat thymus and spleen the localization of dopamine stores by immunohistochemistry and dopamine transporter expression by assessing VMAT type-1 and type-2 transporters with immunochemical, RT-PCR and immunohistochemical techniques. These techniques were also used for characterizing and localizing microanatomically dopamine D1-like and D2-like receptor subtypes.
and killed by decapitation. Striatum, thymus, spleen and adrenal medulla of 8 rats were dissected out and used for immunochemistry or RT-PCR analysis. After removal, tissues were weighed and homogenized as detailed below. Thymus and spleen of the remaining 8 rats were removed, washed in ice-cold 0.9% saline solution, embedded in a cryoprotectant medium and frozen in isopentane cooled with liquid nitrogen. This material was used for immunohistochemistry. 2.2. Western blot analysis Samples of thymus and spleen (0.1 ± 0.02 g) were lysed in a Mixer Mill MM300 (Qiagen, Hilden, Germany) with 800 μl of lysed buffer as previously described (Mignini et al., 2006). Equal amounts of protein (40 μg) were separated by 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane by electro blotting in Towbin buffer. Transblotted membranes were incubated with VMAT-1 and VMAT-2 antibodies and dopamine D1, D2, D3, D4 and D5 receptor antibodies, as summarized in Table 1. Membranes were then incubated with corresponding horseradishperoxidase conjugated secondary antibodies (Table 1). The product of immune reaction was visualized by a chemiluminescence detection system (ECL Chemiluminescent detection system, GE Healthcare U.K.), followed by computer-driven densitometry with an IAAS 2000 image analyzer (Delta Sistemi, Rome, Italy). The specificity of immune reaction was assessed using antibodies pre-adsorbed with specific peptides used for generating them. 2.3. RNA extraction and RT-PCR analysis Total RNA was extracted from striatum, adrenal medulla, thymus and spleen using Trizol reagent according to the manufacturer's instructions as previously described (Mignini et al., 2006). mRNA samples (2 μg) were subjected to reverse transcription using the RevertAid Hminus First Strand cDNA synthesis kit (Fermentas, Life Science, M-Medical srl, Cornaredo, Italy). Oligonucleotide primers used are listed in Table 2. For PCR, 1 μl of each cDNA sample was amplified using HotStartTaq Master Mix (Qiagen), containing 10 pmol of each specific primer, for 40 cycles with thermal cycler profile denaturation at 95 °C for 30 sec, primers annealing at temperature different for each specific pair of primers (Table 2), elongation at 72 °C for 30 sec. PCR reactions were preceded by incubation at 95 °C for 15 min and a post-incubation at 72 °C for 10 min. Samples were separated and analyzed by 1.8% agarose gel electrophoresis with DNA molecular size markers as a reference (Mass Ruler, DNA Low Range, Fermentas, Cat. No. SM0383). Resulting amplicons were visualized with an ultra violet light, photographed and their size was determined by an Image Master VDS, Pharmacia Biotech. Table 2 Primers used to assess vesicular monoamine transporters or dopamine receptor subtypes mRNA expression Gene
Primer
Oligonucleotide sequence
Annealing (°C)
Size bp
VMAT-1
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5′-AGA CAG CAA CTC TTC TCT GC-3′ 5′-CTA TCC CTT GCA AGC AGT TGT-3′ 5′-ATC GCT GCA GCC TCC ATC T-3′ 5′-TGC CAC TTT CGG GAA CAC A-3′ 5′-CAT TCT GAA CCT CTG CGT GA-3′ 5′-GTT GTC ATC CTC GGT GTC CT-3′ 5′-GCA GTC GAG CTT TCA GAG CC-3′ 5′-TCT GCG GCT CAT CGT CTT AGG-3′ 5′-GGG GTC ACT GTC CTG GTC TA-3′ 5′-TGG CCC TTA TTG AAA ACT GC-3′ 5′-GAT GTG TTG GAC GCC TTT CT-3′ 5′-TCG GCA TTG AAG ATG GTG TA-3′ 5′-ACC AAG ACA CGG TCT TCC AC-3′ 5′-CAC AGT CAA GCT CCC AGA CA-3′ 5′-TGT CAC CAA CTG GGA CGA TA-3′ 5′-ACC CTC ATA GAT GGG CAC AG-3′
60
175
57
102
57
229
60 56
404 317 169
56
150
56
140
55
280
2. Materials and methods
VMAT-2
2.1. Animals and tissue treatment
D1 D2
Male Wistar rats (n = 16) of 2 months of age (weight 239 ± 20 g) were used. Animals were handled according to internationally accepted principles for care of laboratory animals (European Community Council Directive 86/609, O.J. No. L358, Dec. 18, 1986). Rats were received 4 weeks before experiments and were kept under a constant light–dark cycle (7:00 a.m. to 7:00 p.m. light period), at an ambient temperature of 22 ± 1 °C, with free access to water and laboratory chow. They were then anesthetized with pentobarbital sodium (50 mg/kg, i.p.)
D3 D4 D5 β-actin
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2.4. Immunohistochemistry Thymus and spleen were cut serially at a −20 °C microtome cryostat. Eight μm thick sections were mounted on polylysinated microscope slides and collected in 8 groups of 5 sections each. The first section of each group was stained with hematoxylin and eosin or with a 0.5% toluidine blue solution to verify microanatomical details. The second and fourth sections were used for the immunohistochemical detection of dopamine or VMAT-1, VMAT-2, dopamine D1, D2, D3, D4 and D5 receptors (test section). The third and fifth sections were used to assess the specificity of immune reaction (control sections). Endogenous peroxidase activity was blocked by H2O2 (20 min). Test sections were pre-incubated for 1 h at 4 °C in a 3% normal donkey serum dissolved in 0.1 M phosphate buffered saline (PBS) and 0.3% TritonX-100 to prevent non-specific binding of IgGs. Sections were then incubated overnight at 4 °C with primary antibodies at the concentrations indicated in Table 1. Slides were rinsed several times with PBS at room temperature, and then incubated for 1 h at room temperature in a biotinylated secondary antibody dissolved in 0.3% PBS–TritonX-100. The product of the immune reaction was then
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revealed using a biotin–streptavidin immunostaining kit with 0.05% diamino benzidine (DAB) in 0.1% H2O2 as a chromogen. Sections were then washed, dehydrated in ethanol, and mounted in a synthetic mounting medium and observed under a light microscope (Mignini et al., 2006). 3. Results 3.1. Dopamine immunohistochemistry Fig. 1 shows the localization of tissue stores of dopamine in rat thymus and spleen. In the thymus dopamine immunoreactivity was developed in the cortico-medullary junction (Fig. 1A) and in the medulla, whereas no immune reaction was observed in the thymic cortex (data not shown). At higher magnification dopamine immunoreactivity displayed a reticular localization (Fig. 1A inset). In the spleen, dopamine immunoreactivity was located primarily in the white pulp border and to a lesser extent in the red pulp (Fig. 1D). Similarly as observed in the thymus, immune reaction displayed a flattened and network-like localization (Fig. 1D inset).
Fig. 1. Dopamine immunohistochemistry in sections of rat thymus (pictures A and C) and spleen (pictures D and F). Sections were exposed to anti-dopamine antibody (pictures A and D) or to the antibody previously adsorbed with dopamine to verify the specificity of the reaction (pictures C and F). Micrographs B and E show sections close to those processed for immunohistochemistry stained with hematoxylin and eosin to verify microanatomical details. Note in thymus and spleen the localization of immune reaction in the medulla (M) or white pulp (W) but not in the cortex (C) or red pulp (R) respectively. The highest immunoreactivity was observed in the thymic cortico-medullary junction (CJM) and in the white pulp border (WPB). Insets are a higher magnification of the two areas showing the localization of dopamine immune reaction in reticular structures probably representing epithelial cells of CMJ and splenic marginal sinus cells. Calibration bars: A–C 25 μm; B–D 50 μm (for the inset 12.5 μm).
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Fig. 2. Western blot analysis of vesicular monoamine transporter (VMAT) type-1 (panel A), and VMAT type-2 (panel B) in membranes obtained from rat thymus (lanes 1), spleen (lane 2), adrenal medulla (panel A, lane 3) or striatum (panel B, lane 3). Panel C shows migration characteristics of actin used as a reference.
3.2. Western blot analysis Immunoblots with anti-VMAT-1 and anti-VMAT-2 antibodies developed a single band of immunoreactivity of approximately 55 kDa and 68 kDa respectively in thymus (Fig. 2, panels A and B lane 1) and spleen (Fig. 2, panels A and B lane 2) lysates. A similar pattern of immunoreactivity was observed for VMAT-1 in the adrenal medulla (Fig. 2, panel a, lane 3) and for VMAT-2 in striatum used as reference tissues (Fig. 2, panel B, lane 3). Immunoblots performed with anti-D1 and anti-D5 dopamine receptor antibodies revealed the development of a single band of approximately 50 kDa and 55 kDa respectively in thymus and spleen (Fig. 3, panels A and E, lanes 1 and 2) as well as in the striatum (Fig. 3, panels A and E, lane 3). Immunoblots exposed to anti-D2 dopamine receptors revealed the development of two bands of approximately 53 and 48 kDa in membrane preparations of thymus, spleen and striatum (Fig. 3, panel B). Exposure of tissue preparations to anti-D3 and anti-D4 dopamine receptors antibodies revealed the development of a single band of approximately 50 and 44 kDa respectively. These bands were similar for thymus, spleen and striatum (Fig. 3, panels C and D lanes 1–3). 3.3. RT-PCR analysis RT-PCR analysis revealed VMAT-1 mRNA expression in rat thymus, spleen and adrenal medulla (Fig. 4, panel A, lanes 1, 2 and 3 respectively) and VMAT-2 mRNA expression in rat thymus, spleen and striatum (Fig. 4, panel B, lanes 1, 2 and 3 respectively). RT-PCR revealed the presence of mRNA for D1 receptor and D5 receptor in thymus, spleen and striatum (Fig. 5, panels A and E, lanes 1, 2 and 3 respectively). Analysis of D2 receptor revealed in the thymus and spleen the predominant expression of the long isoform, whereas striatum expressed to a similar extent both long and short isoforms (Fig. 5, panel B, lanes 1, 2 and 3 respectively). RT-PCR analysis showed the expression of D3 (Fig. 5, panel C) and D4 (Fig. 5, panel D) dopamine
Fig. 3. Western blot analysis of dopamine D1 (panel A), D2 (panel B), D3 (panel C), D4 (panel D) and D5 (panel E) receptors in membranes obtained from rat thymus (lane 1), spleen (lane 2) and striatum (lane 3). For the migration characteristics of actin used as a reference see Fig. 2, panel C.
Fig. 4. RT-PCR of VMAT-1 mRNA (panel A), and VMAT-2 (panel B) in membranes obtained from rat thymus (lanes 1), spleen (lane 2), adrenal medulla (panel A, lane 3) or striatum (panel B, lane 3). Panel C shows migration characteristics of actin used as a reference. LD: DNA ladder; bp: base pair.
receptor mRNAs in the two immune organs examined as well as in the striatum (Fig. 5, panels C and D, lanes 1, 2 and 3 respectively). 3.4. Immunohistochemistry Section of rat thymus exposed to VMAT-1 or VMAT-2 antibodies developed a dark-brown immune reaction in the adventitia–media junction of arteries and arterioles located in the septa of the connective tissue (data not shown). No VMAT-1 (Fig. 6A, C) or VMAT-2 immunoreactivity was observed in the thymic cortex (Fig. 6B). An obvious immune reaction was developed in the medulla and corticomedullary junction (Fig. 6A and B). At higher magnification VMAT-1 and VMAT-2 immunoreactivity was located in structures reticular in shape (Fig. 6C and D). In the spleen, VMAT-1 and VMAT-2 were found primarily in the white pulp border and to a lesser extent in the white pulp (Fig. 7). Sections of rat thymus exposed to dopamine D1 and D5 receptor antibodies developed a faint immune reaction in the medulla (Fig. 8A and B). Sections exposed to dopamine D2-like antibodies developed a dark-brown immune reaction in the adventitia–media border of thymic arteries (data not shown), in cortico-medullary junction and in the medulla (Fig. 8C–E). Comparatively, immune reaction was more
Fig. 5. RT-PCR of dopamine D1 (panel A), D2 (panel B), D3 (panel C), D4 (panel D) and D5 (panel E) mRNA receptors in membranes obtained from rat thymus (lane 1), spleen (lane 2) and striatum (lane 3). For the migration characteristics of actin used as a reference see Fig. 4, panel C. For the significance of abbreviations see legend to Fig. 4.
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Fig. 6. Sections of rat thymus exposed to VMAT-1 (A, C, E) and VMAT-2 (B, D, F) antibodies, processed for immunohistochemistry and counterstained with methyl green. A and B represent lower magnification pictures; C and D are higher magnification fields of the cortico-medullary junction, whereas pictures E and F are sections next to those shown in A and B respectively incubated with primary antibodies adsorbed with peptides used to generate them to verify the specificity of the reaction. No VMAT-1 immunostaining was observed in thymic cortex, whereas an immune reaction was observed in the cortico-medullary junction and to a lesser extent in the medulla. A similar pattern was observed for VAMT-2 immunoreactivity, but with a lighter immunostaining compared to VMAT-1. For the significance of abbreviations see legend to Fig. 1. Calibration bars: A, B, E and F 50 μm; C and D 25 μm.
pronounced in the cortico-medullary junction than in the medulla (Fig. 8C–E). In the spleen sections exposed to dopamine D1 and D5 receptor antibodies developed a faint immunoreaction in the white pulp border (see below). Sections exposed to dopamine D2-like receptor antibodies developed a dark-brown immune reaction in the adventitia–media border of splenic arteries (data not shown). Dopamine D2, D3 and D4 receptor protein immunoreactivity was localized in the white pulp border and to a lesser extent in the white pulp (Fig. 9A–D). A similar localization was observed for both D1 (data not shown) and D5 receptors (Fig. 9E). Comparatively in the spleen, dopamine D2-like receptor immunoreactivity was more pronounced than dopamine D1-like receptor protein immunoreactivity (Fig. 9). 4. Discussion Membrane (DAT) and vesicular (VMAT –1 and –2) transport systems for monoamines (dopamine, noradrenaline and serotonin) are involved in the regulation on synaptic communication and neuronal plasticity (Levitt et al., 1997; Sherren and Pappas, 2005). Increasing evidence indicates that DAT and VMAT-2 transporters are probably the most specific markers of dopaminergic neurons (Masson et al., 1999). The
possibility that immune organs such as thymus and spleen express a dopaminergic system was first suggested by studies identifying dopamine receptors in these organs (for a review see Mignini et al., 2003). In the present work, thymic and splenic stores of dopamine were analyzed immunohistochemically using antibodies raised against dopamine itself (Yang et al., 1991). This approach was pursued in view of the difficulties reported by other studies to demonstrate dopamine localization in the thymus probably due to the low concentrations of the catecholamine (Pilipović et al., 2008). Moreover, VMAT-1 and 2, and D1-like and D2-like dopamine receptors were characterized in thymus and spleen by several techniques. The results of Western blot and RT-PCR analysis are consistent with those obtained by previous investigators characterizing these systems in brain or peripheral tissue preparations, including peripheral blood lymphocytes, thymus and spleen (Andersen, 1987; Hitri et al., 1991; Vaughan et al., 1993; Niznik et al., 1995; Masson et al., 1999; Amenta et al., 2001; Mignini et al., 2006). Dopamine immunohistochemistry, which represents an original contribution of this work, revealed the microanatomical localization of the catecholamine primarily in the thymic cortico-medullary junction and in medulla with a reticular distribution. Owing to the resolution limits of microanatomical techniques used, we were unable to identify the cellular populations positive to dopamine. From their morphology
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Fig. 7. Sections of rat spleen exposed to VMAT-1 (A, C, E) and VMAT-2 (B, D, F) antibodies, processed for immunohistochemistry and counterstained with methyl green. A and B represent lower magnification pictures; C and D are higher magnification fields of the white pulp border, whereas pictures E and F are sections next to those shown in A and B respectively incubated with primary antibodies adsorbed with peptides used to generate them to verify the specificity of the reaction. No VMAT-1 immunostaining was observed in the red pulp, a light immunoreactivity was found in the white pulp and a more pronounced immunostaining was seen in the red–white pulp marginal zone. The pattern of distribution of VMAT-2 immunoreactivity was similar, but the density of immune staining was less pronounced compared to VMAT-1. For the significance of abbreviations see legend to Fig. 1. Calibration bars: A, B, E and F 50 μm; C and D 25 μm.
these structures can correspond to processes of thymic epithelial cells or may represent lymphocytes close to these processes. Further work is in progress to clarify this point. The pattern of dopamine immunoreactivity localization is similar to that of VMAT-1 and VMAT-2, as well as of the other dopamine transporter DAT as previously described (Mignini et al., 2006). Hence, the cortico-medullary junction represents the thymic area with the highest expression of dopaminergic markers. The same is true for the white pulp border of the spleen (see below), which is another area in which a remarkable accumulation of lymphocytes is noticeable (Wang et al., 2000). Thymocytes maturation and activation are complex processes of cell multiplication and differentiation, and their regulation is only partially understood (Hayday and Pennington, 2007). Our microanatomical study has shown that in the thymic cortex the dopaminergic system is not expressed. In this portion of the thymus, epithelial cells interact with the earliest lymphocytes immigrants and immature thymocytes participate in intimate cortical lymphoepithelial complex (Pezzano et al., 2001). The observation that dopamine and the catecholamine vesicular transporters are located in the medulla, where resides most of single-positive (CD4+ and CD8+) lymphocytes suggests that the dopaminergic system may play a role in modulating
T cell maturation and in regulating activity of thymic medulla epithelial cells. VMAT-1 is primarily present in endocrine and paracrine cells of peripheral organs (Masson et al., 1999); VMAT-2 is the predominant monoamine vesicular transporter in central nervous system and has been identified also in histamine-containing cells of the stomach, adrenal medulla and blood cells. In the central nervous system, VMAT2 moves cytoplasmic dopamine into synaptic vesicles for storage and subsequent exocytotic release (Zheng et al., 2006). Reduced function and/or quantity of VMAT-2 probably compromises normal functioning of the nerve terminal (Volz et al., 2006; Fei et al., 2008). An apparent particularity of immune organs investigated is the co-expression of VMAT-1 and VMAT-2 that are considered to be mutually exclusive except than in human or rat chromaffin cells where they are coexpressed (Masson et al., 1999). These results are consistent with those obtained in peripheral blood lymphocyte (Amenta et al., 2001) and with evidence that both VMAT-1 and VMAT-2 are more widely expressed during embryonic development (Hansson et al., 1998). VMAT-2 protects dopamine from oxidation, and likely prevents excessive production of dopamine-related free radicals (Xia et al., 2001). Several in vitro studies have shown that dopamine can cause
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Fig. 8. Dopamine receptor protein immunohistochemistry in sections of rat thymus. Sections were incubated with a dopamine D1 receptor protein antibody alone (A) or plus the peptide fragment used to generate it (B) to define the specificity of immune staining, with a dopamine D2 receptor protein antibody (C), a dopamine D3 receptor protein antibody (D) or with a dopamine D4 receptor protein antibody alone (E) or plus the peptide fragment used to generate it (F). No dopamine receptor protein immunostaining was observed in the thymic cortex. A positive immune reaction was visualized in the cortico-medullary junction and to a lesser extent in the medulla. Comparatively, dopamine D2-like receptor (D2, D3 and D4) immunostaining was more pronounced than dopamine D1-like receptor immunostaining. For the significance of abbreviations see legend to Fig. 1. Calibration bar: 50 μm.
apoptosis and lysis of thymocytes (Offen et al., 1995) through a mechanism similar to that observed in the central nervous system (Jaubert et al., 2006). This effect is mediated partially by dopamine transporters and involves reactive oxygen species (ROS) formation as well as oxidative stress. The pro-apoptotic effect of dopamine in vivo remains unclear (Jaubert et al., 2006). In the spleen, dopamine and VMAT-1 and VMAT-2 immunoreactivity is located primarily in the white pulp border, which is an area containing splenic sinuses and is supplied with a rich sympathetic innervation (for a review see Mignini et al., 2003). Splenic red–white pulp marginal zone is enriched in germ-line encoded T-cells (LopesCarvalho et al., 2005). The localization of dopamine and vesicular monoamine transporter immunoreactivity in this area suggests a possible role of dopamine and of dopaminergic mechanisms in the effector phase of the immunocompetent response. From a receptor point of view, this study has shown that dopamine D1-like and D2-like receptors are expressed in the same areas of thymus and spleen where dopamine stores and transporters are apparently accumulated. These data are consistent with the hypothesis that D2-like activity may stimulate mitogenesis in a number of cell types and is implicated in cell differentiation (Swarzenski et al., 1994). The presence
of the dopamine D2 receptor in rat thymus and spleen suggests a role of this receptor subtype in the interactions between the nervous and immune system. D2 receptor exists in two alternatively spliced isoforms, which differ in the insertion of 20 amino acids in the third cytoplasmic loop, termed D2-S (short) and D2-L (long) (Dal Toso et al., 1989; Giros et al., 1989; Monsma et al., 1989; Wolfe and Morris, 1999). In this study we have observed that in the immune organs investigated the D2-L isoform predominates. In NG108-15 cells D2-L receptor isoform was mainly located in the perinuclear compartment around the Golgi apparatus, whereas the D2-S has a plasma membrane localization (Takeuchi and Fukunaga, 2003). The dopamine D2-S isoform induces calcineurin activation (Takeuchi and Fukunaga, 2003). Calcineurin activity and the duration of both calcineurin and Erk pathways activation are relevant for thymocyte lineage differentiation (Adachi and Iwata, 2002). An absolute requirement for calcineurin in positive selection by deletion of the genetic locus encoding the regulatory B1 subunit of calcineurin in thymocytes was suggested (Neilson et al., 2004). Further molecular studies are necessary to define the relevance of the D2-L isoform expression in thymus. The distribution of vesicular dopamine transporters and dopamine receptors, is consistent with our previous data on DAT localization
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Fig. 9. Dopamine receptor protein immunohistochemistry in sections of rat spleen. Sections were incubated with a dopamine D2 receptor protein antibody (A), dopamine D3 receptor protein antibody (B), a dopamine D4 receptor protein antibody alone (C) or plus the peptide fragment used to generate it (D), or with a dopamine D5 receptor protein antibody alone (E) or plus the peptide fragment used to generate it (F) to define the specificity of immune staining. A positive immunostaining more pronounced for dopamine D2-like than for D1like receptors was observed primarily in the white pulp border and to a lesser extent in the white pulp. For the significance of abbreviations see legend to Fig. 1. Calibration bar: 50 μm.
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